0	Protein Phosphatase 2A Is Targeted to Cell Division Control, otherFeat=[]-->, belongsTo=title
1	Protein 6 by a Calcium-binding Regulatory Subunit*S, otherFeat=[]-->, belongsTo=parrnote
2	Received for publication, December 19, 2007, and in revised form, February 29, 2008 Published, JBC Papers in Press, April 8, 2008, DOI 10.1074/jbc.M710313200, otherFeat=[]-->, belongsTo=parrnote
3	Anthony J. Davis1, Zhen Yan?, Bobbie Martinez, and Marc C. Mumby2, otherFeat=[]-->, belongsTo=title
4	From the Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041, otherFeat=[]-->, belongsTo=title
5	and ?the Division of Cardiology, Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710, otherFeat=[]-->, belongsTo=title
6	The cell division control protein 6 (Cdc6) is essential for for-, otherFeat=[]-->, belongsTo=parrnote
7	mation of pre-replication complexes at origins of DNA replica-, otherFeat=[]-->, belongsTo=parrnote
8	tion. Phosphorylation of Cdc6 by cyclin-dependent kinases, otherFeat=[]-->, belongsTo=parrnote
9	inhibits ubiquitination of Cdc6 by APC/Ccdh1 and degradation, otherFeat=[]-->, belongsTo=parrnote
10	by the proteasome. Experiments described here show that the, otherFeat=[]-->, belongsTo=parrnote
11	PR70 member of the PPP2R3 family of regulatory subunits tar-, otherFeat=[]-->, belongsTo=parrnote
12	gets protein phosphatase 2A (PP2A) to Cdc6. Interaction with, otherFeat=[]-->, belongsTo=parrnote
13	Cdc6 is mediated by residues within the C terminus of PR70,, otherFeat=[]-->, belongsTo=parrnote
14	whereas interaction with PP2A requires N-terminal sequences, otherFeat=[]-->, belongsTo=parrnote
15	conserved within the PPP2R3 family. Two functional EF-hand, otherFeat=[]-->, belongsTo=parrnote
16	calcium-binding motifs mediate a calcium-enhanced interac-, otherFeat=[]-->, belongsTo=parrnote
17	tion of PR70 with PP2A. Calcium has no effect on the interac-, otherFeat=[]-->, belongsTo=parrnote
18	tion of PR70 with Cdc6 but enhances the association of PP2A, otherFeat=[]-->, belongsTo=parrnote
19	with Cdc6 through its effects on PR70. Knockdown of PR70 by, otherFeat=[]-->, belongsTo=parrnote
20	RNA interference results in an accumulation of endogenous and, otherFeat=[]-->, belongsTo=parrnote
21	expressed Cdc6 protein that is dependent on the cyclin-depend-, otherFeat=[]-->, belongsTo=parrnote
22	ent protein kinase phosphorylation sites on Cdc6. Knockdown, otherFeat=[]-->, belongsTo=parrnote
23	of PR70 also causes G1 arrest, suggesting that PR70 function is, otherFeat=[]-->, belongsTo=parrnote
24	critical for progression into S phase. These observations indi-, otherFeat=[]-->, belongsTo=parrnote
25	cate that PP2A can be targeted in a calcium-regulated manner to, otherFeat=[]-->, belongsTo=parrnote
26	Cdc6 via the PR70 subunit, where it plays a role in regulating, otherFeat=[]-->, belongsTo=parrnote
27	protein phosphorylation and stability., otherFeat=[]-->, belongsTo=parrnote
28	Precise regulation of DNA replication is necessary to ensure, otherFeat=[]-->, belongsTo=parr
29	that daughter cells receive a complete and intact genome dur-, otherFeat=[]-->, belongsTo=parr
30	ing mitosis. A crucial step in regulating DNA replication is the, otherFeat=[]-->, belongsTo=parr
31	assembly of pre-replicative complexes at origins of replication, otherFeat=[]-->, belongsTo=parr
32	(1). Coordination of DNA replication with the cell cycle is, otherFeat=[]-->, belongsTo=parr
33	achieved through a periodic accumulation and destruction of, otherFeat=[]-->, belongsTo=parr
34	proteins involved in formation of pre-RCs3 is mediated by, otherFeat=[]-->, belongsTo=parr
35	cyclin-dependent kinases (CDKs) and the E3 ubiquitin ligase,, otherFeat=[]-->, belongsTo=parr
36	anaphase promoting complex/cyclosome (2). The mammalian, otherFeat=[]-->, belongsTo=parr
37	Cdc6 protein is required for DNA replication and acts in con-, otherFeat=[]-->, belongsTo=parr
38	junction with the Cdt1 protein to recruit the mini-chromosome, otherFeat=[]-->, belongsTo=parr
39	maintenance complex into pre-RCs (1, 3, 4). Mammalian cells, otherFeat=[]-->, belongsTo=parr
40	have multiple mechanisms to ensure that pre-RCs only assem-, otherFeat=[]-->, belongsTo=parr
41	ble during late M and G1, including regulation of the levels and, otherFeat=[]-->, belongsTo=parr
42	function of Cdc6 (2)., otherFeat=[]-->, belongsTo=parr
43	Mammalian Cdc6 is regulated by phosphorylation of multi-, otherFeat=[]-->, belongsTo=parr
44	ple sites within its N-terminal domain by cyclin-dependent, otherFeat=[]-->, belongsTo=parr
45	protein kinases. Cdc6 is phosphorylated at canonical CDK sites,, otherFeat=[]-->, belongsTo=parr
46	including serines 54, 74, and 106 of human Cdc6 (5, 6). Exper-, otherFeat=[]-->, belongsTo=parr
47	iments with exogenously expressed protein have shown that, otherFeat=[]-->, belongsTo=parr
48	phosphorylation can regulate the nuclear localization of Cdc6, otherFeat=[]-->, belongsTo=parr
49	(5, 7?9). However, other studies have shown that a subpopula-, otherFeat=[]-->, belongsTo=parr
50	tion of endogenous Cdc6 remains in the nucleus, bound to, otherFeat=[]-->, belongsTo=parr
51	chromatin, throughout the cell cycle (10 ?12). Phosphorylation, otherFeat=[]-->, belongsTo=parr
52	of Cdc6 also plays an important role in regulating the stability of, otherFeat=[]-->, belongsTo=parr
53	Cdc6. The N-terminal domain of Cdc6 contains RXXL (D box), otherFeat=[]-->, belongsTo=parr
54	and KEN (KEN box) destruction motifs, which are binding sites, otherFeat=[]-->, belongsTo=parr
55	for the form of the APC/C containing the cdh1-targeting sub-, otherFeat=[]-->, belongsTo=parr
56	unit (13). Cdc6 is polyubiquitinated and targeted for degrada-, otherFeat=[]-->, belongsTo=parr
57	tion by APC/Ccdh1, which prevents formation of pre-RCs in, otherFeat=[]-->, belongsTo=parr
58	quiescent cells and during early G1 by maintaining low levels of, otherFeat=[]-->, belongsTo=parr
59	Cdc6 (14). Phosphorylation of Cdc6 by CDKs protects the pro-, otherFeat=[]-->, belongsTo=parr
60	tein from degradation by blocking recognition by cdh1 result-, otherFeat=[]-->, belongsTo=parr
61	ing in stabilization of Cdc6 during a window of time that allows, otherFeat=[]-->, belongsTo=parr
62	formation of pre-RCs during G1 (15). The importance of CDK-, otherFeat=[]-->, belongsTo=parr
63	mediated stabilization of Cdc6 is also supported by evidence, otherFeat=[]-->, belongsTo=parr
64	showing that the cell cycle arrest caused by DNA damage is due, otherFeat=[]-->, belongsTo=parr
65	to dephosphorylation and degradation of Cdc6 (16)., otherFeat=[]-->, belongsTo=parr
66	Because the extent of Cdc6 phosphorylation is controlled by, otherFeat=[]-->, belongsTo=parr
67	the opposing actions of cyclin-dependent kinases and protein, otherFeat=[]-->, belongsTo=parr
68	phosphatases, dephosphorylation of Cdc6 can also control for-, otherFeat=[]-->, belongsTo=parr
69	mation of pre-RCs. Much less is known about mechanisms that, otherFeat=[]-->, belongsTo=parr
70	regulate Cdc6 dephosphorylation. A previous study identified a, otherFeat=[]-->, belongsTo=parr
71	fragment of PR70 as a member of the PPP2R3 family of PP2A, otherFeat=[]-->, belongsTo=parr
72	regulatory subunits that interacted with Cdc6 and implicated, otherFeat=[]-->, belongsTo=parr
73	PP2A in regulating Cdc6 phosphorylation (17). The major, otherFeat=[]-->, belongsTo=parr
74	forms of PP2A contain a dimeric core complex composed of a, otherFeat=[]-->, belongsTo=parr
75	scaffold (A) and a catalytic subunit (C). The AC core dimer, otherFeat=[]-->, belongsTo=parr
76	associates with regulatory subunits that form heterotrimeric, otherFeat=[]-->, belongsTo=parr
77	holoenzymes and target the catalytic subunit to specific phos-, otherFeat=[]-->, belongsTo=parr
78	phoprotein substrates (18 ?20). In this study, the mechanism, otherFeat=[]-->, belongsTo=parr
79	and functional consequences of targeting of PP2A to Cdc6 by, otherFeat=[]-->, belongsTo=parr
80	PR70 were investigated. The results show that PR70 interacts, otherFeat=[]-->, belongsTo=parr
81	with PP2A and Cdc6 through distinct regions of the protein,, otherFeat=[]-->, belongsTo=parr
82	* This work was supported, in whole or in part, by National Institutes of Health, otherFeat=[]-->, belongsTo=parrnote
83	Grant GM49505. The costs of publication of this article were defrayed in, otherFeat=[]-->, belongsTo=parrnote
84	part by the payment of page charges. This article must therefore be hereby, otherFeat=[]-->, belongsTo=parrnote
85	marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to, otherFeat=[]-->, belongsTo=parrnote
86	indicate this fact., otherFeat=[]-->, belongsTo=parrnote
87	S The on-line version of this article (available at http://www.jbc.org) contains, otherFeat=[]-->, belongsTo=parrnote
88	supplemental Table S1 and Figs. S1 and S2., otherFeat=[]-->, belongsTo=parrnote
89	1 Supported by National Institutes of Health Pharmacological Sciences Train-, otherFeat=[]-->, belongsTo=parrnote
90	ing Grant T32 GM07062., otherFeat=[]-->, belongsTo=parrnote
91	2 To whom correspondence should be addressed: Dept. of Pharmacology,, otherFeat=[]-->, belongsTo=parrnote
92	University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,, otherFeat=[]-->, belongsTo=parrnote
93	Dallas, TX 75390-9041. Tel.: 214-645-6152; Fax: 214-645-6151; E-mail:, otherFeat=[]-->, belongsTo=parrnote
94	marc.mumby@utsouthwestern.edu., otherFeat=[]-->, belongsTo=parrnote
95	3 The abbreviations used are: pre-RC, pre-replicative complex; PP2A, protein, otherFeat=[]-->, belongsTo=parrnote
96	phosphatases 2A; APC/C, anaphase promoting complex/cyclosome;, otherFeat=[]-->, belongsTo=parrnote
97	siRNA, small interfering RNA; Cdk, cyclin-dependent kinase; E3, ubiquitin-, otherFeat=[]-->, belongsTo=parrnote
98	protein isopeptide ligase; PBS, phosphate-buffered saline; IP, immunopre-, otherFeat=[]-->, belongsTo=parrnote
99	cipitation; GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglu-, otherFeat=[]-->, belongsTo=parrnote
100	tinin; CMV, cytomegalovirus; Cdc6, cell division control protein 6., otherFeat=[]-->, belongsTo=parrnote
101	THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 23, pp. 16104 ?16114, June 6, 2008, otherFeat=[]-->, belongsTo=nota_cab_pie
102	? 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A., otherFeat=[]-->, belongsTo=nota_cab_pie
103	16104 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
104	VOLUME 283 ? NUMBER 23 ? JUNE 6, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie
105	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
106	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
107	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
108	de, otherFeat=[]-->, belongsTo=nota_cab_pie
109	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
110	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
111	on, otherFeat=[]-->, belongsTo=nota_cab_pie
112	May, otherFeat=[]-->, belongsTo=nota_cab_pie
113	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
114	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
115	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
116	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
117	from, otherFeat=[]-->, belongsTo=nota_cab_pie
118	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
119	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
120	that the association of PP2A to Cdc6 is enhanced by calcium, otherFeat=[]-->, belongsTo=parr
121	binding to PR70, and that loss of PR70 causes increased levels of, otherFeat=[]-->, belongsTo=parr
122	Cdc6 and G1 arrest., otherFeat=[]-->, belongsTo=parr
123	EXPERIMENTAL PROCEDURES, otherFeat=['U']-->, belongsTo=title
124	Cloning of Full-length PR70--A human expressed sequence, otherFeat=[]-->, belongsTo=parr
125	tag encoding the PR70 start codon was identified in the human, otherFeat=[]-->, belongsTo=parr
126	expressed sequence tag data base using the MegaBLAST tool, otherFeat=[]-->, belongsTo=parr
127	(www.ncbi.nlm.nih.gov/BLAST/) with the assembled PR70, otherFeat=[]-->, belongsTo=parr
128	sequence (21). A PR70 cDNA was constructed using the PR48, otherFeat=[]-->, belongsTo=parr
129	cDNA and the IMAGE Human Clone ID 5728169 (GenBankTM, otherFeat=[]-->, belongsTo=parr
130	accession number BM544432), purchased from Invitrogen,, otherFeat=[]-->, belongsTo=parr
131	using an internal NcoI restriction site present in the common, otherFeat=[]-->, belongsTo=parr
132	region of BM54432 and PR48. A PCR fragment containing the, otherFeat=[]-->, belongsTo=parr
133	translational start codon, the 5 -end, and the 3 -NcoI site of, otherFeat=[]-->, belongsTo=parr
134	BM54432 was generated using the BM54432 cDNA as template, otherFeat=[]-->, belongsTo=parr
135	with the PCR primers: 5 -CGGGATCCATGCCGCCCGGCA-, otherFeat=[]-->, belongsTo=parr
136	AAGT-3 (sense strand) and 5 -GCGCCTTGATCCGGC-3, otherFeat=[]-->, belongsTo=parr
137	(antisense strand). The PCR product was digested with the, otherFeat=[]-->, belongsTo=parr
138	restriction enzymes BamHI and NcoI. The 3 portion of the, otherFeat=[]-->, belongsTo=parr
139	PR48 cDNA was excised from the PR48 cDNA (17) using NcoI, otherFeat=[]-->, belongsTo=parr
140	and HindIII, and the fragments were ligated and subcloned into, otherFeat=[]-->, belongsTo=parr
141	the pCMV-Tag2B vector (Stratagene) digested with BamHI, otherFeat=[]-->, belongsTo=parr
142	and HindIII. The resulting construct encoded a full-length, otherFeat=[]-->, belongsTo=parr
143	PR70 cDNA fused to an N-terminal FLAG epitope tag. The, otherFeat=[]-->, belongsTo=parr
144	sequence was verified by automated sequencing., otherFeat=[]-->, belongsTo=parr
145	Cell Culture, Transfection, and RNA Interference--COS-7,, otherFeat=[]-->, belongsTo=parr
146	HeLa, and U2OS cells were maintained at 37 ?C in Dulbecco's, otherFeat=[]-->, belongsTo=parr
147	modified Eagle's medium containing 10% fetal bovine serum in, otherFeat=[]-->, belongsTo=parr
148	an atmosphere of 5% CO2. U2OS, obtained from the ATCC, is a, otherFeat=[]-->, belongsTo=parr
149	human osteosarcoma cell line that expresses wild-type p53. For, otherFeat=[]-->, belongsTo=parr
150	transient expression of proteins, cells were transfected with, otherFeat=[]-->, belongsTo=parr
151	expression plasmids using Lipofectamine 2000 (Invitrogen), otherFeat=[]-->, belongsTo=parr
152	according to the manufacturer's protocol. Cells were harvested, otherFeat=[]-->, belongsTo=parr
153	either 24 or 48 h after transfection. Transfection with small, otherFeat=[]-->, belongsTo=parr
154	interfering RNA to knock down PR70 was carried out using, otherFeat=[]-->, belongsTo=parr
155	Oligofectamine (Invitrogen) following the manufacturer's pro-, otherFeat=[]-->, belongsTo=parr
156	tocol. Annealed duplex siRNAs were purchased from Dharma-, otherFeat=[]-->, belongsTo=parr
157	con and had the following sequences: PR70-1, 5 -AGCCGG-, otherFeat=[]-->, belongsTo=parr
158	UCCUGAAGAUGAAdTdT-3 (sense strand) and PR70-2,, otherFeat=[]-->, belongsTo=parr
159	5 -AAAGCAUUCCGACCUUCUAdTdT-3 (sense strand)., otherFeat=[]-->, belongsTo=parr
160	Controls included an siRNA that knocks down the MEKK2, otherFeat=[]-->, belongsTo=parr
161	protein kinase (22), an siRNA that knocks down protein phos-, otherFeat=[]-->, belongsTo=parr
162	phatase 5 (23), and an siRNA corresponding to the sequence of, otherFeat=[]-->, belongsTo=parr
163	firefly luciferase (5 -TCGAAGTATTCCGCGTACGdTdT-3 )., otherFeat=[]-->, belongsTo=parr
164	Cells were lysed and analyzed by immunoblotting 48 h after, otherFeat=[]-->, belongsTo=parr
165	transfection. In some experiments, cells were co-transfected, otherFeat=[]-->, belongsTo=parr
166	with PR70 siRNA and expression plasmids encoding wild-type, otherFeat=[]-->, belongsTo=parr
167	Cdc6 or Cdc6 mutants in which all three N-terminal phospho-, otherFeat=[]-->, belongsTo=parr
168	rylation sites were mutated to alanine (AAA-Cdc6) or aspartic, otherFeat=[]-->, belongsTo=parr
169	acid (DDD-Cdc6) using Lipofectamine 2000 and harvested 48 h, otherFeat=[]-->, belongsTo=parr
170	later. The cDNAs encoding phosphorylation site mutants of, otherFeat=[]-->, belongsTo=parr
171	Cdc6 were prepared using a PCR-based site-directed mutagen-, otherFeat=[]-->, belongsTo=parr
172	esis kit (Invitrogen) according to the manufacturer's protocol., otherFeat=[]-->, belongsTo=parr
173	Wild-type and mutant Cdc6 were expressed as a fusion proteins, otherFeat=[]-->, belongsTo=parr
174	fused to the N terminus of enhanced green fluorescent protein, otherFeat=[]-->, belongsTo=parr
175	using the pEGFP-N expression vector (Clontech)., otherFeat=[]-->, belongsTo=parr
176	Immunoprecipitation and Immunoblotting--Rabbit antisera, otherFeat=[]-->, belongsTo=parr
177	were raised against a synthetic peptide corresponding to the C, otherFeat=[]-->, belongsTo=parr
178	terminus of human PR70 (CDLYEYACGDEDLEPL) conju-, otherFeat=[]-->, belongsTo=parr
179	gated to keyhole limpet hemocyanin. Anti-PR70 antibodies, otherFeat=[]-->, belongsTo=parr
180	were affinity purified on a peptide column made with the same, otherFeat=[]-->, belongsTo=parr
181	peptide using the MicroLink Peptide Coupling Kit (Pierce) fol-, otherFeat=[]-->, belongsTo=parr
182	lowing the manufacturer's protocol. Rabbit antiserum against, otherFeat=[]-->, belongsTo=parr
183	human Cdc6 was generated against a full-length Cdc6 fusion, otherFeat=[]-->, belongsTo=parr
184	protein as described previously (4)., otherFeat=[]-->, belongsTo=parr
185	Proteins were immunoprecipitated following the protocol, otherFeat=[]-->, belongsTo=parr
186	described previously (17). Briefly, the media was aspirated and, otherFeat=[]-->, belongsTo=parr
187	the cells were washed with cold PBS. The cells were incubated, otherFeat=[]-->, belongsTo=parr
188	on ice for 20 min in 300 l of IP lysis buffer containing 20 mM, otherFeat=[]-->, belongsTo=parr
189	Tris-HCl (pH 7.5), 0.2% Nonidet P-40, 20% glycerol, 200 mM, otherFeat=[]-->, belongsTo=parr
190	NaCl, 1 mM EDTA, and protease inhibitor mixture (Roche, otherFeat=[]-->, belongsTo=parr
191	Applied Science). Lysates were centrifuged at 14,000 g for 10, otherFeat=[]-->, belongsTo=parr
192	min, and protein complexes were immunoprecipitated from, otherFeat=[]-->, belongsTo=parr
193	the supernatant. Endogenous PR70 and Cdc6 were immuno-, otherFeat=[]-->, belongsTo=parr
194	precipitated from 1.2 106 HeLa cells lysed in 300 l of IP lysis, otherFeat=[]-->, belongsTo=parr
195	buffer as described above. PR70 was immunoprecipitated using, otherFeat=[]-->, belongsTo=parr
196	a rabbit antiserum generated against the peptide CDLYEY-, otherFeat=[]-->, belongsTo=parr
197	ACGDEDLEPL conjugated to hemocyanin. Cdc6 was immuno-, otherFeat=[]-->, belongsTo=parr
198	precipitated using a rabbit polyclonal antibody generated, otherFeat=[]-->, belongsTo=parr
199	against a full-length Cdc6-GST fusion protein described previ-, otherFeat=[]-->, belongsTo=parr
200	ously. As a negative control, immunoprecipitations were per-, otherFeat=[]-->, belongsTo=parr
201	formed using pre-immune serum collected from the rabbits, otherFeat=[]-->, belongsTo=parr
202	immunized against PR70 or Cdc6. 10 l of antiserum and 40 l, otherFeat=[]-->, belongsTo=parr
203	of protein A-Sepharose (Sigma-Aldrich) were added to 300 l, otherFeat=[]-->, belongsTo=parr
204	of lysate, and the mixture was incubated for2hat4 ?C.The, otherFeat=[]-->, belongsTo=parr
205	protein-A beads were washed three times with IP lysis buffer,, otherFeat=[]-->, belongsTo=parr
206	and protein was solubilized in 60 lof2 SDS-PAGE loading, otherFeat=[]-->, belongsTo=parr
207	buffer. Thirty microliters of solubilized material was resolved, otherFeat=[]-->, belongsTo=parr
208	on a 10% SDS-PAGE gel and transferred to a nitrocellulose, otherFeat=[]-->, belongsTo=parr
209	membrane. The membrane was cut into pieces, which were, otherFeat=[]-->, belongsTo=parr
210	probed with anti-PP2A C-subunit monoclonal antibody 1F6, otherFeat=[]-->, belongsTo=parr
211	(24), anti-PP2A A-subunit antiserum (C-20, Santa Cruz Bio-, otherFeat=[]-->, belongsTo=parr
212	technology), anti-Cdc6 monoclonal antibody (clone DCS-180,, otherFeat=[]-->, belongsTo=parr
213	Upstate), or anti-PR70 antiserum. Following incubation with, otherFeat=[]-->, belongsTo=parr
214	horseradish peroxidase-conjugated secondary antibodies, the, otherFeat=[]-->, belongsTo=parr
215	blots were developed using the enhanced chemiluminescence, otherFeat=[]-->, belongsTo=parr
216	detection system (Amersham Biosciences)., otherFeat=[]-->, belongsTo=parr
217	Transiently expressed FLAG-tagged proteins were immuno-, otherFeat=[]-->, belongsTo=parr
218	precipitated from 1.5 106 cells using 7 g of anti-FLAG poly-, otherFeat=[]-->, belongsTo=parr
219	clonal antibody (Sigma-Aldrich) or 7 g of non-immune rabbit, otherFeat=[]-->, belongsTo=parr
220	IgG (Sigma-Aldrich) and 40 l of protein A-Sepharose (Sigma-, otherFeat=[]-->, belongsTo=parr
221	Aldrich) for 2 h at 4 ?C. The immunoprecipitates were washed, otherFeat=[]-->, belongsTo=parr
222	three times with lysis buffer and solubilized in 60 lof2, otherFeat=[]-->, belongsTo=parr
223	SDS-PAGE loading buffer. 30 l of solubilized protein was, otherFeat=[]-->, belongsTo=parr
224	resolved on a 10% SDS-PAGE gel and transferred to a nitrocel-, otherFeat=[]-->, belongsTo=parr
225	lulose membrane. The membrane was probed with anti-FLAG, otherFeat=[]-->, belongsTo=parr
226	M2 monoclonal (Stratagene), anti-PP2A C-subunit 1F6, and, otherFeat=[]-->, belongsTo=parr
227	anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) anti-, otherFeat=[]-->, belongsTo=parr
228	bodies and developed as described above., otherFeat=[]-->, belongsTo=parr
229	Calcium Overlay Assay--In vitro 45Ca2 overlay assays were, otherFeat=[]-->, belongsTo=parr
230	carried out using a protocol described previously (25). Purified, otherFeat=[]-->, belongsTo=parr
231	GST fusion proteins were resolved by SDS-PAGE and trans-, otherFeat=[]-->, belongsTo=parr
232	ferred to a nitrocellulose membrane. The membrane was, otherFeat=[]-->, belongsTo=parr
233	washed three times in IMK buffer (10 mM imidazole-HCl, pH, otherFeat=[]-->, belongsTo=parr
234	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
235	JUNE 6, 2008 ? VOLUME 283 ? NUMBER 23, otherFeat=['U']-->, belongsTo=nota_cab_pie
236	JOURNAL OF BIOLOGICAL CHEMISTRY 16105, otherFeat=[]-->, belongsTo=nota_cab_pie
237	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
238	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
239	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
240	de, otherFeat=[]-->, belongsTo=nota_cab_pie
241	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
242	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
243	on, otherFeat=[]-->, belongsTo=nota_cab_pie
244	May, otherFeat=[]-->, belongsTo=nota_cab_pie
245	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
246	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
247	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
248	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
249	from, otherFeat=[]-->, belongsTo=nota_cab_pie
250	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
251	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
252	6.8, 5 mM MgCl2, and 60 mM KCl) for1hat room temperature., otherFeat=[]-->, belongsTo=parr
253	The membrane was then incubated in IMK buffer containing 5, otherFeat=[]-->, belongsTo=parr
254	Ci/ml 45Ca2 for 10 min. The membrane was washed three, otherFeat=[]-->, belongsTo=parr
255	times in 30% ethanol for 5 min, dried, and exposed to x-ray film, otherFeat=[]-->, belongsTo=parr
256	for 12 h., otherFeat=[]-->, belongsTo=parr
257	Generation of PR70 Mutants--Point mutations were intro-, otherFeat=[]-->, belongsTo=parr
258	duced into the EF-hands of PR70 as described in the manual for, otherFeat=[]-->, belongsTo=parr
259	the QuikChange Multi Site-directed mutagenesis kit (Strat-, otherFeat=[]-->, belongsTo=parr
260	agene) using the following primers (mutated residues underlined)., otherFeat=[]-->, belongsTo=parr
261	PCR was performed with pCMV-Tag2B containing the full-length, otherFeat=[]-->, belongsTo=parr
262	PR70 cDNA as template with the following primers: EF1(x,y) 5 -, otherFeat=[]-->, belongsTo=parr
263	CAAGTTCTGGGAGCTGGCCACGGCCCACGACCTGCTC-, otherFeat=[]-->, belongsTo=parr
264	ATCG-3 (sense strand) and 5 -CGATGAGCAGGTCGTGGG-, otherFeat=[]-->, belongsTo=parr
265	CCGTGGCCAGCTCCCAGAACTTG-3 (antisense strand),, otherFeat=[]-->, belongsTo=parr
266	EF1(-z) 5 -TTGTGCCGCGCCAGGTTGTCCGCGTCGATGA-, otherFeat=[]-->, belongsTo=parr
267	GCGCTCATCGACGCGGACAACCTGGCGCGGCACAA-3, otherFeat=[]-->, belongsTo=parr
268	(sense strand) and 5 3 3 (antisense strand), EF2(x,y) 5 -TGGT-, otherFeat=[]-->, belongsTo=parr
269	TCCGCTGCATGGCCCTGGCCGGGGACGGCGCCCTG-3, otherFeat=[]-->, belongsTo=parr
270	(sense strand) and 5 -CAGGGCGCCGTCCCCGGCCAGGGC-, otherFeat=[]-->, belongsTo=parr
271	CATGCAGCGGAACCA-3 (antisense strand), and EF2(-z), 5 -, otherFeat=[]-->, belongsTo=parr
272	GCGCCCTGTCCATGTTCCAGCTCGAGTACTTCTAC-3, otherFeat=[]-->, belongsTo=parr
273	(sense strand) and 5 -GTAGAAGTACTCGAGCTGGAACAT-, otherFeat=[]-->, belongsTo=parr
274	GGACAGGGCGC-3 (antisense strand). Mutations in both EF-, otherFeat=[]-->, belongsTo=parr
275	hands were introduced using the EF1 mutant cDNAs as tem-, otherFeat=[]-->, belongsTo=parr
276	plate for PCR with primers for introduction of EF2 point, otherFeat=[]-->, belongsTo=parr
277	mutants. All mutations were verified by automated sequencing., otherFeat=[]-->, belongsTo=parr
278	PR70 truncation mutants were generated by PCR amplifi-, otherFeat=[]-->, belongsTo=parr
279	cation using the PR70 cDNA as template. The N1 (aa 125?, otherFeat=[]-->, belongsTo=parr
280	575) corresponds to the PR48 protein described previously, otherFeat=[]-->, belongsTo=parr
281	(17). N2, N3, and C were generated using the following, otherFeat=[]-->, belongsTo=parr
282	primers: N2 (aa 136 ?575) 5 -CGGGATCCGCCACCATG-, otherFeat=[]-->, belongsTo=parr
283	GATGACATG-3 , N3 (aa 162?575) 5 -CGGGATCCAG-, otherFeat=[]-->, belongsTo=parr
284	GACTCCGTCAACGTG-3 , and C (aa 1? 441) 5 -CCCA-, otherFeat=[]-->, belongsTo=parr
285	AGCTTCATCTGGCAGAGGCAGTC-3 ., otherFeat=[]-->, belongsTo=parr
286	Point mutations were introduced into the FYF motif (aa, otherFeat=[]-->, belongsTo=parr
287	128 ?130) of PR70 using the full-length PR70 cDNA as template, otherFeat=[]-->, belongsTo=parr
288	with the following mutagenic primers (mutated residues under-, otherFeat=[]-->, belongsTo=parr
289	lined): AYF, 5 -GCCAAAGCATTCCGACCGCCTACTTCCC-, otherFeat=[]-->, belongsTo=parr
290	CAGAGGACG-3 (sense strand) and 5 -CGTCCTCTGGGGA-, otherFeat=[]-->, belongsTo=parr
291	AGTAGGCGGTCGGAATGCTTTGGC-3 (antisense strand),, otherFeat=[]-->, belongsTo=parr
292	FAF, 5 -CCAAAGCATTCCGACCTTCGCCTTCCCCAGAG-, otherFeat=[]-->, belongsTo=parr
293	GACGCC-3 (sense strand) and 5 -GGCGTCCTCTGGGGAA-, otherFeat=[]-->, belongsTo=parr
294	GGCGAAGGTCGGAATGCTTTGG-3 (antisense strand),, otherFeat=[]-->, belongsTo=parr
295	FYA, 5 -GCATTCCGACCTTCTACGCCCCCAGAGGACGC-, otherFeat=[]-->, belongsTo=parr
296	CCGC-3 (sense strand) and 5 -GCTTTCGTCCTCTGGGGGC-, otherFeat=[]-->, belongsTo=parr
297	GTAGAAGGTCGGAATGC-3 (antisense strand). To make the, otherFeat=[]-->, belongsTo=parr
298	AYAP mutant, the AYFP cDNA was used as a template, and, otherFeat=[]-->, belongsTo=parr
299	PCR mutagenesis was done with the following primers: AYA,, otherFeat=[]-->, belongsTo=parr
300	5 -CATTCCGACCGCCTACGCCCCCAGAGGACGCCCG-3, otherFeat=[]-->, belongsTo=parr
301	(sense strand) and 5 -CGGGCGTCCTCTGGGGGCGTAGGC-, otherFeat=[]-->, belongsTo=parr
302	GGTCGGAATG-3 (antisense strand). To make the AAAP, otherFeat=[]-->, belongsTo=parr
303	mutant, the AYAP cDNA was used as a template, and PCR, otherFeat=[]-->, belongsTo=parr
304	mutagenesis was done with the following primers AAA, 5 -GCA-, otherFeat=[]-->, belongsTo=parr
305	TTCCGACCGCCGCCGCCCCCAGAGGACG-3, otherFeat=[]-->, belongsTo=parr
306	(sense, otherFeat=[]-->, belongsTo=parr
307	strand) and 5 -CGTCCTCTGGGGGCGGCGGCGGTCGG-, otherFeat=[]-->, belongsTo=parr
308	AATGC-3 (antisense strand). All mutations were verified, otherFeat=[]-->, belongsTo=parr
309	by automated sequencing., otherFeat=[]-->, belongsTo=parr
310	Cloning of GST-tagged PR70 and EF-hand Mutants--PR70, otherFeat=[]-->, belongsTo=parr
311	and PR70 EF-hand mutant cDNA were cloned into the pGEX-, otherFeat=[]-->, belongsTo=parr
312	4T-1 vector (Amersham Biosciences). The cDNAs were ampli-, otherFeat=[]-->, belongsTo=parr
313	fied by PCR using the pCMV-Tag2B vector containing the full-, otherFeat=[]-->, belongsTo=parr
314	length PR70 or EF-hand mutant cDNA as template with the, otherFeat=[]-->, belongsTo=parr
315	following primers: 5 -CGGGATCCATGCCGCCCGGCAA-, otherFeat=[]-->, belongsTo=parr
316	AGT-3 (sense strand) and 5 -ATTTGCGGCCGCTCACAG-, otherFeat=[]-->, belongsTo=parr
317	CGGCTCCAGGTC-3 (antisense strand). The products were, otherFeat=[]-->, belongsTo=parr
318	digested with BamHI and NotI and ligated into pGEX 4T-1,, otherFeat=[]-->, belongsTo=parr
319	which had been cut with the same restriction enzymes. The, otherFeat=[]-->, belongsTo=parr
320	resulting constructs encode the GST protein fused to the N, otherFeat=[]-->, belongsTo=parr
321	terminus of full-length PR70 or EF-hand mutant proteins. The, otherFeat=[]-->, belongsTo=parr
322	sequences were verified by automated sequencing., otherFeat=[]-->, belongsTo=parr
323	Expression and Purification of GST-Cdc6, GST-A, and GST-, otherFeat=[]-->, belongsTo=parr
324	PR70 Fusion Proteins--A GST-Cdc6 fusion protein was pre-, otherFeat=[]-->, belongsTo=parr
325	pared by a modification of a method previously described (6)., otherFeat=[]-->, belongsTo=parr
326	Briefly, 1 liter of Sf9 cells (2 106 cells/ml) was infected with, otherFeat=[]-->, belongsTo=parr
327	recombinant GST-Cdc6 baculovirus (a gift of Dr. Ellen Fan-, otherFeat=[]-->, belongsTo=parr
328	ning, Vanderbilt University) at a Sf9 culture:baculovirus ratio of, otherFeat=[]-->, belongsTo=parr
329	1:20 (v/v) for 60 h. The cells were collected by centrifugation, otherFeat=[]-->, belongsTo=parr
330	and washed once with PBS. Cells were lysed on ice in 40 ml of, otherFeat=[]-->, belongsTo=parr
331	buffer A (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM KCl,, otherFeat=[]-->, belongsTo=note
332	0.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM dithiothreitol, 10 mM, otherFeat=[]-->, belongsTo=parr
333	NaF, 1 mM EGTA, 2 mM EDTA, and a protease inhibitor tablet, otherFeat=[]-->, belongsTo=parr
334	(Roche Applied Science)) using a Dounce homogenizer. Lysates, otherFeat=[]-->, belongsTo=parr
335	were centrifuged at 30,000 g for 30 min at 4 ?C to remove, otherFeat=[]-->, belongsTo=parr
336	cellular debris, and the lysate was mixed with 2 ml of glutathi-, otherFeat=[]-->, belongsTo=parr
337	one-agarose (Sigma-Aldrich) for2hat4 ?C.The resin was, otherFeat=[]-->, belongsTo=parr
338	recovered by centrifugation and washed twice with PBS, once, otherFeat=[]-->, belongsTo=parr
339	with PBS containing 1.5 M NaCl, and once with PBS containing, otherFeat=[]-->, belongsTo=parr
340	1.5 mM NaCl and 0.1% (v/v) Nonidet P-40, and then re-equili-, otherFeat=[]-->, belongsTo=parr
341	brated in PBS. The GST-Cdc6 fusion protein immobilized on, otherFeat=[]-->, belongsTo=parr
342	glutathione agarose beads was resuspended in buffer B (20 mM, otherFeat=[]-->, belongsTo=parr
343	HEPES, pH 7.6, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA,, otherFeat=[]-->, belongsTo=note
344	and 50% glycerol) and stored at 80 ?C., otherFeat=[]-->, belongsTo=parr
345	GST-A fusion protein, GST-PR70, and GST-EF-hand, otherFeat=[]-->, belongsTo=parr
346	mutants were expressed in bacteria and prepared as previously, otherFeat=[]-->, belongsTo=parr
347	described (26). The GST fusion proteins immobilized on gluta-, otherFeat=[]-->, belongsTo=parr
348	thione agarose beads were resuspended in buffer B and stored, otherFeat=[]-->, belongsTo=parr
349	at 80 ?C until use., otherFeat=[]-->, belongsTo=parr
350	GST Pulldown Assays--GST, GST-A, and GST-Cdc6 immo-, otherFeat=[]-->, belongsTo=parr
351	bilized on glutathione-agarose beads were used to assess the, otherFeat=[]-->, belongsTo=parr
352	binding of PR70. Wild-type or mutant FLAG-PR70 was, otherFeat=[]-->, belongsTo=parr
353	expressed by transient transfection of COS-7 cells. The cells, otherFeat=[]-->, belongsTo=parr
354	were lysed on ice in 300 l of IP lysis buffer or in 300 lofIP, otherFeat=[]-->, belongsTo=parr
355	lysis buffer containing 10 mM EDTA or 10 mM CaCl2 for 20 min., otherFeat=[]-->, belongsTo=note
356	GST pulldown assays (26) were conducted by incubating 300 l, otherFeat=[]-->, belongsTo=parr
357	of lysate with either GST, GST-A, or GST-Cdc6. The samples, otherFeat=[]-->, belongsTo=parr
358	were incubated for1hat room temperature with agitation. The, otherFeat=[]-->, belongsTo=parr
359	calpain inhibitor calpeptin (Calbiochem) was added at a con-, otherFeat=[]-->, belongsTo=parr
360	centration of 50 M in some experiments. Following incuba-, otherFeat=[]-->, belongsTo=parr
361	tion, the sample was washed three times with IP lysis buffer, otherFeat=[]-->, belongsTo=parr
362	supplemented with EGTA, CaCl2, or CaCl2 and calpeptin, and, otherFeat=[]-->, belongsTo=parr
363	the beads were collected by centrifugation. After washing, the, otherFeat=[]-->, belongsTo=parr
364	bound proteins were solubilized in 60 lof2 SDS-PAGE, otherFeat=[]-->, belongsTo=parr
365	loading buffer. 30 l of solubilized protein was resolved on a, otherFeat=[]-->, belongsTo=parr
366	10% SDS-PAGE gel and transferred to a nitrocellulose mem-, otherFeat=[]-->, belongsTo=parr
367	brane. The membrane was probed with anti-FLAG monoclonal, otherFeat=[]-->, belongsTo=parr
368	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
369	16106 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
370	VOLUME 283 ? NUMBER 23 ? JUNE 6, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie
371	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
372	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
373	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
374	de, otherFeat=[]-->, belongsTo=nota_cab_pie
375	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
376	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
377	on, otherFeat=[]-->, belongsTo=nota_cab_pie
378	May, otherFeat=[]-->, belongsTo=nota_cab_pie
379	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
380	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
381	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
382	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
383	from, otherFeat=[]-->, belongsTo=nota_cab_pie
384	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
385	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
386	(M2, Stratagene), anti-PP2A C-subunit 1F6, and anti-PP2A, otherFeat=[]-->, belongsTo=parr
387	A-subunit (C-20, Santa Cruz Biotechnology) antibodies and, otherFeat=[]-->, belongsTo=parr
388	developed as described above., otherFeat=[]-->, belongsTo=parr
389	Flow Cytometry--U2OS cells (4, otherFeat=[]-->, belongsTo=parr
390	105) were seeded into, otherFeat=[]-->, belongsTo=parr
391	60-mm dishes and transfected with PR70 or control siRNA 24 h, otherFeat=[]-->, belongsTo=parr
392	later. The cells were then incubated for 48 h and harvested by, otherFeat=[]-->, belongsTo=parr
393	trypsinization, washed once with PBS, and resuspended in 0.5, otherFeat=[]-->, belongsTo=parr
394	ml of PBS. The cell suspension was then added to 4.5 ml of 70%, otherFeat=[]-->, belongsTo=parr
395	ethanol and incubated on ice for 2 h. Cells were collected by, otherFeat=[]-->, belongsTo=parr
396	centrifugation, washed once with PBS, and suspended in 1 ml of, otherFeat=[]-->, belongsTo=parr
397	propidium iodide/Triton X-100 staining solution with RNase, otherFeat=[]-->, belongsTo=parr
398	(0.1% Triton X-100, 0.2 mg/ml DNase-free RNase, and 10, otherFeat=[]-->, belongsTo=parr
399	g/ml propidium iodide in PBS). The DNA content of 10,000, otherFeat=[]-->, belongsTo=parr
400	cells was determined using a BD Biosciences FACScan flow, otherFeat=[]-->, belongsTo=parr
401	cytometer and FlowJo software. Single cells were gated away, otherFeat=[]-->, belongsTo=parr
402	from clumped cells using an FL3 width versus FL3 height dot, otherFeat=[]-->, belongsTo=parr
403	plot, and the DNA content of individual cells was plotted as FL3, otherFeat=[]-->, belongsTo=parr
404	area versus cell number., otherFeat=[]-->, belongsTo=parr
405	Experimental Reproducibility--The data shown in the fig-, otherFeat=[]-->, belongsTo=parr
406	ures are from individual experiments that were representative, otherFeat=[]-->, belongsTo=parr
407	of common results obtained in at least three independent, otherFeat=[]-->, belongsTo=parr
408	experiments., otherFeat=[]-->, belongsTo=parr
409	RESULTS, otherFeat=[]-->, belongsTo=title
410	Interaction of PR70 with Cdc6--The original cDNA for PR70,, otherFeat=[]-->, belongsTo=parr
411	termed PR48, was identified in a yeast two-hybrid screen using, otherFeat=[]-->, belongsTo=parr
412	the human Cdc6 protein as bait (17) and subsequently shown to, otherFeat=[]-->, belongsTo=parr
413	be a fragment of a longer cDNA (27). A full-length human PR70, otherFeat=[]-->, belongsTo=parr
414	cDNA was constructed by ligating expressed sequence tag, otherFeat=[]-->, belongsTo=parr
415	BM54432 to the PR48 cDNA using an internal NcoI restriction, otherFeat=[]-->, belongsTo=parr
416	site. The predicted open reading frame of the PR70 cDNA, otherFeat=[]-->, belongsTo=parr
417	encodes a protein with a calculated molecular mass of 65.1-kDa, otherFeat=[]-->, belongsTo=parr
418	and corresponds to the longer transcript (variant 1) of the, otherFeat=[]-->, belongsTo=parr
419	PPP2R3B gene (GeneID: 28227). The predicted amino acid, otherFeat=[]-->, belongsTo=parr
420	sequence of PR70 is highly similar to the human PR72 and, otherFeat=[]-->, belongsTo=parr
421	mouse PR59 members of the PPP2R3 gene family, but more, otherFeat=[]-->, belongsTo=parr
422	distantly related to the G5PR protein (supplemental Table  S1)., otherFeat=[]-->, belongsTo=parr
423	An alignment of the PPP2R3 family (supplemental Fig.  S1), otherFeat=[]-->, belongsTo=parr
424	revealed a highly conserved central domain, termed the R3, otherFeat=[]-->, belongsTo=parr
425	domain, that contains two conserved EF-hand calcium binding, otherFeat=[]-->, belongsTo=parr
426	motifs previously identified in PR72 (27). Rabbit antisera were, otherFeat=[]-->, belongsTo=parr
427	raised against a peptide corresponding to the unique C termi-, otherFeat=[]-->, belongsTo=parr
428	nus of PR70 and affinity purified on a peptide column. The, otherFeat=[]-->, belongsTo=parr
429	purified antibodies recognized a protein band of Mr 70,000 in, otherFeat=[]-->, belongsTo=parr
430	lysates of HeLa cells. The 70-kDa protein recognized by the, otherFeat=[]-->, belongsTo=parr
431	antibody was greatly reduced in cells treated with two different, otherFeat=[]-->, belongsTo=parr
432	siRNAs corresponding to sequences within PR70 but not with, otherFeat=[]-->, belongsTo=parr
433	control siRNA (supplemental Fig.  S2)., otherFeat=[]-->, belongsTo=parr
434	To verify that PR70 associates with PP2A and Cdc6, HeLa, otherFeat=[]-->, belongsTo=parr
435	lysates were immunoprecipitated with PR70 and Cdc6 antibod-, otherFeat=[]-->, belongsTo=parr
436	ies. Immunoprecipitation of Cdc6 co-precipitated a diffuse, otherFeat=[]-->, belongsTo=parr
437	protein band that migrated at the position of PR70 that was not, otherFeat=[]-->, belongsTo=parr
438	FIGURE 1. PR70 interacts with Cdc6 and PP2A. A, Cdc6 was immunoprecipi-, otherFeat=[]-->, belongsTo=fig_caption
439	tated from exponentially growing HeLa cells using a polyclonal antiserum, otherFeat=[]-->, belongsTo=fig_caption
440	specific for Cdc6 (Cdc6) or pre-immune serum (Pre). The immunoprecipitates, otherFeat=[]-->, belongsTo=fig_caption
441	and supernatant fractions were analyzed by immunoblotting using anti-Cdc6, otherFeat=[]-->, belongsTo=fig_caption
442	(Cdc6), anti-PR70, anti-A-subunit, or anti-C-subunit antibodies. B, PR70 was, otherFeat=[]-->, belongsTo=fig_caption
443	immunoprecipitated from HeLa cells using an anti-peptide antiserum against, otherFeat=[]-->, belongsTo=fig_caption
444	PR70 (PR70) or pre-immune serum (Pre). The immunoprecipitates (IP) or the, otherFeat=[]-->, belongsTo=fig_caption
445	supernatants remaining after immunoprecipitation (S) were resolved by SDS-, otherFeat=[]-->, belongsTo=fig_caption
446	PAGE and analyzed by immunoblotting with anti-PR70 (PR70), anti-A-subunit, otherFeat=[]-->, belongsTo=fig_caption
447	(A), and anti-C-subunit (C) antibodies as indicated on the left. C, HeLa cells, otherFeat=[]-->, belongsTo=fig_caption
448	were transiently transfected with empty expression vector (Emp Vec), plas-, otherFeat=[]-->, belongsTo=fig_caption
449	mids expressing the FLAG-PR70 N mutant ( N1), full-length FLAG-PR70, or a, otherFeat=[]-->, belongsTo=fig_caption
450	combination of plasmids expressing FLAG-PR70 and HA-Cdc6 (Cdc6). Cells, otherFeat=[]-->, belongsTo=fig_caption
451	were harvested after 24 h, and lysates were immunoprecipitated with anti-, otherFeat=[]-->, belongsTo=fig_caption
452	FLAG antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE, otherFeat=[]-->, belongsTo=fig_caption
453	and immunoblotted with anti-FLAG (FLAG-PR70), anti-Cdc6, anti-A-subunit,, otherFeat=[]-->, belongsTo=fig_caption
454	and anti-C-subunit antibodies as indicated on the left., otherFeat=[]-->, belongsTo=fig_caption
455	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
456	JUNE 6, 2008 ? VOLUME 283 ? NUMBER 23, otherFeat=['U']-->, belongsTo=nota_cab_pie
457	JOURNAL OF BIOLOGICAL CHEMISTRY 16107, otherFeat=[]-->, belongsTo=nota_cab_pie
458	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
459	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
460	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
461	de, otherFeat=[]-->, belongsTo=nota_cab_pie
462	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
463	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
464	on, otherFeat=[]-->, belongsTo=nota_cab_pie
465	May, otherFeat=[]-->, belongsTo=nota_cab_pie
466	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
467	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
468	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
469	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
470	from, otherFeat=[]-->, belongsTo=nota_cab_pie
471	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
472	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
473	present in immunoprecipitates obtained with the preimmune, otherFeat=[]-->, belongsTo=parr
474	serum (Fig. 1A). The anti-Cdc6 serum also co-precipitated the, otherFeat=[]-->, belongsTo=parr
475	A- and C-subunits of PP2A. Although the anti-PR70 antibodies, otherFeat=[]-->, belongsTo=parr
476	co-precipitated the A- and C-subunits of PP2A (Fig. 1B), a com-, otherFeat=[]-->, belongsTo=parr
477	plex between PR70 and Cdc6 could not be detected. The inabil-, otherFeat=[]-->, belongsTo=parr
478	ity to detect Cdc6 may be due to steric hindrance by the anti-, otherFeat=[]-->, belongsTo=parr
479	C-terminal antibody, because the C terminus of PR70 is, otherFeat=[]-->, belongsTo=parr
480	required for interaction with Cdc6 (see below). The association, otherFeat=[]-->, belongsTo=parr
481	of PR70 with PP2A and Cdc6 was also tested in co-immunopre-, otherFeat=[]-->, belongsTo=parr
482	cipitation experiments using exogenously expressed pro-, otherFeat=[]-->, belongsTo=parr
483	teins. FLAG-tagged PR70 was expressed in HeLa cells and, otherFeat=[]-->, belongsTo=parr
484	immunoprecipitated with anti-FLAG antibodies. Analysis of, otherFeat=[]-->, belongsTo=parr
485	the immunoprecipitates by immunoblotting showed that the, otherFeat=[]-->, belongsTo=parr
486	endogenous A- and C-subunits of PP2A and HA-tagged, otherFeat=[]-->, belongsTo=parr
487	Cdc6 co-precipitated with FLAG-PR70 (Fig. 1C). These, otherFeat=[]-->, belongsTo=parr
488	results indicate that PR70 can interact with both the PP2A, otherFeat=[]-->, belongsTo=parr
489	core dimer and Cdc6 in intact cells., otherFeat=[]-->, belongsTo=parr
490	Calcium Enhances the Interaction of PR70 with the AC Core, otherFeat=[]-->, belongsTo=parr
491	Dimer and Recruits PP2A to Cdc6--Analysis of the amino acid, otherFeat=[]-->, belongsTo=parr
492	sequence of PR70 identified two EF-hand calcium binding, otherFeat=[]-->, belongsTo=parr
493	motifs that are conserved within the PPP2R3 family (supple-, otherFeat=[]-->, belongsTo=parr
494	mental Fig.  S1). The roles of these motifs were tested in a gel, otherFeat=[]-->, belongsTo=parr
495	overlay assay with wild-type PR70 and PR70 containing inacti-, otherFeat=[]-->, belongsTo=parr
496	vating mutations of the EF-hand motifs. Point mutants were, otherFeat=[]-->, belongsTo=parr
497	constructed that had substitutions of amino acids involved in, otherFeat=[]-->, belongsTo=parr
498	calcium binding (28), including alanine substitutions at both, otherFeat=[]-->, belongsTo=parr
499	the x and y coordinates and a conservative change at the -z, otherFeat=[]-->, belongsTo=parr
500	coordinate (Fig. 2A). Wild-type PR70 bound calcium in the in, otherFeat=[]-->, belongsTo=parr
501	vitro 45Ca2 overlay assay (Fig. 2B). Mutation of the first EF-, otherFeat=[]-->, belongsTo=parr
502	hand (EF1) resulted in reduced binding of calcium compared, otherFeat=[]-->, belongsTo=parr
503	with wild-type PR70. Mutation of the second EF-hand (EF2), otherFeat=[]-->, belongsTo=parr
504	severely reduced calcium binding, whereas the double muta-, otherFeat=[]-->, belongsTo=parr
505	tion of EF1 and EF2 nearly abolished the ability of PR70 to bind, otherFeat=[]-->, belongsTo=parr
506	calcium., otherFeat=[]-->, belongsTo=parr
507	Calcium binding causes a conformational change in the PR72, otherFeat=[]-->, belongsTo=parr
508	member of the PPP2R3 family that is associated with enhanced, otherFeat=[]-->, belongsTo=parr
509	interaction with the A-subunit of PP2A (27). Therefore, the, otherFeat=[]-->, belongsTo=parr
510	effects of calcium on the interaction of PR70 with PP2A and, otherFeat=[]-->, belongsTo=parr
511	Cdc6 were determined. FLAG-tagged PR70 was expressed in, otherFeat=[]-->, belongsTo=parr
512	COS-7 cells, which were lysed in buffer containing EGTA or, otherFeat=[]-->, belongsTo=parr
513	calcium. The lysates were incubated with either GST-A or, otherFeat=[]-->, belongsTo=parr
514	GST-Cdc6 and bound proteins detected by immunoblotting., otherFeat=[]-->, belongsTo=parr
515	FLAG-PR70 interacted with both GST-A and GST-Cdc6 but, otherFeat=[]-->, belongsTo=parr
516	not GST alone (Fig. 2C, lanes 1?2 and 6?7). Compared with, otherFeat=[]-->, belongsTo=parr
517	lysates prepared with standard buffer or EGTA, the addition of, otherFeat=[]-->, belongsTo=parr
518	calcium enhanced the binding of PR70 to GST-A, but not to, otherFeat=[]-->, belongsTo=parr
519	GST-Cdc6 (Fig. 2C, lanes 4 and 9). Although calcium did not, otherFeat=[]-->, belongsTo=parr
520	enhance the binding of PR70 to GST-Cdc6, it did cause a sig-, otherFeat=[]-->, belongsTo=parr
521	nificant increase in the amount of A- and C-subunits associated, otherFeat=[]-->, belongsTo=parr
522	with GST-Cdc6 (Fig. 2C, lanes 9 and 10). Although an excess of, otherFeat=[]-->, belongsTo=parr
523	calcium was used in the experiments shown in Fig. 2C, other, otherFeat=[]-->, belongsTo=parr
524	experiments showed that enhanced binding of the AC core, otherFeat=[]-->, belongsTo=parr
525	dimer was also observed at calcium concentrations of 100 M, otherFeat=[]-->, belongsTo=parr
526	(not shown)., otherFeat=[]-->, belongsTo=parr
527	To test the function of the individual EF-hand motifs in the, otherFeat=[]-->, belongsTo=parr
528	calcium-enhanced interaction with PP2A, GST pulldown, otherFeat=[]-->, belongsTo=parr
529	experiments were performed with the calcium-binding, otherFeat=[]-->, belongsTo=parr
530	mutants. Compared with assays in the presence of EGTA, the, otherFeat=[]-->, belongsTo=parr
531	addition of calcium resulted in enhanced binding of wild-type, otherFeat=[]-->, belongsTo=parr
532	PR70 and the EF1 mutant to GST-A, but not to GST-Cdc6 (Fig., otherFeat=[]-->, belongsTo=parr
533	3A and B, lanes 2?5). Although it did not increase the amount of, otherFeat=[]-->, belongsTo=parr
534	PR70 or the EF1 mutant associated with Cdc6, calcium did, otherFeat=[]-->, belongsTo=parr
535	increase the association of the A- and C-subunits with GST-, otherFeat=[]-->, belongsTo=parr
536	Cdc6 (Fig. 3B, lanes 2?5). Mutation of EF2 or mutation of both, otherFeat=[]-->, belongsTo=parr
537	EF1 and EF2 resulted in loss of the calcium-enhanced binding, otherFeat=[]-->, belongsTo=parr
538	of PR70 to GST-A (Fig. 3A, lanes 6 ?9) and the calcium-depend-, otherFeat=[]-->, belongsTo=parr
539	ent association of the A- and C-subunits with GST-Cdc6 (Fig., otherFeat=[]-->, belongsTo=parr
540	3B, lanes 6 ?9)., otherFeat=[]-->, belongsTo=parr
541	The effect of calcium on the interaction of PR70 with PP2A, otherFeat=[]-->, belongsTo=parr
542	was also assessed by expression and immunoprecipitation in, otherFeat=[]-->, belongsTo=parr
543	COS-7 cells. Both wild-type PR70 and the EF1 mutants inter-, otherFeat=[]-->, belongsTo=parr
544	acted with endogenous PP2A (Fig. 3C, lanes 2? 4). The EF1(-z), otherFeat=[]-->, belongsTo=parr
545	mutant interacted as well as wild-type PR70, but interaction of, otherFeat=[]-->, belongsTo=parr
546	the EF1(x,y) mutant was reduced suggesting that mutation of, otherFeat=[]-->, belongsTo=parr
547	the x and y residues causes a structural defect in PR70. Muta-, otherFeat=[]-->, belongsTo=parr
548	tion of EF2, or both EF1 and EF2, resulted in a nearly complete, otherFeat=[]-->, belongsTo=parr
549	PR70, otherFeat=[]-->, belongsTo=??
550	EF1 EF2, otherFeat=[]-->, belongsTo=??
551	WT, otherFeat=[]-->, belongsTo=??
552	166-DTDHDLLIDADD-177 240-DLDGDGALSMFE-251, otherFeat=[]-->, belongsTo=??
553	EF1(x,y), otherFeat=[]-->, belongsTo=??
554	DLDGDGALSMFE, otherFeat=[]-->, belongsTo=??
555	EF1(-z), otherFeat=[]-->, belongsTo=??
556	DTDHDLLIDADN, otherFeat=[]-->, belongsTo=??
557	DLDGDGALSMFE, otherFeat=[]-->, belongsTo=??
558	EF2(x,y), otherFeat=[]-->, belongsTo=??
559	DTDHDLLIDADD, otherFeat=[]-->, belongsTo=??
560	ALAGDGALSMFE, otherFeat=[]-->, belongsTo=??
561	EF2(-z), otherFeat=[]-->, belongsTo=??
562	DTDHDLLIDADD, otherFeat=[]-->, belongsTo=??
563	DLDGDGALSMFQ, otherFeat=[]-->, belongsTo=??
564	EF1/EF2(x,y) ATAHDLLIDADD, otherFeat=[]-->, belongsTo=??
565	ALAGDGALSMFE, otherFeat=[]-->, belongsTo=??
566	EF1/EF2(-z) DTDHDLLIDADN, otherFeat=[]-->, belongsTo=??
567	DLDGDGALSMFQ, otherFeat=[]-->, belongsTo=??
568	45Ca2+, otherFeat=[]-->, belongsTo=??
569	CBB, otherFeat=[]-->, belongsTo=??
570	EF1/EF2(-z), otherFeat=[]-->, belongsTo=??
571	EF1/EF2(x,y), otherFeat=[]-->, belongsTo=??
572	EF2(-z), otherFeat=[]-->, belongsTo=??
573	EF2(x,y), otherFeat=[]-->, belongsTo=??
574	EF1(, otherFeat=[]-->, belongsTo=??
575	x,y), otherFeat=[]-->, belongsTo=??
576	PR70, otherFeat=[]-->, belongsTo=??
577	N, otherFeat=[]-->, belongsTo=??
578	N, otherFeat=[]-->, belongsTo=??
579	Ca+CP, otherFeat=[]-->, belongsTo=??
580	Ca, otherFeat=[]-->, belongsTo=??
581	E, otherFeat=[]-->, belongsTo=??
582	N, otherFeat=[]-->, belongsTo=??
583	Ca+CP, otherFeat=[]-->, belongsTo=??
584	Ca, otherFeat=[]-->, belongsTo=??
585	E, otherFeat=[]-->, belongsTo=??
586	N, otherFeat=[]-->, belongsTo=??
587	GST-A, otherFeat=[]-->, belongsTo=??
588	GST-Cdc6, otherFeat=[]-->, belongsTo=??
589	GST, otherFeat=[]-->, belongsTo=??
590	FLAG-PR70, otherFeat=[]-->, belongsTo=??
591	C, otherFeat=[]-->, belongsTo=??
592	GST, otherFeat=[]-->, belongsTo=??
593	GST, otherFeat=[]-->, belongsTo=??
594	A, otherFeat=[]-->, belongsTo=??
595	EF1(-z), otherFeat=[]-->, belongsTo=??
596	A, otherFeat=[]-->, belongsTo=??
597	B, otherFeat=[]-->, belongsTo=??
598	C, otherFeat=[]-->, belongsTo=??
599	ATAHDLLIDADD, otherFeat=[]-->, belongsTo=??
600	X Y Z-X-Y -Z, otherFeat=['U']-->, belongsTo=??
601	X Y Z-X-Y -Z, otherFeat=['U']-->, belongsTo=??
602	12 3 4 5, otherFeat=[]-->, belongsTo=??
603	6 7 8 9 10, otherFeat=[]-->, belongsTo=??
604	FIGURE 2. Interaction of PR70 with PP2A is enhanced by calcium-binding., otherFeat=[]-->, belongsTo=fig_caption
605	A, the figure shows a diagram of the location and sequences of wild-type, otherFeat=[]-->, belongsTo=fig_caption
606	PR70 (WT) and PR70 mutants containing substitutions of calcium-binding, otherFeat=[]-->, belongsTo=fig_caption
607	residues within the EF-hand motifs (EF mutants). The canonical EF-hand resi-, otherFeat=[]-->, belongsTo=fig_caption
608	dues involved in coordination of calcium are indicated by the letters x, y, z, -x,, otherFeat=[]-->, belongsTo=fig_caption
609	-y, and -z using standard nomenclature (28). The x, y, and -z residues that were, otherFeat=[]-->, belongsTo=fig_caption
610	mutated are shown in bold type. B, GST fusions of wild-type PR70 (PR70) and, otherFeat=[]-->, belongsTo=fig_caption
611	the EF-hand mutant were analyzed for calcium binding by 45Ca2 overlay, otherFeat=[]-->, belongsTo=fig_caption
612	assay. The amounts of GST-PR70 in each lane were determined by staining the, otherFeat=[]-->, belongsTo=fig_caption
613	gel with Coomassie Brilliant Blue (CBB). C, calcium enhances binding of PR70, otherFeat=[]-->, belongsTo=fig_caption
614	to the A-subunit of PP2A but not to Cdc6. FLAG-PR70 and the indicated EF-, otherFeat=[]-->, belongsTo=fig_caption
615	hand mutants were transiently expressed in COS-7 cells, and the cells were, otherFeat=[]-->, belongsTo=fig_caption
616	lysed in standard buffer (N) or lysis buffer containing EGTA (E) or CaCl2 (Ca)., otherFeat=[]-->, belongsTo=fig_caption
617	Calpeptin (50 M) was included in some experiments (Ca CP). The lysates, otherFeat=[]-->, belongsTo=fig_caption
618	were incubated with GST alone (GST), GST-A, or GST-Cdc6, and bound pro-, otherFeat=[]-->, belongsTo=fig_caption
619	teins were detected by immunoblotting with anti-FLAG (FLAG-PR70), anti-A-, otherFeat=[]-->, belongsTo=fig_caption
620	subunit (A), anti-C-subunit (C), and anti-GST (GST) antibodies., otherFeat=[]-->, belongsTo=fig_caption
621	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
622	16108 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
623	VOLUME 283 ? NUMBER 23 ? JUNE 6, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie
624	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
625	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
626	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
627	de, otherFeat=[]-->, belongsTo=nota_cab_pie
628	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
629	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
630	on, otherFeat=[]-->, belongsTo=nota_cab_pie
631	May, otherFeat=[]-->, belongsTo=nota_cab_pie
632	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
633	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
634	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
635	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
636	from, otherFeat=[]-->, belongsTo=nota_cab_pie
637	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
638	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
639	loss of interaction with PP2A (Fig. 3C, lanes 5? 8). A longer, otherFeat=[]-->, belongsTo=parr
640	exposure of the blot showed that a weak interaction of the A-, otherFeat=[]-->, belongsTo=parr
641	and C-subunits with the EF2 and EF1/EF2 double mutants, otherFeat=[]-->, belongsTo=parr
642	could still be detected (Fig. 3C, OE). The combination of intact, otherFeat=[]-->, belongsTo=parr
643	cell data and in vitro binding assays provide evidence that PR70, otherFeat=[]-->, belongsTo=parr
644	is a calcium binding protein and that interaction with the core, otherFeat=[]-->, belongsTo=parr
645	dimer of PP2A is enhanced by binding of calcium to the second, otherFeat=[]-->, belongsTo=parr
646	EF-hand motif. Calcium does not affect interaction of PR70, otherFeat=[]-->, belongsTo=parr
647	with Cdc6 but increases the association of the PP2A core dimer, otherFeat=[]-->, belongsTo=parr
648	with Cdc6 in a manner dependent upon binding of calcium to, otherFeat=[]-->, belongsTo=parr
649	the second EF-hand of PR70., otherFeat=[]-->, belongsTo=parr
650	PP2A and Cdc6 Bind to Distinct Regions of PR70--Compari-, otherFeat=[]-->, belongsTo=parr
651	son of the amino acid sequences of the PPP2R3 regulatory sub-, otherFeat=[]-->, belongsTo=parr
652	units identified a conserved domain in the central region of, otherFeat=[]-->, belongsTo=parr
653	PR70 (supplemental Figs.  S1 and  S4A). The R3 domain is 66%, otherFeat=[]-->, belongsTo=parr
654	identical and 82% conserved between human PR70 (PPP2R3B), otherFeat=[]-->, belongsTo=parr
655	and PR72 (PPP2R3A). A series of truncation mutants were con-, otherFeat=[]-->, belongsTo=parr
656	structed to identify regions within PR70 that were important, otherFeat=[]-->, belongsTo=parr
657	for interaction with PP2A and Cdc6. FLAG-tagged mutants, otherFeat=[]-->, belongsTo=parr
658	were expressed in COS-7 cells and immunoprecipitated with, otherFeat=[]-->, belongsTo=parr
659	anti-FLAG antibody. The ability of the mutants to incorporate, otherFeat=[]-->, belongsTo=parr
660	into endogenous PP2A heterotrimers was determined by, otherFeat=[]-->, belongsTo=parr
661	immunoblotting for associated A- and C-subunits. The N1, otherFeat=[]-->, belongsTo=parr
662	mutant contains a deletion of the entire N-terminal PR70-, otherFeat=[]-->, belongsTo=parr
663	unique region and interacted with endogenous PP2A subunits, otherFeat=[]-->, belongsTo=parr
664	to the same extent as full-length PR70 (Fig. 4B). Deletion of a, otherFeat=[]-->, belongsTo=parr
665	C-terminal segment that included the PR70-unique region, otherFeat=[]-->, belongsTo=parr
666	( C) had little, if any, effect on the interaction with PP2A. How-, otherFeat=[]-->, belongsTo=parr
667	ever, deletions of N-terminal regions of the conserved R3, otherFeat=[]-->, belongsTo=parr
668	domain, N2 and N3, resulted in proteins that failed to inter-, otherFeat=[]-->, belongsTo=parr
669	act with PP2A. These data indicated that the region between, otherFeat=[]-->, belongsTo=parr
670	amino acids 125 and 136 of PR70 were necessary for interaction, otherFeat=[]-->, belongsTo=parr
671	with the PP2A core dimer., otherFeat=[]-->, belongsTo=parr
672	The sequence between residues 125 and 136 of PR70 con-, otherFeat=[]-->, belongsTo=parr
673	tains a hydrophobic motif (FYF) that was conserved in PPP2R3, otherFeat=[]-->, belongsTo=parr
674	proteins from humans to flies (Fig. 5A). The role of the FYF, otherFeat=[]-->, belongsTo=parr
675	motif was tested by mutating these residues to alanines (Fig. 5B), otherFeat=[]-->, belongsTo=parr
676	and determining the effects on interaction with the AC core, otherFeat=[]-->, belongsTo=parr
677	dimer. Mutation of any one of these residues resulted in a sig-, otherFeat=[]-->, belongsTo=parr
678	nificant loss of interaction with endogenous PP2A (Fig. 5C). A, otherFeat=[]-->, belongsTo=parr
679	longer exposure of the immunoblot showed that small amounts, otherFeat=[]-->, belongsTo=parr
680	of the A- and C-subunits could be detected in immunoprecipi-, otherFeat=[]-->, belongsTo=parr
681	tates of each of the mutants (not shown). Although the interac-, otherFeat=[]-->, belongsTo=parr
682	tion of the FYF mutants was severely compromised in intact, otherFeat=[]-->, belongsTo=parr
683	cells, these mutants still bound to PP2A when assayed in vitro, otherFeat=[]-->, belongsTo=parr
684	by GST pulldown experiments (not shown). These results indi-, otherFeat=[]-->, belongsTo=parr
685	cate that the FYF motif contributes to the interaction of PR70, otherFeat=[]-->, belongsTo=parr
686	with the A-subunit., otherFeat=[]-->, belongsTo=parr
687	The N- and C-terminal truncation mutants of PR70 were also, otherFeat=[]-->, belongsTo=parr
688	tested for their ability to interact with Cdc6. Pulldown experi-, otherFeat=[]-->, belongsTo=parr
689	ments with GST-Cdc6 were performed with full-length PR70,, otherFeat=[]-->, belongsTo=parr
690	the N3, and the C mutants in the presence and absence of, otherFeat=[]-->, belongsTo=parr
691	calcium. As expected, full-length PR70 and the C mutant, otherFeat=[]-->, belongsTo=parr
692	interacted with the A-subunit of PP2A, whereas the N3, otherFeat=[]-->, belongsTo=parr
693	GST PR70, otherFeat=[]-->, belongsTo=??
694	FL-PR70, otherFeat=[]-->, belongsTo=??
695	C, otherFeat=[]-->, belongsTo=??
696	FL-PR70, otherFeat=[]-->, belongsTo=??
697	C, otherFeat=[]-->, belongsTo=??
698	A, otherFeat=[]-->, belongsTo=??
699	FL-PR70, otherFeat=[]-->, belongsTo=??
700	C OE, otherFeat=[]-->, belongsTo=??
701	A OE, otherFeat=[]-->, belongsTo=??
702	C, otherFeat=[]-->, belongsTo=??
703	A, otherFeat=[]-->, belongsTo=??
704	Em, otherFeat=[]-->, belongsTo=??
705	pVec, otherFeat=[]-->, belongsTo=??
706	EF1/EF2(-z), otherFeat=[]-->, belongsTo=??
707	EF1/EF2, otherFeat=[]-->, belongsTo=??
708	(x,, otherFeat=[]-->, belongsTo=??
709	y), otherFeat=[]-->, belongsTo=??
710	EF2(-z), otherFeat=[]-->, belongsTo=??
711	EF2(x,, otherFeat=[]-->, belongsTo=??
712	y), otherFeat=[]-->, belongsTo=??
713	EF1(-z), otherFeat=[]-->, belongsTo=??
714	EF1(x,, otherFeat=[]-->, belongsTo=??
715	y), otherFeat=[]-->, belongsTo=??
716	PR70, otherFeat=[]-->, belongsTo=??
717	B, otherFeat=[]-->, belongsTo=??
718	A, otherFeat=[]-->, belongsTo=??
719	EF1/2(-z), otherFeat=[]-->, belongsTo=??
720	EF1(-z) EF2(-z), otherFeat=[]-->, belongsTo=??
721	NE Ca, otherFeat=[]-->, belongsTo=??
722	12 3, otherFeat=[]-->, belongsTo=??
723	ECa, otherFeat=[]-->, belongsTo=??
724	45, otherFeat=[]-->, belongsTo=??
725	ECa, otherFeat=[]-->, belongsTo=??
726	6 7, otherFeat=[]-->, belongsTo=??
727	ECa, otherFeat=[]-->, belongsTo=??
728	89, otherFeat=[]-->, belongsTo=??
729	C, otherFeat=[]-->, belongsTo=??
730	12 3 4 5 6 7 8, otherFeat=[]-->, belongsTo=??
731	GST-A, otherFeat=[]-->, belongsTo=??
732	pulldown, otherFeat=[]-->, belongsTo=??
733	GST-Cdc6, otherFeat=[]-->, belongsTo=??
734	pulldown, otherFeat=[]-->, belongsTo=??
735	Anti-FLAG, otherFeat=[]-->, belongsTo=??
736	IP, otherFeat=[]-->, belongsTo=??
737	FIGURE 3. The calcium-enhanced association of PP2A with PR70 requires, otherFeat=[]-->, belongsTo=fig_caption
738	EF2. FLAG-PR70 and the indicated EF-hand mutants were transiently, otherFeat=[]-->, belongsTo=fig_caption
739	expressed in COS-7 cells, and the cells were lysed in standard lysis buffer (N)or, otherFeat=[]-->, belongsTo=fig_caption
740	lysis buffer containing EGTA (E) or CaCl2 (Ca). A, GST pulldown assays were, otherFeat=[]-->, belongsTo=fig_caption
741	performed with the different lysates using immobilized GST-A. Bound pro-, otherFeat=[]-->, belongsTo=fig_caption
742	teins were detected by immunoblotting with anti-FLAG (FL-PR70), anti-A-sub-, otherFeat=[]-->, belongsTo=fig_caption
743	unit (A), and anti-C-subunit (C) antibodies. B, GST pulldown assays were per-, otherFeat=[]-->, belongsTo=fig_caption
744	formed using immobilized GST-Cdc6 as described for A. Lane 1 of panels A and, otherFeat=[]-->, belongsTo=fig_caption
745	B shows a control pulldown assay using GST alone. C, COS-7 cells were tran-, otherFeat=[]-->, belongsTo=fig_caption
746	siently transfected with FLAG-tagged wild-type PR70 or the indicated EF-, otherFeat=[]-->, belongsTo=fig_caption
747	hand mutants, and lysates prepared with standard buffer were immunopre-, otherFeat=[]-->, belongsTo=fig_caption
748	cipitated with anti-FLAG antibody. The immunoprecipitates were resolved by, otherFeat=[]-->, belongsTo=fig_caption
749	SDS-PAGE and immunoblotted as described in A. Overexposures of the anti-, otherFeat=[]-->, belongsTo=fig_caption
750	A-subunit (AOE) and anti-C-subunit (COE) immunoblots are also shown., otherFeat=[]-->, belongsTo=fig_caption
751	Em, otherFeat=[]-->, belongsTo=??
752	pVec, otherFeat=[]-->, belongsTo=??
753	C, otherFeat=[]-->, belongsTo=??
754	N3, otherFeat=[]-->, belongsTo=??
755	N2, otherFeat=[]-->, belongsTo=??
756	N1, otherFeat=[]-->, belongsTo=??
757	PR70, otherFeat=[]-->, belongsTo=??
758	FLAG, otherFeat=[]-->, belongsTo=??
759	Conserved R3 domain, otherFeat=[]-->, belongsTo=??
760	EF1 EF2, otherFeat=[]-->, belongsTo=??
761	PR70, otherFeat=[]-->, belongsTo=??
762	unique, otherFeat=[]-->, belongsTo=??
763	PR70, otherFeat=[]-->, belongsTo=??
764	unique, otherFeat=[]-->, belongsTo=??
765	FYF motif, otherFeat=[]-->, belongsTo=??
766	PR70, otherFeat=[]-->, belongsTo=??
767	C, otherFeat=[]-->, belongsTo=??
768	N1, otherFeat=[]-->, belongsTo=??
769	N2, otherFeat=[]-->, belongsTo=??
770	N3, otherFeat=[]-->, belongsTo=??
771	1-575, otherFeat=[]-->, belongsTo=??
772	125-575, otherFeat=[]-->, belongsTo=??
773	136-575, otherFeat=[]-->, belongsTo=??
774	162-575, otherFeat=[]-->, belongsTo=??
775	1-441, otherFeat=[]-->, belongsTo=??
776	A, otherFeat=[]-->, belongsTo=??
777	C, otherFeat=[]-->, belongsTo=??
778	A, otherFeat=[]-->, belongsTo=??
779	B, otherFeat=[]-->, belongsTo=??
780	Anti-FLAG, otherFeat=[]-->, belongsTo=??
781	IP, otherFeat=[]-->, belongsTo=??
782	FIGURE 4. Mapping of PP2A binding domains in PR70. A, a schematic dia-, otherFeat=[]-->, belongsTo=fig_caption
783	gram of PR70 showing the region containing the conserved R3 domain and, otherFeat=[]-->, belongsTo=fig_caption
784	PR70-unique regions. The truncation mutants used in binding assays are, otherFeat=[]-->, belongsTo=fig_caption
785	shown below with their corresponding designations on the left and amino, otherFeat=[]-->, belongsTo=fig_caption
786	acid numbers on the right. The conserved FYF (FYF motif) and EF-hand motifs, otherFeat=[]-->, belongsTo=fig_caption
787	(EF1 and EF2) are also depicted. B, interaction of PR70 truncation mutants with, otherFeat=[]-->, belongsTo=fig_caption
788	the A- and C-subunits of PP2A. FLAG-tagged PR70 (PR70) and the indicated, otherFeat=[]-->, belongsTo=fig_caption
789	truncation mutants were transiently expressed in COS-7 cells. The cells were, otherFeat=[]-->, belongsTo=fig_caption
790	lysed and the FLAG-tagged proteins immunoprecipitated with anti-FLAG, otherFeat=[]-->, belongsTo=fig_caption
791	antibody (Anti-FLAG IP). The immunoprecipitates were resolved by SDS-PAGE, otherFeat=[]-->, belongsTo=fig_caption
792	and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A), and anti-C-, otherFeat=[]-->, belongsTo=fig_caption
793	subunit (C) antibodies. A control immunoprecipitate using lysate from cells, otherFeat=[]-->, belongsTo=fig_caption
794	transfected with the empty expression vector (Emp Vec) is shown in the first, otherFeat=[]-->, belongsTo=fig_caption
795	lane., otherFeat=[]-->, belongsTo=fig_caption
796	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
797	JUNE 6, 2008 ? VOLUME 283 ? NUMBER 23, otherFeat=['U']-->, belongsTo=nota_cab_pie
798	JOURNAL OF BIOLOGICAL CHEMISTRY 16109, otherFeat=[]-->, belongsTo=nota_cab_pie
799	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
800	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
801	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
802	de, otherFeat=[]-->, belongsTo=nota_cab_pie
803	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
804	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
805	on, otherFeat=[]-->, belongsTo=nota_cab_pie
806	May, otherFeat=[]-->, belongsTo=nota_cab_pie
807	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
808	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
809	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
810	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
811	from, otherFeat=[]-->, belongsTo=nota_cab_pie
812	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
813	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
814	mutant did not (Fig. 6A). Furthermore, there was an enhanced, otherFeat=[]-->, belongsTo=parr
815	interaction of PR70 and the C mutant in the presence of cal-, otherFeat=[]-->, belongsTo=parr
816	cium. As shown previously with full-length PR70 (Fig. 2C), the, otherFeat=[]-->, belongsTo=parr
817	enhanced binding of PR70 and the C mutant was accompa-, otherFeat=[]-->, belongsTo=parr
818	nied by a decrease in the amount of C-subunit associated with, otherFeat=[]-->, belongsTo=parr
819	GST-A (Fig. 6A, lanes 3 and 9). This decrease in associated, otherFeat=[]-->, belongsTo=parr
820	C-subunit was not observed with the N3 mutant, which did, otherFeat=[]-->, belongsTo=parr
821	not bind to GST-A. These observations suggest that the, otherFeat=[]-->, belongsTo=parr
822	decrease in C-subunit in the presence of calcium is due to dis-, otherFeat=[]-->, belongsTo=parr
823	placement of endogenous regulatory and catalytic subunits, otherFeat=[]-->, belongsTo=parr
824	from GST-A by excess free PR70., otherFeat=[]-->, belongsTo=parr
825	Both full-length PR70 and the N3 mutant bound to GST-, otherFeat=[]-->, belongsTo=parr
826	Cdc6 (Fig. 6B). However, only full-length PR70 was able to, otherFeat=[]-->, belongsTo=parr
827	recruit additional A- and C-subunits in the presence of calcium., otherFeat=[]-->, belongsTo=parr
828	In contrast, the C mutant bound very poorly to Cdc6 in the, otherFeat=[]-->, belongsTo=parr
829	presence or absence of calcium. A low level of the A- and C-sub-, otherFeat=[]-->, belongsTo=parr
830	units was pulled down with GST-Cdc6 from lysates expressing, otherFeat=[]-->, belongsTo=parr
831	the N3 or C mutants (Fig. 6B, lanes 5? 6 and 8?9). Similar, otherFeat=[]-->, belongsTo=parr
832	amounts of these subunits were also bound to GST-Cdc6 in, otherFeat=[]-->, belongsTo=parr
833	lysates from non-transfected cells (not shown) suggesting that, otherFeat=[]-->, belongsTo=parr
834	GST-Cdc6 can interact with endogenous A- and C-subunits in, otherFeat=[]-->, belongsTo=parr
835	the absence of expressed PR70 (presumably by binding to, otherFeat=[]-->, belongsTo=parr
836	endogenous PR70). The amounts of A- and C-subunits bound, otherFeat=[]-->, belongsTo=parr
837	to GST-Cdc6 in experiments with the N3 or C mutants were, otherFeat=[]-->, belongsTo=parr
838	increased in the presence of calcium. This observation provides, otherFeat=[]-->, belongsTo=parr
839	additional support for the conclusion that calcium can regulate, otherFeat=[]-->, belongsTo=parr
840	the association of PP2A with Cdc6 and shows that a C-terminal, otherFeat=[]-->, belongsTo=parr
841	region of PR70, which includes the PR70-unique domain, is, otherFeat=[]-->, belongsTo=parr
842	necessary for interaction with Cdc6., otherFeat=[]-->, belongsTo=parr
843	PR70 Regulates Cdc6 Levels--Because phosphorylation of the, otherFeat=[]-->, belongsTo=parr
844	N-terminal regulatory sites of Cdc6 inhibits degradation, loss of, otherFeat=[]-->, belongsTo=parr
845	the phosphatase that dephosphorylates these sites should pro-, otherFeat=[]-->, belongsTo=parr
846	mote accumulation of Cdc6. Therefore, RNA interference was, otherFeat=[]-->, belongsTo=parr
847	used to determine if knockdown of PR70 affected the levels of, otherFeat=[]-->, belongsTo=parr
848	Cdc6. Knocking down the catalytic subunit of PP2A increased, otherFeat=[]-->, belongsTo=parr
849	the levels of endogenous Cdc6 in HeLa cells (Fig. 7A). Treat-, otherFeat=[]-->, belongsTo=parr
850	ment of cells with either a control siRNA or an siRNA that, otherFeat=[]-->, belongsTo=parr
851	knocks down protein phosphatase 5 had no effect on Cdc6 lev-, otherFeat=[]-->, belongsTo=parr
852	els. Knocking down the PR70 subunit also caused a substantial, otherFeat=[]-->, belongsTo=parr
853	increase in the levels of Cdc6 (Fig. 7B). The increase in Cdc6, otherFeat=[]-->, belongsTo=parr
854	levels occurred with two PR70 siRNAs targeted to distinct, otherFeat=[]-->, belongsTo=parr
855	regions of the mRNA. The accumulation of Cdc6 following, otherFeat=[]-->, belongsTo=parr
856	knockdown with PR70-1 siRNA appeared to be greater than, otherFeat=[]-->, belongsTo=parr
857	that with PR70-2 siRNA, which is consistent with the greater, otherFeat=[]-->, belongsTo=parr
858	efficiency of the PR70-1 siRNA in reducing PR70 levels (supple-, otherFeat=[]-->, belongsTo=parr
859	mental Fig.  S2). Phosphorylation site mutants of Cdc6 were, otherFeat=[]-->, belongsTo=parr
860	then used to test the role of phosphorylation in the accumula-, otherFeat=[]-->, belongsTo=parr
861	tion of Cdc6 caused by knockdown of PR70. Knockdown of, otherFeat=[]-->, belongsTo=parr
862	PR70 caused an increase in the levels of expressed wild-type, otherFeat=[]-->, belongsTo=parr
863	GFP-Cdc6 compared with transfection with a control siRNA, otherFeat=[]-->, belongsTo=parr
864	(Fig. 7B). Transfection with a mutant of Cdc6 in which all three, otherFeat=[]-->, belongsTo=parr
865	N-terminal phosphorylation sites had been mutated to phos-, otherFeat=[]-->, belongsTo=parr
866	pho-mimicking aspartic acid residues (DDD-Cdc6) resulted in, otherFeat=[]-->, belongsTo=parr
867	substantially higher levels of expression than those observed, otherFeat=[]-->, belongsTo=parr
868	with the wild-type protein as previously reported (15). PR70-1, otherFeat=[]-->, belongsTo=parr
869	siRNA had little or no effect on the levels of DDD-Cdc6. The, otherFeat=[]-->, belongsTo=parr
870	ability of PR70 knockdown to cause accumulation of Cdc6 was, otherFeat=[]-->, belongsTo=parr
871	also greatly diminished when the phosphorylation sites were, otherFeat=[]-->, belongsTo=parr
872	C, otherFeat=[]-->, belongsTo=??
873	A, otherFeat=[]-->, belongsTo=??
874	FLAG, otherFeat=[]-->, belongsTo=??
875	Em, otherFeat=[]-->, belongsTo=??
876	pVec, otherFeat=[]-->, belongsTo=??
877	AAA, otherFeat=[]-->, belongsTo=??
878	AYA, otherFeat=[]-->, belongsTo=??
879	FYA, otherFeat=[]-->, belongsTo=??
880	FAF, otherFeat=[]-->, belongsTo=??
881	AYF, otherFeat=[]-->, belongsTo=??
882	PR70, otherFeat=[]-->, belongsTo=??
883	DN3, otherFeat=[]-->, belongsTo=??
884	IPTFYFPRGRP, otherFeat=[]-->, belongsTo=??
885	IPRFYFGEGLP, otherFeat=[]-->, belongsTo=??
886	VPAFYFPCGRP, otherFeat=[]-->, belongsTo=??
887	IPKFYFPKGCP, otherFeat=[]-->, belongsTo=??
888	IPRFYFPHGKP, otherFeat=[]-->, belongsTo=??
889	Human PR70, otherFeat=[]-->, belongsTo=??
890	Human PR72, otherFeat=[]-->, belongsTo=??
891	Mouse PR59, otherFeat=[]-->, belongsTo=??
892	Xenopus PR70, otherFeat=[]-->, belongsTo=??
893	Drosophila PR72, otherFeat=[]-->, belongsTo=??
894	EF1 EF2, otherFeat=[]-->, belongsTo=??
895	FYF motif, otherFeat=[]-->, belongsTo=??
896	PR70, otherFeat=[]-->, belongsTo=??
897	125-IPTFYFPRGRP-135, otherFeat=[]-->, belongsTo=??
898	IPTAYFPRGRP, otherFeat=[]-->, belongsTo=??
899	IPTFAFPRGRP, otherFeat=[]-->, belongsTo=??
900	IPTFYAPRGRP, otherFeat=[]-->, belongsTo=??
901	IPTAYAPRGRP, otherFeat=[]-->, belongsTo=??
902	IPTAAAPRGRP, otherFeat=[]-->, belongsTo=??
903	WT, otherFeat=[]-->, belongsTo=??
904	AYF, otherFeat=[]-->, belongsTo=??
905	FAF, otherFeat=[]-->, belongsTo=??
906	FYA, otherFeat=[]-->, belongsTo=??
907	AYA, otherFeat=[]-->, belongsTo=??
908	AAA, otherFeat=[]-->, belongsTo=??
909	A, otherFeat=[]-->, belongsTo=??
910	B, otherFeat=[]-->, belongsTo=??
911	C, otherFeat=[]-->, belongsTo=??
912	Anti-FLAG, otherFeat=[]-->, belongsTo=??
913	IP, otherFeat=[]-->, belongsTo=??
914	FIGURE 5. A conserved hydrophobic motif is involved in the interac-, otherFeat=[]-->, belongsTo=fig_caption
915	tion of PR70 with PP2A. A, an alignment of the N-terminal region of the, otherFeat=[]-->, belongsTo=fig_caption
916	conserved R3 domains (residues 125?135 of PR70) of PPP2R3 subunits, otherFeat=[]-->, belongsTo=fig_caption
917	from various species. Conserved FYF residues are shown in bold. B, a dia-, otherFeat=[]-->, belongsTo=fig_caption
918	gram showing the residues within the PR70 that were changed in the, otherFeat=[]-->, belongsTo=fig_caption
919	mutant forms of PR70 listed on the left. Amino acid substitutions are, otherFeat=[]-->, belongsTo=fig_caption
920	shown in bold. C, FLAG-tagged PR70 (PR70), the N3 mutant ( N3), and, otherFeat=[]-->, belongsTo=fig_caption
921	the indicated FYF mutants were transiently expressed in COS-7 cells. The, otherFeat=[]-->, belongsTo=fig_caption
922	cells were lysed, and tagged proteins were immunoprecipitated with anti-, otherFeat=[]-->, belongsTo=fig_caption
923	FLAG antibody (Anti-FLAG IP). The immunoprecipitates were resolved by, otherFeat=[]-->, belongsTo=fig_caption
924	SDS-PAGE and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A),, otherFeat=[]-->, belongsTo=fig_caption
925	and anti-C-subunit (C) antibodies. A control immunoprecipitate using, otherFeat=[]-->, belongsTo=fig_caption
926	lysate from cells transfected with the empty expression vector (Emp Vec)is, otherFeat=[]-->, belongsTo=fig_caption
927	shown in the first lane., otherFeat=[]-->, belongsTo=fig_caption
928	FLAG, otherFeat=[]-->, belongsTo=??
929	FLAG, otherFeat=[]-->, belongsTo=??
930	-A, otherFeat=[]-->, belongsTo=??
931	GST, otherFeat=[]-->, belongsTo=??
932	PR70, otherFeat=[]-->, belongsTo=??
933	GST, otherFeat=[]-->, belongsTo=??
934	C, otherFeat=[]-->, belongsTo=??
935	N3, otherFeat=[]-->, belongsTo=??
936	9, otherFeat=[]-->, belongsTo=??
937	C, otherFeat=[]-->, belongsTo=??
938	-, otherFeat=[]-->, belongsTo=??
939	GST, otherFeat=[]-->, belongsTo=??
940	12 3 4 56 78, otherFeat=[]-->, belongsTo=??
941	C, otherFeat=[]-->, belongsTo=??
942	EE, otherFeat=[]-->, belongsTo=??
943	E, otherFeat=[]-->, belongsTo=??
944	Ca, otherFeat=[]-->, belongsTo=??
945	Ca, otherFeat=[]-->, belongsTo=??
946	Ca, otherFeat=[]-->, belongsTo=??
947	A, otherFeat=[]-->, belongsTo=??
948	B, otherFeat=[]-->, belongsTo=??
949	GST-A, otherFeat=[]-->, belongsTo=??
950	pulldown, otherFeat=[]-->, belongsTo=??
951	GST-Cdc6, otherFeat=[]-->, belongsTo=??
952	pulldown, otherFeat=[]-->, belongsTo=??
953	FIGURE 6. The C-terminal region of PR70 mediates interaction with, otherFeat=[]-->, belongsTo=fig_caption
954	Cdc6. FLAG-PR70 (lanes 1?3), the N3 mutant (lanes 4 ? 6), and the C, otherFeat=[]-->, belongsTo=fig_caption
955	mutant (lanes 7?9) were transiently expressed in COS-7 cells, and the cells, otherFeat=[]-->, belongsTo=fig_caption
956	were lysed in the presence of EGTA (E) or CaCl2 (Ca). GST pulldown assays, otherFeat=[]-->, belongsTo=fig_caption
957	were performed using immobilized GST-A (panel A) or GST-Cdc6 (panel B),, otherFeat=[]-->, belongsTo=fig_caption
958	and bound proteins were detected by immunoblotting with anti-FLAG, otherFeat=[]-->, belongsTo=fig_caption
959	(FLAG), anti-A-subunit (A) or anti-C-subunit (C) antibodies. Control pull-, otherFeat=[]-->, belongsTo=fig_caption
960	downs with GST alone (GST) were carried out with all three expressed, otherFeat=[]-->, belongsTo=fig_caption
961	proteins using lysates prepared with standard buffer (lanes 1, 4, and 7)., otherFeat=[]-->, belongsTo=fig_caption
962	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
963	16110 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
964	VOLUME 283 ? NUMBER 23 ? JUNE 6, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie
965	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
966	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
967	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
968	de, otherFeat=[]-->, belongsTo=nota_cab_pie
969	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
970	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
971	on, otherFeat=[]-->, belongsTo=nota_cab_pie
972	May, otherFeat=[]-->, belongsTo=nota_cab_pie
973	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
974	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
975	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
976	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
977	from, otherFeat=[]-->, belongsTo=nota_cab_pie
978	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
979	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
980	mutated to non-phosphorylatable alanine residues (AAA-, otherFeat=[]-->, belongsTo=parr
981	Cdc6). Similar results were seen in U2OS cells. These data indi-, otherFeat=[]-->, belongsTo=parr
982	cate that knockdown of PR70 results in an increase in the levels, otherFeat=[]-->, belongsTo=parr
983	of endogenous and exogenous Cdc6 that is dependent on the, otherFeat=[]-->, belongsTo=parr
984	presence of phosphorylatable residues at the N-terminal phos-, otherFeat=[]-->, belongsTo=parr
985	phorylation sites., otherFeat=[]-->, belongsTo=parr
986	The effects of overexpressing PR70 on Cdc6 levels were also, otherFeat=[]-->, belongsTo=parr
987	determined. When HeLa cells were transfected with expression, otherFeat=[]-->, belongsTo=parr
988	plasmids containing the CMV promoter, FLAG-tagged PR70, otherFeat=[]-->, belongsTo=parr
989	was expressed at levels 5- to 10-fold higher than the endoge-, otherFeat=[]-->, belongsTo=parr
990	nous protein (not shown). Co-expression of CDK2 and Cdc6, otherFeat=[]-->, belongsTo=parr
991	caused a substantial increase in Cdc6 levels as reported previ-, otherFeat=[]-->, belongsTo=parr
992	ously (15). Expression of wild-type PR70 caused an increase in, otherFeat=[]-->, belongsTo=parr
993	the levels of both co-transfected and endogenous Cdc6 (Fig., otherFeat=[]-->, belongsTo=parr
994	8A). Expression of the N3 or EF1/2 mutants, which cannot, otherFeat=[]-->, belongsTo=parr
995	interact with the AC core dimer but bind to Cdc6, also, otherFeat=[]-->, belongsTo=parr
996	increased the levels of Cdc6. In contrast, expression of the C, otherFeat=[]-->, belongsTo=parr
997	mutant, which binds to the AC core dimer but not to Cdc6, had, otherFeat=[]-->, belongsTo=parr
998	Luc, otherFeat=[]-->, belongsTo=??
999	PR70, otherFeat=[]-->, belongsTo=??
1000	Luc, otherFeat=[]-->, belongsTo=??
1001	Luc, otherFeat=[]-->, belongsTo=??
1002	Luc, otherFeat=[]-->, belongsTo=??
1003	PR70, otherFeat=[]-->, belongsTo=??
1004	PR70, otherFeat=[]-->, belongsTo=??
1005	PR70, otherFeat=[]-->, belongsTo=??
1006	EV, otherFeat=[]-->, belongsTo=??
1007	wtCdc6 DDD-Cdc6 AAA-Cdc6, otherFeat=[]-->, belongsTo=??
1008	GPDH, otherFeat=[]-->, belongsTo=??
1009	siRNA, otherFeat=[]-->, belongsTo=??
1010	plasmid, otherFeat=[]-->, belongsTo=??
1011	GFP-Cdc6, otherFeat=[]-->, belongsTo=??
1012	B, otherFeat=[]-->, belongsTo=??
1013	EV, otherFeat=[]-->, belongsTo=??
1014	wtCdc6 DDD-Cdc6 AAA-Cdc6, otherFeat=[]-->, belongsTo=??
1015	PR70 siRNA, otherFeat=[]-->, belongsTo=??
1016	C, otherFeat=[]-->, belongsTo=??
1017	Luc siRNA, otherFeat=[]-->, belongsTo=??
1018	plasmid, otherFeat=[]-->, belongsTo=??
1019	D, otherFeat=[]-->, belongsTo=??
1020	A, otherFeat=[]-->, belongsTo=??
1021	Luc, otherFeat=[]-->, belongsTo=??
1022	PP2A-C, otherFeat=[]-->, belongsTo=??
1023	Mock, otherFeat=[]-->, belongsTo=??
1024	PP5, otherFeat=[]-->, belongsTo=??
1025	Cdc6, otherFeat=[]-->, belongsTo=??
1026	PP2A-C, otherFeat=[]-->, belongsTo=??
1027	PP5, otherFeat=[]-->, belongsTo=??
1028	siRNA, otherFeat=[]-->, belongsTo=??
1029	PR70, otherFeat=[]-->, belongsTo=??
1030	Cdc6, otherFeat=[]-->, belongsTo=??
1031	PR70, otherFeat=[]-->, belongsTo=??
1032	Actin, otherFeat=[]-->, belongsTo=??
1033	Luc, otherFeat=[]-->, belongsTo=??
1034	Mock, otherFeat=[]-->, belongsTo=??
1035	PR70-1, otherFeat=[]-->, belongsTo=??
1036	PR70-2, otherFeat=[]-->, belongsTo=??
1037	siRNA, otherFeat=[]-->, belongsTo=??
1038	FIGURE 7. Knockdown of PR70 increases the levels of Cdc6. A, HeLa cells were, otherFeat=[]-->, belongsTo=fig_caption
1039	mock transfected (Mock) or transfected with control (Luc), PP2A catalytic subunit, otherFeat=[]-->, belongsTo=fig_caption
1040	(PP2A-C), or PP5 (PP5) siRNA. Cells were harvested 48 h after transfection and, otherFeat=[]-->, belongsTo=fig_caption
1041	immunoblotted with anti-Cdc6, PP2A-C, PR70, or PP5 antibodies. B, HeLa cells, otherFeat=[]-->, belongsTo=fig_caption
1042	were mock transfected (Mock) or transfected with control (Luc), PR70-1, or PR70-2, otherFeat=[]-->, belongsTo=fig_caption
1043	siRNA. Cells were harvested 48 h after transfection and immunoblotted with anti-, otherFeat=[]-->, belongsTo=fig_caption
1044	Cdc6, PR70, or actin (as a loading control) antibodies. C, HeLa cells were co-trans-, otherFeat=[]-->, belongsTo=fig_caption
1045	fected with control or PR70-1 siRNA and plasmids encoding GFP-tagged versions, otherFeat=[]-->, belongsTo=fig_caption
1046	of wild type Cdc6 (wtCdc6) or Cdc6 in which the N-terminal phosphorylation sites, otherFeat=[]-->, belongsTo=fig_caption
1047	were mutated to aspartic acid (DDD-Cdc6) or alanine (AAA-Cdc6). Forty-eight, otherFeat=[]-->, belongsTo=fig_caption
1048	hours later, the cells were harvested and lysates were analyzed by immunoblot-, otherFeat=[]-->, belongsTo=fig_caption
1049	ting with antibodies against Cdc6 or glyceraldehyde-3 phosphate dehydrogen-, otherFeat=[]-->, belongsTo=fig_caption
1050	ase (GPDH) as a loading control. D, duplicate samples of the lysates described in, otherFeat=[]-->, belongsTo=fig_caption
1051	B, from cells co-transfected with Cdc6 and either luciferase control (Luc siRNA)or, otherFeat=[]-->, belongsTo=fig_caption
1052	PR70-1 (PR70) siRNAs were immunoblotted with anti-PR70 antibodies to confirm, otherFeat=[]-->, belongsTo=fig_caption
1053	knockdown of PR70., otherFeat=[]-->, belongsTo=fig_caption
1054	Empty Vec, otherFeat=[]-->, belongsTo=??
1055	tor, otherFeat=[]-->, belongsTo=??
1056	Cdc6, otherFeat=[]-->, belongsTo=??
1057	+ CDK2, otherFeat=[]-->, belongsTo=??
1058	Cdc6, otherFeat=[]-->, belongsTo=??
1059	Cdc6, otherFeat=[]-->, belongsTo=??
1060	+ PR70EF1/2, otherFeat=[]-->, belongsTo=??
1061	Cdc6, otherFeat=[]-->, belongsTo=??
1062	+ PR70, otherFeat=[]-->, belongsTo=??
1063	C, otherFeat=[]-->, belongsTo=??
1064	Cdc6, otherFeat=[]-->, belongsTo=??
1065	+ PR70, otherFeat=[]-->, belongsTo=??
1066	N3, otherFeat=[]-->, belongsTo=??
1067	Cdc6, otherFeat=[]-->, belongsTo=??
1068	+ PR70, otherFeat=[]-->, belongsTo=??
1069	FLAG-PR70, otherFeat=[]-->, belongsTo=??
1070	Myc-CDK2, otherFeat=[]-->, belongsTo=??
1071	Actin, otherFeat=[]-->, belongsTo=??
1072	Empty Vector, otherFeat=[]-->, belongsTo=??
1073	DDD-Cdc, otherFeat=[]-->, belongsTo=??
1074	6 + PR7, otherFeat=[]-->, belongsTo=??
1075	0, otherFeat=[]-->, belongsTo=??
1076	DDD-Cdc, otherFeat=[]-->, belongsTo=??
1077	6, otherFeat=[]-->, belongsTo=??
1078	AAA-Cdc, otherFeat=[]-->, belongsTo=??
1079	6 + PR70, otherFeat=[]-->, belongsTo=??
1080	AAA-Cdc, otherFeat=[]-->, belongsTo=??
1081	6, otherFeat=[]-->, belongsTo=??
1082	wtCdc, otherFeat=[]-->, belongsTo=??
1083	6 + PR, otherFeat=[]-->, belongsTo=??
1084	70, otherFeat=[]-->, belongsTo=??
1085	wtCdc, otherFeat=[]-->, belongsTo=??
1086	6, otherFeat=[]-->, belongsTo=??
1087	A, otherFeat=[]-->, belongsTo=??
1088	B, otherFeat=[]-->, belongsTo=??
1089	GFP-Cdc6, otherFeat=[]-->, belongsTo=??
1090	Endo-Cdc6, otherFeat=[]-->, belongsTo=??
1091	GFP-Cdc6, otherFeat=[]-->, belongsTo=??
1092	FLAG-PR70, otherFeat=[]-->, belongsTo=??
1093	Actin, otherFeat=[]-->, belongsTo=??
1094	Endo-Cdc6, otherFeat=[]-->, belongsTo=??
1095	FIGURE 8. Overexpression of PR70 increases the levels of Cdc6. A, U2OS, otherFeat=[]-->, belongsTo=fig_caption
1096	cells were transfected with empty vector, or plasmids encoding GFP-Cdc6, otherFeat=[]-->, belongsTo=fig_caption
1097	(Cdc6), myc-tagged CDK2 (CDK2), or FLAG-tagged constructs of wild-type, otherFeat=[]-->, belongsTo=fig_caption
1098	PR70 (PR70) or the indicated PR70 mutants. Cells were harvested 24 h after, otherFeat=[]-->, belongsTo=fig_caption
1099	transfection, and lysates were analyzed by immunoblotting with antibodies, otherFeat=[]-->, belongsTo=fig_caption
1100	against Cdc6, actin, FLAG, or myc as indicated at the right. The Cdc6 antibod-, otherFeat=[]-->, belongsTo=fig_caption
1101	ies detected both the expressed Cdc6 (GFP-Cdc6) and endogenous Cdc6, otherFeat=[]-->, belongsTo=fig_caption
1102	(Endo-Cdc6). B, U2OS cells were co-transfected with empty vector or plasmids, otherFeat=[]-->, belongsTo=fig_caption
1103	expressing FLAG-PR70 and plasmids expressing GFP-tagged versions of wild-, otherFeat=[]-->, belongsTo=fig_caption
1104	type Cdc6 (wtCdc6), or the AAA (AAA-Cdc6), or DDD (DDD-Cdc6) triple phos-, otherFeat=[]-->, belongsTo=fig_caption
1105	phorylation site mutants of Cdc6. Cells were harvested 24 h later, and lysates, otherFeat=[]-->, belongsTo=fig_caption
1106	were analyzed by immunoblotting with antibodies against Cdc6, actin, or the, otherFeat=[]-->, belongsTo=fig_caption
1107	FLAG epitope as indicated at the right., otherFeat=[]-->, belongsTo=fig_caption
1108	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
1109	JUNE 6, 2008 ? VOLUME 283 ? NUMBER 23, otherFeat=['U']-->, belongsTo=nota_cab_pie
1110	JOURNAL OF BIOLOGICAL CHEMISTRY 16111, otherFeat=[]-->, belongsTo=nota_cab_pie
1111	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
1112	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
1113	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1114	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1115	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1116	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1117	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1118	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1119	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1120	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
1121	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
1122	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
1123	from, otherFeat=[]-->, belongsTo=nota_cab_pie
1124	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
1125	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
1126	relatively little effect on the levels of exogenous or endogenous, otherFeat=[]-->, belongsTo=parr
1127	Cdc6. The potential role of phosphorylation in the effects of, otherFeat=[]-->, belongsTo=parr
1128	overexpressed PR70 was tested using the phosphorylation site, otherFeat=[]-->, belongsTo=parr
1129	mutants of Cdc6. Although co-expression of PR70 caused some, otherFeat=[]-->, belongsTo=parr
1130	increase in the levels of the AAA mutant of Cdc6, the effect was, otherFeat=[]-->, belongsTo=parr
1131	much less than its effect on wild-type Cdc6 (Fig. 8B). Similarly,, otherFeat=[]-->, belongsTo=parr
1132	co-expression of PR70 had little effect on the levels of the, otherFeat=[]-->, belongsTo=parr
1133	DDD mutant of Cdc6 even though endogenous Cdc6 was, otherFeat=[]-->, belongsTo=parr
1134	increased. Thus, the ability of overexpressed PR70 to cause, otherFeat=[]-->, belongsTo=parr
1135	accumulation of the protein was inhibited when the phos-, otherFeat=[]-->, belongsTo=parr
1136	phorylation sites in Cdc6 were mutated. The effects of PR70, otherFeat=[]-->, belongsTo=parr
1137	overexpression to cause accumulation of Cdc6 suggest it acts, otherFeat=[]-->, belongsTo=parr
1138	in a dominant-negative manner to block Cdc6 dephospho-, otherFeat=[]-->, belongsTo=parr
1139	rylation (see "Discussion")., otherFeat=[]-->, belongsTo=parr
1140	Knockdown of PR70 Causes G1 Arrest--The potential role of, otherFeat=[]-->, belongsTo=parr
1141	PR70 in progression through G1 was determined by determin-, otherFeat=[]-->, belongsTo=parr
1142	ing the cell cycle distribution of cells in which PR70 was, otherFeat=[]-->, belongsTo=parr
1143	depleted by RNA interference. Knockdown of PR70 caused, otherFeat=[]-->, belongsTo=parr
1144	accumulation of cells in G0/G1 and depletion of cells in S and, otherFeat=[]-->, belongsTo=parr
1145	G2/M (Fig. 9). The apparent G1 arrest occurred with either of, otherFeat=[]-->, belongsTo=parr
1146	two siRNAs that target distinct regions of PR70. The level of G1, otherFeat=[]-->, belongsTo=parr
1147	arrest correlated with the extent of PR70 knockdown. The, otherFeat=[]-->, belongsTo=parr
1148	lower levels of PR70 achieved with the PR70 siRNA-1 com-, otherFeat=[]-->, belongsTo=parr
1149	pared with PR70 siRNA-2 corresponded to a greater increase in, otherFeat=[]-->, belongsTo=parr
1150	the number of G1 cells (76% versus 65%). The G1 arrest caused, otherFeat=[]-->, belongsTo=parr
1151	by knockdown of PR70 supports a role for this PP2A regulatory, otherFeat=[]-->, belongsTo=parr
1152	subunit in progression through G1 phase., otherFeat=[]-->, belongsTo=parr
1153	DISCUSSION, otherFeat=[]-->, belongsTo=title
1154	The formation of pre-replicative complexes during the initi-, otherFeat=[]-->, belongsTo=parr
1155	ation of DNA replication is regulated, in part, by the availability, otherFeat=[]-->, belongsTo=parr
1156	of Cdc6. Cyclin-dependent kinases phosphorylate regulatory, otherFeat=[]-->, belongsTo=parr
1157	sites within the N-terminal domain of Cdc6 and block ubiquiti-, otherFeat=[]-->, belongsTo=parr
1158	nation by APC/Ccdh1 and subsequent degradation by the pro-, otherFeat=[]-->, belongsTo=parr
1159	teasome (15). The results reported here help establish the form, otherFeat=[]-->, belongsTo=parr
1160	of PP2A complexed with the PR70 regulatory subunit as a phys-, otherFeat=[]-->, belongsTo=parr
1161	iological Cdc6 phosphatase and are consistent with a model in, otherFeat=[]-->, belongsTo=parr
1162	which PR70 targets PP2A to Cdc6 through direct protein-pro-, otherFeat=[]-->, belongsTo=parr
1163	tein interactions. Knockdown of PR70 by RNA interference, otherFeat=[]-->, belongsTo=parr
1164	results in an increase in the levels of Cdc6 protein, consistent, otherFeat=[]-->, belongsTo=parr
1165	with a role for this subunit in regulating the stability of Cdc6., otherFeat=[]-->, belongsTo=parr
1166	Overexpression of PR70 appeared to act in a dominant-nega-, otherFeat=[]-->, belongsTo=parr
1167	tive manner to also increase the levels of Cdc6. The observa-, otherFeat=[]-->, belongsTo=parr
1168	tions that increased protein levels did not occur with phospho-, otherFeat=[]-->, belongsTo=parr
1169	rylation site mutants of Cdc6 are consistent with a role for PR70, otherFeat=[]-->, belongsTo=parr
1170	in regulating Cdc6 phosphorylation and stability. A novel, otherFeat=[]-->, belongsTo=parr
1171	aspect of this model is the potential regulation of Cdc6 dephos-, otherFeat=[]-->, belongsTo=parr
1172	phorylation by calcium. Calcium enhances the recruitment of, otherFeat=[]-->, belongsTo=parr
1173	the core dimer of PP2A to Cdc6 by binding to the EF-hand, otherFeat=[]-->, belongsTo=parr
1174	motifs of PR70, raising the possibility that changes in intracel-, otherFeat=[]-->, belongsTo=parr
1175	lular calcium can regulate the accumulation of Cdc6 and initi-, otherFeat=[]-->, belongsTo=parr
1176	ation of DNA replication. However, it remains to be deter-, otherFeat=[]-->, belongsTo=parr
1177	mined if physiological changes in intracellular calcium, otherFeat=[]-->, belongsTo=parr
1178	No., otherFeat=[]-->, belongsTo=title
1179	of, otherFeat=[]-->, belongsTo=??
1180	Cells, otherFeat=[]-->, belongsTo=??
1181	PR70, otherFeat=[]-->, belongsTo=??
1182	GPDH, otherFeat=[]-->, belongsTo=??
1183	Non-transfectedLuc, otherFeat=[]-->, belongsTo=??
1184	control, otherFeat=[]-->, belongsTo=??
1185	PR70, otherFeat=[]-->, belongsTo=??
1186	siRNA-2, otherFeat=[]-->, belongsTo=??
1187	PR70, otherFeat=[]-->, belongsTo=??
1188	siRNA-1, otherFeat=[]-->, belongsTo=??
1189	Luc control, otherFeat=[]-->, belongsTo=??
1190	0, otherFeat=[]-->, belongsTo=??
1191	100, otherFeat=[]-->, belongsTo=??
1192	200, otherFeat=[]-->, belongsTo=??
1193	300, otherFeat=[]-->, belongsTo=??
1194	400, otherFeat=[]-->, belongsTo=??
1195	G0/G1 55.3, otherFeat=[]-->, belongsTo=??
1196	G2/M 20.9, otherFeat=[]-->, belongsTo=??
1197	S 22.2, otherFeat=[]-->, belongsTo=??
1198	Non-transfected, otherFeat=[]-->, belongsTo=??
1199	0, otherFeat=[]-->, belongsTo=??
1200	100, otherFeat=[]-->, belongsTo=??
1201	200, otherFeat=[]-->, belongsTo=??
1202	300, otherFeat=[]-->, belongsTo=??
1203	400, otherFeat=[]-->, belongsTo=??
1204	500, otherFeat=[]-->, belongsTo=??
1205	G0/G1 52.1, otherFeat=[]-->, belongsTo=??
1206	G2/M 24.4, otherFeat=[]-->, belongsTo=??
1207	S 22.2, otherFeat=[]-->, belongsTo=??
1208	PR70 siRNA-2, otherFeat=[]-->, belongsTo=??
1209	0, otherFeat=[]-->, belongsTo=??
1210	200, otherFeat=[]-->, belongsTo=??
1211	400, otherFeat=[]-->, belongsTo=??
1212	600, otherFeat=[]-->, belongsTo=??
1213	G0/G1 65, otherFeat=[]-->, belongsTo=??
1214	G2/M 17.9, otherFeat=[]-->, belongsTo=??
1215	S 16.3, otherFeat=[]-->, belongsTo=??
1216	PR70 siRNA-1, otherFeat=[]-->, belongsTo=??
1217	0, otherFeat=[]-->, belongsTo=??
1218	200, otherFeat=[]-->, belongsTo=??
1219	400, otherFeat=[]-->, belongsTo=??
1220	600, otherFeat=[]-->, belongsTo=??
1221	800, otherFeat=[]-->, belongsTo=??
1222	FL3 Area, otherFeat=[]-->, belongsTo=??
1223	0, otherFeat=[]-->, belongsTo=??
1224	200, otherFeat=[]-->, belongsTo=??
1225	400, otherFeat=[]-->, belongsTo=??
1226	600, otherFeat=[]-->, belongsTo=??
1227	800, otherFeat=[]-->, belongsTo=??
1228	G0/G1 76.5, otherFeat=[]-->, belongsTo=??
1229	G2/M 10.7, otherFeat=[]-->, belongsTo=??
1230	S 12.1, otherFeat=[]-->, belongsTo=??
1231	A, otherFeat=[]-->, belongsTo=??
1232	B, otherFeat=[]-->, belongsTo=??
1233	C, otherFeat=[]-->, belongsTo=??
1234	D, otherFeat=[]-->, belongsTo=??
1235	E, otherFeat=[]-->, belongsTo=??
1236	FIGURE 9. Knockdown of PR70 causes G1 arrest. A?D, U2OS cells were left, otherFeat=[]-->, belongsTo=fig_caption
1237	untreated (A), or transfected with control siRNA (B), PR70-2 siRNA (C), or, otherFeat=[]-->, belongsTo=fig_caption
1238	PR70-1 siRNA (D). Forty-eight hours later, the cells were harvested and, otherFeat=[]-->, belongsTo=fig_caption
1239	analyzed by flow cytometry. The data are plotted as the number of cells versus, otherFeat=[]-->, belongsTo=fig_caption
1240	DNA content determined by FL3 area. The percentages of cells in G0/G1,S,and, otherFeat=[]-->, belongsTo=fig_caption
1241	G2/M phases are indicated. E, duplicate transfections were harvested after, otherFeat=[]-->, belongsTo=fig_caption
1242	48 h and immunoblotted with anti-PR70 antibody to confirm knockdown., otherFeat=[]-->, belongsTo=fig_caption
1243	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
1244	16112 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
1245	VOLUME 283 ? NUMBER 23 ? JUNE 6, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie
1246	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
1247	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
1248	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1249	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1250	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1251	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1252	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1253	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1254	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1255	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
1256	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
1257	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
1258	from, otherFeat=[]-->, belongsTo=nota_cab_pie
1259	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
1260	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
1261	concentrations are sufficient to regulate association of PP2A, otherFeat=[]-->, belongsTo=parr
1262	with Cdc6., otherFeat=[]-->, belongsTo=parr
1263	Biochemical analysis of the interaction of PR70 with the AC, otherFeat=[]-->, belongsTo=parr
1264	core dimer suggested an unanticipated mechanism for regulat-, otherFeat=[]-->, belongsTo=parr
1265	ing PP2A activity. Experiments with N-terminal truncation, otherFeat=[]-->, belongsTo=parr
1266	mutants showed that PR70 can associate with Cdc6 independ-, otherFeat=[]-->, belongsTo=parr
1267	ently of the A- and C-subunits. This observation contrasts with, otherFeat=[]-->, belongsTo=parr
1268	the prevailing view of PP2A in which the regulatory subunits, otherFeat=[]-->, belongsTo=parr
1269	have been thought to be constitutively associated with the core, otherFeat=[]-->, belongsTo=parr
1270	dimer (18 ?20). The importance of the existence of PP2A in, otherFeat=[]-->, belongsTo=parr
1271	heterotrimeric forms is supported by data showing that some, otherFeat=[]-->, belongsTo=parr
1272	PP2A regulatory subunits are only stable when incorporated, otherFeat=[]-->, belongsTo=parr
1273	into holoenzymes (29 ?31). Overexpression of the PPP2R2 fam-, otherFeat=[]-->, belongsTo=parr
1274	ily member, B , leads to proteasome-dependent degradation of, otherFeat=[]-->, belongsTo=parr
1275	the free protein but not protein incorporation into holoen-, otherFeat=[]-->, belongsTo=parr
1276	zymes (31). In contrast, several lines of evidence indicate that, otherFeat=[]-->, belongsTo=parr
1277	members of the PPP2R3/PR72 family are stable regardless of, otherFeat=[]-->, belongsTo=parr
1278	whether or not they are incorporated into holoenzymes. As, otherFeat=[]-->, belongsTo=parr
1279	shown here, mutants of PR70 that cannot bind to PP2A accu-, otherFeat=[]-->, belongsTo=parr
1280	mulate to the same levels as the wild-type protein. The apparent, otherFeat=[]-->, belongsTo=parr
1281	stability of PR70 is also independent of interaction with Cdc6,, otherFeat=[]-->, belongsTo=parr
1282	because a mutant ( C) that interacts poorly with Cdc6 accu-, otherFeat=[]-->, belongsTo=parr
1283	mulates to similar levels. Similarly, mutations of the related, otherFeat=[]-->, belongsTo=parr
1284	PR72 subunit that block interaction with the AC core dimer, otherFeat=[]-->, belongsTo=parr
1285	have no effect on the levels of expressed protein (27). In addi-, otherFeat=[]-->, belongsTo=parr
1286	tion, the PR59 subunit is not degraded following loss of the, otherFeat=[]-->, belongsTo=parr
1287	A-subunit (31). Thus, in contrast to the PPP2R2/B and, otherFeat=[]-->, belongsTo=parr
1288	PPP2R5/B56 families of PP2A regulatory subunits, members of, otherFeat=[]-->, belongsTo=parr
1289	the PPP2R3/PR72 family are stable proteins whose levels and, otherFeat=[]-->, belongsTo=parr
1290	functional interactions with substrates and other proteins may, otherFeat=[]-->, belongsTo=parr
1291	be independent of the core dimer of PP2A., otherFeat=[]-->, belongsTo=parr
1292	The stability of expressed PR70 may also account for its abil-, otherFeat=[]-->, belongsTo=parr
1293	ity to act in an apparent dominant-negative manner to increase, otherFeat=[]-->, belongsTo=parr
1294	the levels of Cdc6. Excess free PR70 would associate with Cdc6, otherFeat=[]-->, belongsTo=parr
1295	and displace endogenous PR70-AC holoenzyme. Loss of the, otherFeat=[]-->, belongsTo=parr
1296	active AC core dimer from Cdc6 would inhibit dephosphoryl-, otherFeat=[]-->, belongsTo=parr
1297	ation leading to decreased ubiquitination by APC/Ccdh1 and, otherFeat=[]-->, belongsTo=parr
1298	increase protein levels. A dominant-negative action of overex-, otherFeat=[]-->, belongsTo=parr
1299	pressed PR70 is supported by observations that the effects on, otherFeat=[]-->, belongsTo=parr
1300	Cdc6 levels are not dependent on interaction of PR70 with the, otherFeat=[]-->, belongsTo=parr
1301	AC core dimer (e.g. the N3 and EF1/2 mutants) but are, otherFeat=[]-->, belongsTo=parr
1302	dependent on interaction with Cdc6 (e.g. the C mutant). Like, otherFeat=[]-->, belongsTo=parr
1303	knockdown of PR70, the dominant-negative actions of PR70 to, otherFeat=[]-->, belongsTo=parr
1304	increase Cdc6 levels appear to be dependent on intact phospho-, otherFeat=[]-->, belongsTo=parr
1305	rylation sites, because the levels of co-expressed DDD and AAA, otherFeat=[]-->, belongsTo=parr
1306	mutants of Cdc6 were not significantly affected. Forced over-, otherFeat=[]-->, belongsTo=parr
1307	expression of the related PR72 subunit has also been reported, otherFeat=[]-->, belongsTo=parr
1308	to act in a dominant-negative manner. Expression of either, otherFeat=[]-->, belongsTo=parr
1309	wild-type PR72 or an EF-hand 2 mutant, which cannot bind the, otherFeat=[]-->, belongsTo=parr
1310	AC core dimer, both cause G1 arrest in U2OS cells (27)., otherFeat=[]-->, belongsTo=parr
1311	The accumulation of cells with G0/G1 DNA content follow-, otherFeat=[]-->, belongsTo=parr
1312	ing knockdown by RNA interference is consistent with an, otherFeat=[]-->, belongsTo=parr
1313	important role for PR70 in progression of cells through into S, otherFeat=[]-->, belongsTo=parr
1314	phase. Similarly, overexpression of a fragment that contains the, otherFeat=[]-->, belongsTo=parr
1315	complete R3 domain and the C terminus of PR70 (termed PR48, otherFeat=[]-->, belongsTo=parr
1316	or N1 in this study) also causes G1 arrest, presumably through, otherFeat=[]-->, belongsTo=parr
1317	a dominant-negative action (17). The G1 arrest in cells depleted, otherFeat=[]-->, belongsTo=parr
1318	of PR70 coincides with an increase in Cdc6 protein levels. A, otherFeat=[]-->, belongsTo=parr
1319	previous study showed that increases in phosphorylation and, otherFeat=[]-->, belongsTo=parr
1320	stability of Cdc6 enhance formation of pre-replicative com-, otherFeat=[]-->, belongsTo=parr
1321	plexes (15). The increase in pre-replicative complex formation, otherFeat=[]-->, belongsTo=parr
1322	would be expected to enhance entry into S phase. Consistent, otherFeat=[]-->, belongsTo=parr
1323	with this idea, expression of exogenous wild-type Cdc6 leads to, otherFeat=[]-->, belongsTo=parr
1324	accelerated entry into S phase (14). However, exogenous, otherFeat=[]-->, belongsTo=parr
1325	expression of a non-phosphorylatable (AAA) mutant of Cdc6, otherFeat=[]-->, belongsTo=parr
1326	(5) or an N-terminally truncated version of Cdc6, missing the, otherFeat=[]-->, belongsTo=parr
1327	CDK phosphorylation sites and destruction motifs recognized, otherFeat=[]-->, belongsTo=parr
1328	by APC/Ccdh1 (14), inhibit initiation of DNA replication and, otherFeat=[]-->, belongsTo=parr
1329	entry into S phase. It is possible that, even though phosphoryl-, otherFeat=[]-->, belongsTo=parr
1330	ation is required for stabilization of Cdc6 and assembly of pre-, otherFeat=[]-->, belongsTo=parr
1331	replicative complexes during G1, an additional Cdc6 dephos-, otherFeat=[]-->, belongsTo=parr
1332	phorylation or degradation step is needed to initiate DNA, otherFeat=[]-->, belongsTo=parr
1333	replication. Knockdown or overexpression of PR70 might, otherFeat=[]-->, belongsTo=parr
1334	inhibit this step and retard entry into S phase. Although the, otherFeat=[]-->, belongsTo=parr
1335	ability of PR70 knockdown to cause G1 arrest is consistent with, otherFeat=[]-->, belongsTo=parr
1336	regulation of Cdc6, an equally likely possibility is that PR70, otherFeat=[]-->, belongsTo=parr
1337	plays other roles during G1. PR70 may regulate the activity of, otherFeat=[]-->, belongsTo=parr
1338	other proteins involved in cell cycle control, either through, otherFeat=[]-->, belongsTo=parr
1339	PP2A-mediated dephosphorylation or actions that are inde-, otherFeat=[]-->, belongsTo=parr
1340	pendent of PP2A., otherFeat=[]-->, belongsTo=parr
1341	The effects of calcium on the PR70-dependent association of, otherFeat=[]-->, belongsTo=parr
1342	PP2A with Cdc6 are consistent with a more general role for the, otherFeat=[]-->, belongsTo=parr
1343	PPP2R3/PR72 family in mediating calcium-regulated dephos-, otherFeat=[]-->, belongsTo=parr
1344	phorylation. All four members of this family contain conserved, otherFeat=[]-->, belongsTo=parr
1345	EF-hand sequence motifs (supplemental Fig.  S1). The EF-hands, otherFeat=[]-->, belongsTo=parr
1346	of PR72 are also functional calcium binding sites, and, similar to, otherFeat=[]-->, belongsTo=parr
1347	PR70, calcium binding to the second EF-hand enhances inter-, otherFeat=[]-->, belongsTo=parr
1348	action with the A-subunit (27). PR72 has been shown to medi-, otherFeat=[]-->, belongsTo=parr
1349	ate calcium-dependent dephosphorylation of threonine-75 in, otherFeat=[]-->, belongsTo=parr
1350	the dopamine- and cAMP-regulated phosphoprotein of 32 kDa, otherFeat=[]-->, belongsTo=parr
1351	(DARPP-32). This report showed that, in addition to the role of, otherFeat=[]-->, belongsTo=parr
1352	EF-hand 2 in interaction with the AC core dimer, calcium bind-, otherFeat=[]-->, belongsTo=parr
1353	ing to EF-hand 1 increased the phosphatase activity of the, otherFeat=[]-->, belongsTo=parr
1354	PR72-holoenzyme toward DARPP-32 (32). Both PR72 and its, otherFeat=[]-->, belongsTo=parr
1355	alternative splice variant (PR130) have been reported to inter-, otherFeat=[]-->, belongsTo=parr
1356	act with the mammalian Naked cuticle protein and regulate, otherFeat=[]-->, belongsTo=parr
1357	Wnt signaling (33, 34). Calcium may therefore influence Wnt, otherFeat=[]-->, belongsTo=parr
1358	signaling through recruitment and/or regulation of PP2A asso-, otherFeat=[]-->, belongsTo=parr
1359	ciated with Naked cuticle. The other member of the PPP2R3, otherFeat=[]-->, belongsTo=parr
1360	family, PR59, targets PP2A to the retinoblastoma-related p107, otherFeat=[]-->, belongsTo=parr
1361	protein (35) and may provide a mechanism for calcium regula-, otherFeat=[]-->, belongsTo=parr
1362	tion of the cell cycle functions of p107., otherFeat=[]-->, belongsTo=parr
1363	The sites involved in the interaction with PP2A and Cdc6, otherFeat=[]-->, belongsTo=parr
1364	mapped to distinct regions of PR70, consistent with a role in, otherFeat=[]-->, belongsTo=parr
1365	bridging the two proteins. The N-terminal domain of PR70,, otherFeat=[]-->, belongsTo=parr
1366	which is not conserved with other members of the PPP2R3, otherFeat=[]-->, belongsTo=parr
1367	family, is not required for interaction with either PP2A or Cdc6., otherFeat=[]-->, belongsTo=parr
1368	Deletion of the C-terminal region, including the PR70-unique, otherFeat=[]-->, belongsTo=parr
1369	sequence and a portion of the conserved R3 domain, had no, otherFeat=[]-->, belongsTo=parr
1370	effect on interaction with the PP2A core dimer but severely, otherFeat=[]-->, belongsTo=parr
1371	inhibited binding to Cdc6. Conversely, deletion of N-terminal, otherFeat=[]-->, belongsTo=parr
1372	sequences within the conserved R3 domain blocked binding to, otherFeat=[]-->, belongsTo=parr
1373	the A-subunit but had no effect on interaction with Cdc6. The, otherFeat=[]-->, belongsTo=parr
1374	N-terminal region required for interaction with the A-subunit, otherFeat=[]-->, belongsTo=parr
1375	contains an FYF amino acid motif that plays a role in the inter-, otherFeat=[]-->, belongsTo=parr
1376	action and is conserved between members of the PPP2R3 fam-, otherFeat=[]-->, belongsTo=parr
1377	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
1378	JUNE 6, 2008 ? VOLUME 283 ? NUMBER 23, otherFeat=['U']-->, belongsTo=nota_cab_pie
1379	JOURNAL OF BIOLOGICAL CHEMISTRY 16113, otherFeat=[]-->, belongsTo=nota_cab_pie
1380	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
1381	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
1382	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1383	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1384	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1385	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1386	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1387	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1388	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1389	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
1390	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
1391	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
1392	from, otherFeat=[]-->, belongsTo=nota_cab_pie
1393	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
1394	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
1395	ily. The A-subunit of PP2A is a HEAT repeat protein (36). The, otherFeat=[]-->, belongsTo=parr
1396	FYF motif in PR70 resembles the FG amino acid repeats (FXFG, otherFeat=[]-->, belongsTo=parr
1397	and GLFG) within the nucleoporin family of nuclear pore pro-, otherFeat=[]-->, belongsTo=parr
1398	teins. The nucleoporins interact with nuclear transport factors,, otherFeat=[]-->, belongsTo=parr
1399	including importin- , which are also HEAT repeat proteins., otherFeat=[]-->, belongsTo=parr
1400	The FG repeats of nucleoporins bind to shallow hydrophobic, otherFeat=[]-->, belongsTo=parr
1401	pockets in importin- (37, 38). The A-subunit of PP2A contains, otherFeat=[]-->, belongsTo=parr
1402	exposed hydrophobic surfaces, predicted to play a role in inter-, otherFeat=[]-->, belongsTo=parr
1403	action with the regulatory subunits (36), that are possible sites, otherFeat=[]-->, belongsTo=parr
1404	of interaction with the FYF motif of PR70., otherFeat=[]-->, belongsTo=parr
1405	The requirement for the N-terminal region of the R3 domain, otherFeat=[]-->, belongsTo=parr
1406	of PR70 for binding to the A-subunit is distinct from results, otherFeat=[]-->, belongsTo=parr
1407	observed with the PR72 protein. A fragment of PR72 consisting, otherFeat=[]-->, belongsTo=parr
1408	of amino acids 219 ? 473 (corresponding to residues 257?509 of, otherFeat=[]-->, belongsTo=parr
1409	PR70) interacts with the A-subunit in the yeast two-hybrid, otherFeat=[]-->, belongsTo=parr
1410	assay (27). This fragment of PR72 is missing the N-terminal, otherFeat=[]-->, belongsTo=parr
1411	region of the R3 domain. Two fragments of PR72 containing, otherFeat=[]-->, belongsTo=parr
1412	putative A-subunit binding domains prepared by in vitro trans-, otherFeat=[]-->, belongsTo=parr
1413	lation (corresponding to residues 234 ?339 and 378 ? 436 of, otherFeat=[]-->, belongsTo=parr
1414	PR70) interacted with the A-subunit in vitro using GST pull-, otherFeat=[]-->, belongsTo=parr
1415	down assays (39). These PR72 fragments also do not contain the, otherFeat=[]-->, belongsTo=parr
1416	conserved N-terminal region of the R3 domain that was neces-, otherFeat=[]-->, belongsTo=parr
1417	sary for interaction of PR70 with the AC core dimer in our, otherFeat=[]-->, belongsTo=parr
1418	assays. These observations indicate that additional regions of, otherFeat=[]-->, belongsTo=parr
1419	PR70, beyond those required in PR72, are required for binding, otherFeat=[]-->, belongsTo=parr
1420	to the A-subunit, or that the differences observed are due to, otherFeat=[]-->, belongsTo=parr
1421	different assays employed., otherFeat=[]-->, belongsTo=parr
1422	In summary, the present study shows that the PR70 regula-, otherFeat=[]-->, belongsTo=parr
1423	tory subunit targets PP2A to Cdc6 and that PP2A is likely to be, otherFeat=[]-->, belongsTo=parr
1424	a physiological Cdc6 phosphatase. The targeting of PP2A to, otherFeat=[]-->, belongsTo=parr
1425	Cdc6 is enhanced by binding of calcium to PR70 raising the, otherFeat=[]-->, belongsTo=parr
1426	possibility that changes in intracellular calcium can influence, otherFeat=[]-->, belongsTo=parr
1427	formation of pre-replicative complexes through regulation of, otherFeat=[]-->, belongsTo=parr
1428	Cdc6 dephosphorylation., otherFeat=[]-->, belongsTo=parr
1429	REFERENCES, otherFeat=[]-->, belongsTo=parrnote
1430	1. Bell, S. P., and Dutta, A. (2002) Annu. Rev. Biochem. 71, 333?374, otherFeat=[]-->, belongsTo=parrnote
1431	2. Diffley, J. F. (2004) Curr. Biol. 14, R778 ?R786, otherFeat=[]-->, belongsTo=parrnote
1432	3. Williams, R. S., Shohet, R. V., and Stillman, B. (1997) Proc. Natl. Acad. Sci., otherFeat=[]-->, belongsTo=parrnote
1433	U. S. A. 94, 142?147, otherFeat=[]-->, belongsTo=parrnote
1434	4. Yan, Z., DeGregori, J., Shohet, R., Leone, G., Stillman, B., Nevins, J. R., and, otherFeat=[]-->, belongsTo=parrnote
1435	Williams, R. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3603?3608, otherFeat=[]-->, belongsTo=parrnote
1436	5. Jiang, W., Wells, N. J., and Hunter, T. (1999) Proc. Natl. Acad. Sci. U. S. A., otherFeat=[]-->, belongsTo=parrnote
1437	96, 6193? 6198, otherFeat=[]-->, belongsTo=parrnote
1438	6. Herbig, U., Griffith, J. W., and Fanning, E. (2000) Mol. Biol. Cell 11,, otherFeat=[]-->, belongsTo=parrnote
1439	4117? 4130, otherFeat=[]-->, belongsTo=parrnote
1440	7. Petersen, B. O., Lukas, J., Sorensen, C. S., Bartek, J., and Helin, K. (1999), otherFeat=[]-->, belongsTo=parrnote
1441	EMBO J. 18, 396 ? 410, otherFeat=[]-->, belongsTo=parrnote
1442	8. Pelizon, C., Madine, M. A., Romanowski, P., and Laskey, R. A. (2000), otherFeat=[]-->, belongsTo=parrnote
1443	Genes Dev. 14, 2526 ?2533, otherFeat=[]-->, belongsTo=parrnote
1444	9. Delmolino, L. M., Saha, P., and Dutta, A. (2001) J. Biol. Chem. 276,, otherFeat=[]-->, belongsTo=parrnote
1445	26947?26954, otherFeat=[]-->, belongsTo=parrnote
1446	10. Coverley, D., Pelizon, C., Trewick, S., and Laskey, R. A. (2000) J. Cell Sci., otherFeat=[]-->, belongsTo=parrnote
1447	113, 1929 ?1938, otherFeat=[]-->, belongsTo=parrnote
1448	11. Mendez, J., and Stillman, B. (2000) Mol. Cell Biol. 20, 8602? 8612, otherFeat=[]-->, belongsTo=parrnote
1449	12. Alexandrow, M. G., and Hamlin, J. L. (2004) Mol. Cell Biol. 24, 1614 ?1627, otherFeat=[]-->, belongsTo=parrnote
1450	13. Harper, J. W., Burton, J. L., and Solomon, M. J. (2002) Genes Dev. 16,, otherFeat=[]-->, belongsTo=parrnote
1451	2179 ?2206, otherFeat=[]-->, belongsTo=parrnote
1452	14. Petersen, B. O., Wagener, C., Marinoni, F., Kramer, E. R., Melixetian, M.,, otherFeat=[]-->, belongsTo=parrnote
1453	Denchi, E. L., Gieffers, C., Matteucci, C., Peters, J. M., and Helin, K. (2000), otherFeat=[]-->, belongsTo=parrnote
1454	Genes Dev. 14, 2330 ?2343, otherFeat=[]-->, belongsTo=parrnote
1455	15. Mailand, N., and Diffley, J. F. (2005) Cell 122, 915?926, otherFeat=[]-->, belongsTo=parrnote
1456	16. Duursma, A., and Agami, R. (2005) Mol. Cell Biol. 25, 6937? 6947, otherFeat=[]-->, belongsTo=parrnote
1457	17. Yan, Z., Fedorov, S. A., Mumby, M. C., and Williams, R. S. (2000) Mol. Cell, otherFeat=[]-->, belongsTo=parrnote
1458	Biol. 20, 1021?1029, otherFeat=[]-->, belongsTo=parrnote
1459	18. Virshup, D. M. (2000) Curr. Opin. Cell Biol. 12, 180 ?185, otherFeat=[]-->, belongsTo=parrnote
1460	19. Janssens, V., and Goris, J. (2001) Biochem. J. 353, 417? 439, otherFeat=[]-->, belongsTo=parrnote
1461	20. Silverstein, A. M., Davis, A. J., Bielinski, V. A., Esplin, E. D., Mahmood,, otherFeat=[]-->, belongsTo=parrnote
1462	N. A., and Mumby, M. C. (2003) in Handbook of Cellular Signaling (Brad-, otherFeat=[]-->, belongsTo=parrnote
1463	shaw, R. A., and Dennis, E. A., eds) pp. 405? 415, Academic Press, San, otherFeat=[]-->, belongsTo=parrnote
1464	Diego, CA, otherFeat=[]-->, belongsTo=parrnote
1465	21. Stevens, I., Janssens, V., Martens, E., Dilworth, S., Goris, J., and Van Hoof,, otherFeat=[]-->, belongsTo=parrnote
1466	C. (2003) Eur. J. Biochem. 270, 376 ?387, otherFeat=[]-->, belongsTo=parrnote
1467	22. Pearson, G. W., Earnest, S., and Cobb, M. H. (2006) Mol. Cell Biol. 26,, otherFeat=[]-->, belongsTo=parrnote
1468	3039 ?3047, otherFeat=[]-->, belongsTo=parrnote
1469	23. Ali, A., Zhang, J., Bao, S., Liu, I., Otterness, D., Dean, N. M., Abraham,, otherFeat=[]-->, belongsTo=parrnote
1470	R. T., and Wang, X. F. (2004) Genes Dev. 18, 249 ?254, otherFeat=[]-->, belongsTo=parrnote
1471	24. Mumby, M. C., Green, D. D., and Russell, K. R. (1985) J. Biol. Chem. 260,, otherFeat=[]-->, belongsTo=parrnote
1472	13763?13770, otherFeat=[]-->, belongsTo=parrnote
1473	25. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. (Tokyo) 95,, otherFeat=[]-->, belongsTo=parrnote
1474	511?519, otherFeat=[]-->, belongsTo=parrnote
1475	26. Sontag, E., Fedorov, S., Kamibayashi, C., Robbins, D., Cobb, M., and, otherFeat=[]-->, belongsTo=parrnote
1476	Mumby, M. (1993) Cell 75, 887? 897, otherFeat=[]-->, belongsTo=parrnote
1477	27. Janssens, V., Jordens, J., Stevens, I., Van Hoof, C., Martens, E., De Smedt,, otherFeat=[]-->, belongsTo=parrnote
1478	H., Engelborghs, Y., Waelkens, E., and Goris, J. (2003) J. Biol. Chem. 278,, otherFeat=[]-->, belongsTo=parrnote
1479	10697?10706, otherFeat=[]-->, belongsTo=parrnote
1480	28. Strynadka, N. C., and James, M. N. (1989) Annu. Rev. Biochem. 58,, otherFeat=[]-->, belongsTo=parrnote
1481	951?998, otherFeat=[]-->, belongsTo=parrnote
1482	29. Silverstein, A. M., Barrow, C. A., Davis, A. J., and Mumby, M. C. (2002), otherFeat=[]-->, belongsTo=parrnote
1483	Proc. Natl. Acad. Sci. U. S. A. 99, 4221? 4226, otherFeat=[]-->, belongsTo=parrnote
1484	30. Li, X., Scuderi, A., Letsou, A., and Virshup, D. M. (2002) Mol. Cell Biol. 22,, otherFeat=[]-->, belongsTo=parrnote
1485	3674 ?3684, otherFeat=[]-->, belongsTo=parrnote
1486	31. Strack, S., Ruediger, R., Walter, G., Dagda, R. K., Barwacz, C. A., and, otherFeat=[]-->, belongsTo=parrnote
1487	Cribbs, J. T. (2002) J. Biol. Chem. 277, 20750 ?20755, otherFeat=[]-->, belongsTo=parrnote
1488	32. Ahn, J. H., Sung, J. Y., McAvoy, T., Nishi, A., Janssens, V., Goris, J., Green-, otherFeat=[]-->, belongsTo=parrnote
1489	gard, P., and Nairn, A. C. (2007) Proc. Natl. Acad. Sci. U. S. A. 104,, otherFeat=[]-->, belongsTo=parrnote
1490	9876 ?9881, otherFeat=[]-->, belongsTo=parrnote
1491	33. Creyghton, M. P., Roel, G., Eichhorn, P. J., Hijmans, E. M., Maurer, I.,, otherFeat=[]-->, belongsTo=parrnote
1492	Destree, O., and Bernards, R. (2005) Genes Dev. 19, 376 ?386, otherFeat=[]-->, belongsTo=parrnote
1493	34. Creyghton, M. P., Roel, G., Eichhorn, P. J., Vredeveld, L. C., Destree, O.,, otherFeat=[]-->, belongsTo=parrnote
1494	and Bernards, R. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 5397?5402, otherFeat=[]-->, belongsTo=parrnote
1495	35. Voorhoeve, P. M., Hijmans, E. M., and Bernards, R. (1999) Oncogene 18,, otherFeat=[]-->, belongsTo=parrnote
1496	515?524, otherFeat=[]-->, belongsTo=parrnote
1497	36. Groves, M. R., Hanlon, N., Turowski, P., Hemmings, B. A., and Barford, D., otherFeat=[]-->, belongsTo=parrnote
1498	(1999) Cell 96, 99 ?110, otherFeat=[]-->, belongsTo=parrnote
1499	37. Bayliss, R., Littlewood, T., and Stewart, M. (2000) Cell 102, 99 ?108, otherFeat=[]-->, belongsTo=parrnote
1500	38. Bayliss, R., Littlewood, T., Strawn, L. A., Wente, S. R., and Stewart, M., otherFeat=[]-->, belongsTo=parrnote
1501	(2002) J. Biol. Chem. 277, 50597?50606, otherFeat=[]-->, belongsTo=parrnote
1502	39. Li, X., and Virshup, D. M. (2002) Eur. J. Biochem. 269, 546 ?552, otherFeat=[]-->, belongsTo=parrnote
1503	PR70 Targets PP2A to Cdc6, otherFeat=[]-->, belongsTo=nota_cab_pie
1504	16114 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
1505	VOLUME 283 ? NUMBER 23 ? JUNE 6, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie
1506	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
1507	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
1508	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1509	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1510	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1511	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1512	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1513	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1514	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1515	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
1516	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
1517	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
1518	from, otherFeat=[]-->, belongsTo=nota_cab_pie
1519	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
1520	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
==============================
0	Protein Phosphatase 2A Is Targeted to Cell Division Control-->id=0, page=0, size=27, fam=Times, col=#231f20, type=title, textLines=1--->[]--->title	Protein Phosphatase >>>ell Division Control
1	Protein 6 by a Calcium-binding Regulatory Subunit*S-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	Protein 6 by a Calci>>>Regulatory Subunit*S
2	Received for publication, December 19, 2007, and in revised form, February 29, 2008 Published, JBC Papers in Press, April 8, 2008, DOI 10.1074/jbc.M710313200-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Received for publica>>>.1074/jbc.M710313200
3	Anthony J. Davis1, Zhen Yan§, Bobbie Martinez, and Marc C. Mumby2 From the Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041 and §the Division of Cardiology, Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Anthony J. Davis1, Z>>>North Carolina 27710
4	The cell division control protein 6 (Cdc6) is essential for formation of pre-replication complexes at origins of DNA replication. Phosphorylation of Cdc6 by cyclin-dependent kinases inhibits ubiquitination of Cdc6 by APC/Ccdh1 and degradation by the proteasome. Experiments described here show that the PR70 member of the PPP2R3 family of regulatory subunits targets protein phosphatase 2A (PP2A) to Cdc6. Interaction with Cdc6 is mediated by residues within the C terminus of PR70, whereas interaction with PP2A requires N-terminal sequences conserved within the PPP2R3 family. Two functional EF-hand calcium-binding motifs mediate a calcium-enhanced interaction of PR70 with PP2A. Calcium has no effect on the interaction of PR70 with Cdc6 but enhances the association of PP2A with Cdc6 through its effects on PR70. Knockdown of PR70 by RNA interference results in an accumulation of endogenous and expressed Cdc6 protein that is dependent on the cyclin-dependent protein kinase phosphorylation sites on Cdc6. Knockdown of PR70 also causes G1 arrest, suggesting that PR70 function is critical for progression into S phase. These observations indicate that PP2A can be targeted in a calcium-regulated manner to Cdc6 via the PR70 subunit, where it plays a role in regulating protein phosphorylation and stability.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->parr	The cell division co>>>ation and stability.
5	Precise regulation of DNA replication is necessary to ensure that daughter cells receive a complete and intact genome during mitosis. A crucial step in regulating DNA replication is the assembly of pre-replicative complexes at origins of replication (1). Coordination of DNA replication with the cell cycle is achieved through a periodic accumulation and destruction of proteins involved in formation of pre-RCs3 is mediated by cyclin-dependent kinases (CDKs) and the E3 ubiquitin ligase, anaphase promoting complex/cyclosome (2). The mammalian Cdc6 protein is required for DNA replication and acts in conjunction with the Cdt1 protein to recruit the mini-chromosome maintenance complex into pre-RCs (1, 3, 4). Mammalian cells have multiple mechanisms to ensure that pre-RCs only assemble during late M and G1, including regulation of the levels and function of Cdc6 (2).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Precise regulation o>>>unction of Cdc6 (2).
6	Mammalian Cdc6 is regulated by phosphorylation of multiple sites within its N-terminal domain by cyclin-dependent protein kinases. Cdc6 is phosphorylated at canonical CDK sites, including serines 54, 74, and 106 of human Cdc6 (5, 6). Experiments with exogenously expressed protein have shown that phosphorylation can regulate the nuclear localization of Cdc6 (5, 7­9). However, other studies have shown that a subpopulation of endogenous Cdc6 remains in the nucleus, bound to chromatin, throughout the cell cycle (10 ­12). Phosphorylation of Cdc6 also plays an important role in regulating the stability of Cdc6. The N-terminal domain of Cdc6 contains RXXL (D box) and KEN (KEN box) destruction motifs, which are binding sites for the form of the APC/C containing the cdh1-targeting subunit (13). Cdc6 is polyubiquitinated and targeted for degradation by APC/Ccdh1, which prevents formation of pre-RCs in quiescent cells and during early G1 by maintaining low levels of Cdc6 (14). Phosphorylation of Cdc6 by CDKs protects the protein from degradation by blocking recognition by cdh1 resulting in stabilization of Cdc6 during a window of time that allows formation of pre-RCs during G1 (15). The importance of CDKmediated stabilization of Cdc6 is also supported by evidence showing that the cell cycle arrest caused by DNA damage is due to dephosphorylation and degradation of Cdc6 (16).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Mammalian Cdc6 is re>>>dation of Cdc6 (16).
7	Because the extent of Cdc6 phosphorylation is controlled by the opposing actions of cyclin-dependent kinases and protein phosphatases, dephosphorylation of Cdc6 can also control formation of pre-RCs. Much less is known about mechanisms that regulate Cdc6 dephosphorylation. A previous study identified a fragment of PR70 as a member of the PPP2R3 family of PP2A regulatory subunits that interacted with Cdc6 and implicated PP2A in regulating Cdc6 phosphorylation (17). The major forms of PP2A contain a dimeric core complex composed of a scaffold (A) and a catalytic subunit (C). The AC core dimer associates with regulatory subunits that form heterotrimeric holoenzymes and target the catalytic subunit to specific phosphoprotein substrates (18 ­20). In this study, the mechanism and functional consequences of targeting of PP2A to Cdc6 by PR70 were investigated. The results show that PR70 interacts with PP2A and Cdc6 through distinct regions of the protein,-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Because the extent o>>>ions of the protein,
8	* This work was supported, in whole or in part, by National Institutes of Health Grant GM49505. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	* This work was supp>>> indicate this fact.
9	S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	S The on-line versio>>>and Figs. S1 and S2.
10	1 Supported by National Institutes of Health Pharmacological Sciences Training Grant T32 GM07062.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	1 Supported by Natio>>>g Grant T32 GM07062.
11	2 To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-645-6152; Fax: 214-645-6151; E-mail: marc.mumby@utsouthwestern.edu.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	2 To whom correspond>>>@utsouthwestern.edu.
12	3 The abbreviations used are: pre-RC, pre-replicative complex; PP2A, protein phosphatases 2A; APC/C, anaphase promoting complex/cyclosome; siRNA, small interfering RNA; Cdk, cyclin-dependent kinase; E3, ubiquitinprotein isopeptide ligase; PBS, phosphate-buffered saline; IP, immunoprecipitation; GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglutinin; CMV, cytomegalovirus; Cdc6, cell division control protein 6.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	3 The abbreviations >>>n control protein 6.
13	THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 23, pp. 16104 ­16114, June 6, 2008-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	THE JOURNAL OF BIOLO>>>­16114, June 6, 2008
14	© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	© 2008 by The Americ>>>rinted in the U.S.A.
15	16104 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	16104 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
16	VOLUME 283 · NUMBER 23 · JUNE 6, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	VOLUME 283 · NUMBER >>>ER 23 · JUNE 6, 2008
17	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
18	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
19	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
20	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
21	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
22	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
23	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
24	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
25	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
26	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
27	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
28	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
29	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
30	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
31	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
32	that the association of PP2A to Cdc6 is enhanced by calcium binding to PR70, and that loss of PR70 causes increased levels of Cdc6 and G1 arrest.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	that the association>>> Cdc6 and G1 arrest.
33	EXPERIMENTAL PROCEDURES-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->['U']--->title	EXPERIMENTAL PROCEDU>>>ERIMENTAL PROCEDURES
34	Cloning of Full-length PR70--A human expressed sequence tag encoding the PR70 start codon was identified in the human expressed sequence tag data base using the MegaBLAST tool (www.ncbi.nlm.nih.gov/BLAST/) with the assembled PR70 sequence (21). A PR70 cDNA was constructed using the PR48 cDNA and the IMAGE Human Clone ID 5728169 (GenBankTM accession number BM544432), purchased from Invitrogen, using an internal NcoI restriction site present in the common region of BM54432 and PR48. A PCR fragment containing the translational start codon, the 5 -end, and the 3 -NcoI site of BM54432 was generated using the BM54432 cDNA as template with the PCR primers: 5 -CGGGATCCATGCCGCCCGGCAAAGT-3 (sense strand) and 5 -GCGCCTTGATCCGGC-3 (antisense strand). The PCR product was digested with the restriction enzymes BamHI and NcoI. The 3 portion of the PR48 cDNA was excised from the PR48 cDNA (17) using NcoI and HindIII, and the fragments were ligated and subcloned into the pCMV-Tag2B vector (Stratagene) digested with BamHI and HindIII. The resulting construct encoded a full-length PR70 cDNA fused to an N-terminal FLAG epitope tag. The sequence was verified by automated sequencing.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Cloning of Full-leng>>>utomated sequencing.
35	Cell Culture, Transfection, and RNA Interference--COS-7, HeLa, and U2OS cells were maintained at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in an atmosphere of 5% CO2. U2OS, obtained from the ATCC, is a human osteosarcoma cell line that expresses wild-type p53. For transient expression of proteins, cells were transfected with expression plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were harvested either 24 or 48 h after transfection. Transfection with small interfering RNA to knock down PR70 was carried out using Oligofectamine (Invitrogen) following the manufacturer's protocol. Annealed duplex siRNAs were purchased from Dharmacon and had the following sequences: PR70-1, 5 -AGCCGGUCCUGAAGAUGAAdTdT-3 (sense strand) and PR70-2, 5 -AAAGCAUUCCGACCUUCUAdTdT-3 (sense strand). Controls included an siRNA that knocks down the MEKK2 protein kinase (22), an siRNA that knocks down protein phosphatase 5 (23), and an siRNA corresponding to the sequence of firefly luciferase (5 -TCGAAGTATTCCGCGTACGdTdT-3 ). Cells were lysed and analyzed by immunoblotting 48 h after transfection. In some experiments, cells were co-transfected with PR70 siRNA and expression plasmids encoding wild-type Cdc6 or Cdc6 mutants in which all three N-terminal phosphorylation sites were mutated to alanine (AAA-Cdc6) or aspartic acid (DDD-Cdc6) using Lipofectamine 2000 and harvested 48 h later. The cDNAs encoding phosphorylation site mutants of Cdc6 were prepared using a PCR-based site-directed mutagenesis kit (Invitrogen) according to the manufacturer's protocol. Wild-type and mutant Cdc6 were expressed as a fusion proteins fused to the N terminus of enhanced green fluorescent protein using the pEGFP-N expression vector (Clontech).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Cell Culture, Transf>>>n vector (Clontech).
36	Immunoprecipitation and Immunoblotting--Rabbit antisera were raised against a synthetic peptide corresponding to the C terminus of human PR70 (CDLYEYACGDEDLEPL) conjugated to keyhole limpet hemocyanin. Anti-PR70 antibodies were affinity purified on a peptide column made with the same peptide using the MicroLink Peptide Coupling Kit (Pierce) following the manufacturer's protocol. Rabbit antiserum against human Cdc6 was generated against a full-length Cdc6 fusion protein as described previously (4).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Immunoprecipitation >>>ibed previously (4).
37	Proteins were immunoprecipitated following the protocol described previously (17). Briefly, the media was aspirated and the cells were washed with cold PBS. The cells were incubated on ice for 20 min in 300 l of IP lysis buffer containing 20 mM Tris-HCl (pH 7.5), 0.2% Nonidet P-40, 20% glycerol, 200 mM NaCl, 1 mM EDTA, and protease inhibitor mixture (Roche Applied Science). Lysates were centrifuged at 14,000 g for 10 min, and protein complexes were immunoprecipitated from the supernatant. Endogenous PR70 and Cdc6 were immunoprecipitated from 1.2 106 HeLa cells lysed in 300 l of IP lysis buffer as described above. PR70 was immunoprecipitated using a rabbit antiserum generated against the peptide CDLYEYACGDEDLEPL conjugated to hemocyanin. Cdc6 was immunoprecipitated using a rabbit polyclonal antibody generated against a full-length Cdc6-GST fusion protein described previously. As a negative control, immunoprecipitations were performed using pre-immune serum collected from the rabbits immunized against PR70 or Cdc6. 10 l of antiserum and 40 l of protein A-Sepharose (Sigma-Aldrich) were added to 300 l of lysate, and the mixture was incubated for2hat4 °C.The protein-A beads were washed three times with IP lysis buffer, and protein was solubilized in 60 lof2 SDS-PAGE loading buffer. Thirty microliters of solubilized material was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was cut into pieces, which were probed with anti-PP2A C-subunit monoclonal antibody 1F6 (24), anti-PP2A A-subunit antiserum (C-20, Santa Cruz Biotechnology), anti-Cdc6 monoclonal antibody (clone DCS-180, Upstate), or anti-PR70 antiserum. Following incubation with horseradish peroxidase-conjugated secondary antibodies, the blots were developed using the enhanced chemiluminescence detection system (Amersham Biosciences).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Proteins were immuno>>>ersham Biosciences).
38	Transiently expressed FLAG-tagged proteins were immunoprecipitated from 1.5 106 cells using 7 g of anti-FLAG polyclonal antibody (Sigma-Aldrich) or 7 g of non-immune rabbit IgG (Sigma-Aldrich) and 40 l of protein A-Sepharose (SigmaAldrich) for 2 h at 4 °C. The immunoprecipitates were washed three times with lysis buffer and solubilized in 60 lof2 6.8, 5 mM MgCl2, and 60 mM KCl) for1hat room temperature. The membrane was then incubated in IMK buffer containing 5 Ci/ml 45Ca2 for 10 min. The membrane was washed three times in 30% ethanol for 5 min, dried, and exposed to x-ray film for 12 h.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Transiently expresse>>>x-ray film for 12 h.
39	SDS-PAGE loading buffer. 30 l of solubilized protein was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with anti-FLAG M2 monoclonal (Stratagene), anti-PP2A C-subunit 1F6, and anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) antibodies and developed as described above.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	SDS-PAGE loading buf>>> as described above.
40	Calcium Overlay Assay--In vitro 45Ca2 overlay assays were carried out using a protocol described previously (25). Purified GST fusion proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was washed three times in IMK buffer (10 mM imidazole-HCl, pH-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Calcium Overlay Assa>>>mM imidazole-HCl, pH
41	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
42	JUNE 6, 2008 · VOLUME 283 · NUMBER 23-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	JUNE 6, 2008 · VOLUM>>>LUME 283 · NUMBER 23
43	JOURNAL OF BIOLOGICAL CHEMISTRY 16105-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16105
44	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
45	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
46	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
47	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
48	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
49	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
50	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
51	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
52	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
53	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
54	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
55	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
56	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
57	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
58	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
59	Generation of PR70 Mutants--Point mutations were introduced into the EF-hands of PR70 as described in the manual for the QuikChange Multi Site-directed mutagenesis kit (Stratagene) using the following primers (mutated residues underlined). PCR was performed with pCMV-Tag2B containing the full-length PR70 cDNA as template with the following primers: EF1(x,y) 5 CAAGTTCTGGGAGCTGGCCACGGCCCACGACCTGCTCATCG-3 (sense strand) and 5 -CGATGAGCAGGTCGTGGGCCGTGGCCAGCTCCCAGAACTTG-3 (antisense strand), EF1(-z) 5 -TTGTGCCGCGCCAGGTTGTCCGCGTCGATGAGCGCTCATCGACGCGGACAACCTGGCGCGGCACAA-3 (sense strand) and 5 3 3 (antisense strand), EF2(x,y) 5 -TGGTTCCGCTGCATGGCCCTGGCCGGGGACGGCGCCCTG-3 (sense strand) and 5 -CAGGGCGCCGTCCCCGGCCAGGGCCATGCAGCGGAACCA-3 (antisense strand), and EF2(-z), 5 GCGCCCTGTCCATGTTCCAGCTCGAGTACTTCTAC-3 (sense strand) and 5 -GTAGAAGTACTCGAGCTGGAACATGGACAGGGCGC-3 (antisense strand). Mutations in both EFhands were introduced using the EF1 mutant cDNAs as template for PCR with primers for introduction of EF2 point mutants. All mutations were verified by automated sequencing. PR70 truncation mutants were generated by PCR amplification using the PR70 cDNA as template. The N1 (aa 125­ 575) corresponds to the PR48 protein described previously (17). N2, N3, and C were generated using the following primers: N2 (aa 136 ­575) 5 -CGGGATCCGCCACCATGGATGACATG-3 , N3 (aa 162­575) 5 -CGGGATCCAGGACTCCGTCAACGTG-3 , and C (aa 1­ 441) 5 -CCCAAGCTTCATCTGGCAGAGGCAGTC-3 .-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Generation of PR70 M>>>TCTGGCAGAGGCAGTC-3 .
60	Point mutations were introduced into the FYF motif (aa 128 ­130) of PR70 using the full-length PR70 cDNA as template with the following mutagenic primers (mutated residues underlined): AYF, 5 -GCCAAAGCATTCCGACCGCCTACTTCCCCAGAGGACG-3 (sense strand) and 5 -CGTCCTCTGGGGAAGTAGGCGGTCGGAATGCTTTGGC-3 (antisense strand), FAF, 5 -CCAAAGCATTCCGACCTTCGCCTTCCCCAGAGGACGCC-3 (sense strand) and 5 -GGCGTCCTCTGGGGAAGGCGAAGGTCGGAATGCTTTGG-3 (antisense strand), FYA, 5 -GCATTCCGACCTTCTACGCCCCCAGAGGACGCCCGC-3 (sense strand) and 5 -GCTTTCGTCCTCTGGGGGCGTAGAAGGTCGGAATGC-3 (antisense strand). To make the AYAP mutant, the AYFP cDNA was used as a template, and PCR mutagenesis was done with the following primers: AYA, 5 -CATTCCGACCGCCTACGCCCCCAGAGGACGCCCG-3 (sense strand) and 5 -CGGGCGTCCTCTGGGGGCGTAGGCGGTCGGAATG-3 (antisense strand). To make the AAAP mutant, the AYAP cDNA was used as a template, and PCR mutagenesis was done with the following primers AAA, 5 -GCATTCCGACCGCCGCCGCCCCCAGAGGACG-3 (sense strand) and 5 -CGTCCTCTGGGGGCGGCGGCGGTCGGAATGC-3 (antisense strand). All mutations were verified by automated sequencing.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Point mutations were>>>utomated sequencing.
61	Cloning of GST-tagged PR70 and EF-hand Mutants--PR70 and PR70 EF-hand mutant cDNA were cloned into the pGEX4T-1 vector (Amersham Biosciences). The cDNAs were amplified by PCR using the pCMV-Tag2B vector containing the fulllength PR70 or EF-hand mutant cDNA as template with the following primers: 5 -CGGGATCCATGCCGCCCGGCAAAGT-3 (sense strand) and 5 -ATTTGCGGCCGCTCACAGCGGCTCCAGGTC-3 (antisense strand). The products were digested with BamHI and NotI and ligated into pGEX 4T-1, which had been cut with the same restriction enzymes. The resulting constructs encode the GST protein fused to the N terminus of full-length PR70 or EF-hand mutant proteins. The sequences were verified by automated sequencing.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Cloning of GST-tagge>>>utomated sequencing.
62	Expression and Purification of GST-Cdc6, GST-A, and GSTPR70 Fusion Proteins--A GST-Cdc6 fusion protein was prepared by a modification of a method previously described (6). Briefly, 1 liter of Sf9 cells (2 106 cells/ml) was infected with recombinant GST-Cdc6 baculovirus (a gift of Dr. Ellen Fanning, Vanderbilt University) at a Sf9 culture:baculovirus ratio of 1:20 (v/v) for 60 h. The cells were collected by centrifugation and washed once with PBS. Cells were lysed on ice in 40 ml of (M2, Stratagene), anti-PP2A C-subunit 1F6, and anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) antibodies and developed as described above.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Expression and Purif>>> as described above.
63	buffer A (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM KCl,-->id=3, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	buffer A (100 mM Tri>>>0 mM NaCl, 5 mM KCl,
64	0.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM dithiothreitol, 10 mM NaF, 1 mM EGTA, 2 mM EDTA, and a protease inhibitor tablet (Roche Applied Science)) using a Dounce homogenizer. Lysates were centrifuged at 30,000 g for 30 min at 4 °C to remove cellular debris, and the lysate was mixed with 2 ml of glutathione-agarose (Sigma-Aldrich) for2hat4 °C.The resin was recovered by centrifugation and washed twice with PBS, once with PBS containing 1.5 M NaCl, and once with PBS containing 1.5 mM NaCl and 0.1% (v/v) Nonidet P-40, and then re-equilibrated in PBS. The GST-Cdc6 fusion protein immobilized on glutathione agarose beads was resuspended in buffer B (20 mM-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	0.5 mM MgCl2, 0.5% N>>>d in buffer B (20 mM
65	HEPES, pH 7.6, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA,-->id=3, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	HEPES, pH 7.6, 100 m>>>threitol, 1 mM EDTA,
66	and 50% glycerol) and stored at 80 °C.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	and 50% glycerol) an>>>and stored at 80 °C.
67	GST-A fusion protein, GST-PR70, and GST-EF-hand mutants were expressed in bacteria and prepared as previously described (26). The GST fusion proteins immobilized on glutathione agarose beads were resuspended in buffer B and stored at 80 °C until use.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	GST-A fusion protein>>> at 80 °C until use.
68	GST Pulldown Assays--GST, GST-A, and GST-Cdc6 immobilized on glutathione-agarose beads were used to assess the binding of PR70. Wild-type or mutant FLAG-PR70 was expressed by transient transfection of COS-7 cells. The cells were lysed on ice in 300 l of IP lysis buffer or in 300 lofIP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	GST Pulldown Assays->>>ffer or in 300 lofIP
69	lysis buffer containing 10 mM EDTA or 10 mM CaCl2 for 20 min.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	lysis buffer contain>>>mM CaCl2 for 20 min.
70	GST pulldown assays (26) were conducted by incubating 300 l of lysate with either GST, GST-A, or GST-Cdc6. The samples were incubated for1hat room temperature with agitation. The calpain inhibitor calpeptin (Calbiochem) was added at a concentration of 50 M in some experiments. Following incubation, the sample was washed three times with IP lysis buffer supplemented with EGTA, CaCl2, or CaCl2 and calpeptin, and the beads were collected by centrifugation. After washing, the bound proteins were solubilized in 60 lof2 SDS-PAGE loading buffer. 30 l of solubilized protein was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with anti-FLAG monoclonal-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	GST pulldown assays >>>anti-FLAG monoclonal
71	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
72	16106 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	16106 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
73	VOLUME 283 · NUMBER 23 · JUNE 6, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	VOLUME 283 · NUMBER >>>ER 23 · JUNE 6, 2008
74	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
75	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
76	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
77	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
78	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
79	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
80	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
81	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
82	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
83	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
84	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
85	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
86	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
87	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
88	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
89	Flow Cytometry--U2OS cells (4 105) were seeded into 60-mm dishes and transfected with PR70 or control siRNA 24 h later. The cells were then incubated for 48 h and harvested by trypsinization, washed once with PBS, and resuspended in 0.5 ml of PBS. The cell suspension was then added to 4.5 ml of 70% ethanol and incubated on ice for 2 h. Cells were collected by centrifugation, washed once with PBS, and suspended in 1 ml of propidium iodide/Triton X-100 staining solution with RNase (0.1% Triton X-100, 0.2 mg/ml DNase-free RNase, and 10 g/ml propidium iodide in PBS). The DNA content of 10,000 cells was determined using a BD Biosciences FACScan flow cytometer and FlowJo software. Single cells were gated away from clumped cells using an FL3 width versus FL3 height dot plot, and the DNA content of individual cells was plotted as FL3 area versus cell number.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Flow Cytometry--U2OS>>> versus cell number.
90	Experimental Reproducibility--The data shown in the figures are from individual experiments that were representative of common results obtained in at least three independent experiments.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Experimental Reprodu>>>pendent experiments.
91	RESULTS-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->title	RESULTS>>>RESULTS
92	Interaction of PR70 with Cdc6--The original cDNA for PR70, termed PR48, was identified in a yeast two-hybrid screen using the human Cdc6 protein as bait (17) and subsequently shown to be a fragment of a longer cDNA (27). A full-length human PR70 cDNA was constructed by ligating expressed sequence tag BM54432 to the PR48 cDNA using an internal NcoI restriction site. The predicted open reading frame of the PR70 cDNA encodes a protein with a calculated molecular mass of 65.1-kDa and corresponds to the longer transcript (variant 1) of the PPP2R3B gene (GeneID: 28227). The predicted amino acid sequence of PR70 is highly similar to the human PR72 and mouse PR59 members of the PPP2R3 gene family, but more distantly related to the G5PR protein (supplemental Table  S1). An alignment of the PPP2R3 family (supplemental Fig.  S1) revealed a highly conserved central domain, termed the R3 domain, that contains two conserved EF-hand calcium binding motifs previously identified in PR72 (27). Rabbit antisera were raised against a peptide corresponding to the unique C terminus of PR70 and affinity purified on a peptide column. The purified antibodies recognized a protein band of Mr 70,000 in lysates of HeLa cells. The 70-kDa protein recognized by the antibody was greatly reduced in cells treated with two different siRNAs corresponding to sequences within PR70 but not with control siRNA (supplemental Fig.  S2).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Interaction of PR70 >>>plemental Fig.  S2).
93	To verify that PR70 associates with PP2A and Cdc6, HeLa lysates were immunoprecipitated with PR70 and Cdc6 antibodies. Immunoprecipitation of Cdc6 co-precipitated a diffuse protein band that migrated at the position of PR70 that was not present in immunoprecipitates obtained with the preimmune serum (Fig. 1A). The anti-Cdc6 serum also co-precipitated the A- and C-subunits of PP2A. Although the anti-PR70 antibodies co-precipitated the A- and C-subunits of PP2A (Fig. 1B), a complex between PR70 and Cdc6 could not be detected. The inability to detect Cdc6 may be due to steric hindrance by the antiC-terminal antibody, because the C terminus of PR70 is required for interaction with Cdc6 (see below). The association of PR70 with PP2A and Cdc6 was also tested in co-immunoprecipitation experiments using exogenously expressed proteins. FLAG-tagged PR70 was expressed in HeLa cells and immunoprecipitated with anti-FLAG antibodies. Analysis of the immunoprecipitates by immunoblotting showed that the endogenous A- and C-subunits of PP2A and HA-tagged Cdc6 co-precipitated with FLAG-PR70 (Fig. 1C). These results indicate that PR70 can interact with both the PP2A core dimer and Cdc6 in intact cells.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	To verify that PR70 >>>dc6 in intact cells.
94	FIGURE 1. PR70 interacts with Cdc6 and PP2A. A, Cdc6 was immunoprecipitated from exponentially growing HeLa cells using a polyclonal antiserum specific for Cdc6 (Cdc6) or pre-immune serum (Pre). The immunoprecipitates and supernatant fractions were analyzed by immunoblotting using anti-Cdc6 (Cdc6), anti-PR70, anti-A-subunit, or anti-C-subunit antibodies. B, PR70 was immunoprecipitated from HeLa cells using an anti-peptide antiserum against PR70 (PR70) or pre-immune serum (Pre). The immunoprecipitates (IP) or the supernatants remaining after immunoprecipitation (S) were resolved by SDSPAGE and analyzed by immunoblotting with anti-PR70 (PR70), anti-A-subunit (A), and anti-C-subunit (C) antibodies as indicated on the left. C, HeLa cells were transiently transfected with empty expression vector (Emp Vec), plasmids expressing the FLAG-PR70 N mutant ( N1), full-length FLAG-PR70, or a combination of plasmids expressing FLAG-PR70 and HA-Cdc6 (Cdc6). Cells were harvested after 24 h, and lysates were immunoprecipitated with antiFLAG antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-FLAG (FLAG-PR70), anti-Cdc6, anti-A-subunit, and anti-C-subunit antibodies as indicated on the left.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 1. PR70 inter>>>dicated on the left.
95	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
96	JUNE 6, 2008 · VOLUME 283 · NUMBER 23-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	JUNE 6, 2008 · VOLUM>>>LUME 283 · NUMBER 23
97	JOURNAL OF BIOLOGICAL CHEMISTRY 16107-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16107
98	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
99	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
100	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
101	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
102	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
103	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
104	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
105	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
106	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
107	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
108	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
109	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
110	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
111	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
112	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
113	Calcium Enhances the Interaction of PR70 with the AC Core Dimer and Recruits PP2A to Cdc6--Analysis of the amino acid sequence of PR70 identified two EF-hand calcium binding motifs that are conserved within the PPP2R3 family (supplemental Fig.  S1). The roles of these motifs were tested in a gel overlay assay with wild-type PR70 and PR70 containing inactivating mutations of the EF-hand motifs. Point mutants were constructed that had substitutions of amino acids involved in calcium binding (28), including alanine substitutions at both the x and y coordinates and a conservative change at the -z coordinate (Fig. 2A). Wild-type PR70 bound calcium in the in vitro 45Ca2 overlay assay (Fig. 2B). Mutation of the first EFhand (EF1) resulted in reduced binding of calcium compared with wild-type PR70. Mutation of the second EF-hand (EF2) severely reduced calcium binding, whereas the double mutation of EF1 and EF2 nearly abolished the ability of PR70 to bind calcium.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Calcium Enhances the>>>R70 to bind calcium.
114	Calcium binding causes a conformational change in the PR72 member of the PPP2R3 family that is associated with enhanced interaction with the A-subunit of PP2A (27). Therefore, the effects of calcium on the interaction of PR70 with PP2A and Cdc6 were determined. FLAG-tagged PR70 was expressed in COS-7 cells, which were lysed in buffer containing EGTA or calcium. The lysates were incubated with either GST-A or GST-Cdc6 and bound proteins detected by immunoblotting. FLAG-PR70 interacted with both GST-A and GST-Cdc6 but not GST alone (Fig. 2C, lanes 1­2 and 6­7). Compared with lysates prepared with standard buffer or EGTA, the addition of calcium enhanced the binding of PR70 to GST-A, but not to GST-Cdc6 (Fig. 2C, lanes 4 and 9). Although calcium did not enhance the binding of PR70 to GST-Cdc6, it did cause a significant increase in the amount of A- and C-subunits associated with GST-Cdc6 (Fig. 2C, lanes 9 and 10). Although an excess of calcium was used in the experiments shown in Fig. 2C, other experiments showed that enhanced binding of the AC core dimer was also observed at calcium concentrations of 100 M (not shown).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Calcium binding caus>>>f 100 M (not shown).
115	To test the function of the individual EF-hand motifs in the calcium-enhanced interaction with PP2A, GST pulldown experiments were performed with the calcium-binding mutants. Compared with assays in the presence of EGTA, the addition of calcium resulted in enhanced binding of wild-type PR70 and the EF1 mutant to GST-A, but not to GST-Cdc6 (Fig. 3A and B, lanes 2­5). Although it did not increase the amount of PR70 or the EF1 mutant associated with Cdc6, calcium did increase the association of the A- and C-subunits with GSTCdc6 (Fig. 3B, lanes 2­5). Mutation of EF2 or mutation of both EF1 and EF2 resulted in loss of the calcium-enhanced binding of PR70 to GST-A (Fig. 3A, lanes 6 ­9) and the calcium-dependent association of the A- and C-subunits with GST-Cdc6 (Fig. 3B, lanes 6 ­9).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	To test the function>>>ig. 3B, lanes 6 ­9).
116	The effect of calcium on the interaction of PR70 with PP2A was also assessed by expression and immunoprecipitation in COS-7 cells. Both wild-type PR70 and the EF1 mutants interacted with endogenous PP2A (Fig. 3C, lanes 2­ 4). The EF1(-z) mutant interacted as well as wild-type PR70, but interaction of the EF1(x,y) mutant was reduced suggesting that mutation of the x and y residues causes a structural defect in PR70. Mutation of EF2, or both EF1 and EF2, resulted in a nearly complete loss of interaction with PP2A (Fig. 3C, lanes 5­ 8). A longer exposure of the blot showed that a weak interaction of the Aand C-subunits with the EF2 and EF1/EF2 double mutants could still be detected (Fig. 3C, OE). The combination of intact cell data and in vitro binding assays provide evidence that PR70 is a calcium binding protein and that interaction with the core dimer of PP2A is enhanced by binding of calcium to the second EF-hand motif. Calcium does not affect interaction of PR70 with Cdc6 but increases the association of the PP2A core dimer with Cdc6 in a manner dependent upon binding of calcium to the second EF-hand of PR70.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The effect of calciu>>>ond EF-hand of PR70.
117	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
118	EF1 EF2-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1 EF2>>>EF1 EF2
119	WT-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	WT>>>WT
120	166-DTDHDLLIDADD-177 240-DLDGDGALSMFE-251-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	166-DTDHDLLIDADD-177>>>240-DLDGDGALSMFE-251
121	EF1(x,y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(x,y)>>>EF1(x,y)
122	DLDGDGALSMFE-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DLDGDGALSMFE>>>DLDGDGALSMFE
123	EF1(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(-z)>>>EF1(-z)
124	DTDHDLLIDADN-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DTDHDLLIDADN>>>DTDHDLLIDADN
125	DLDGDGALSMFE-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DLDGDGALSMFE>>>DLDGDGALSMFE
126	EF2(x,y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF2(x,y)>>>EF2(x,y)
127	DTDHDLLIDADD-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DTDHDLLIDADD>>>DTDHDLLIDADD
128	ALAGDGALSMFE-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	ALAGDGALSMFE>>>ALAGDGALSMFE
129	EF2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF2(-z)>>>EF2(-z)
130	DTDHDLLIDADD-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DTDHDLLIDADD>>>DTDHDLLIDADD
131	DLDGDGALSMFQ-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DLDGDGALSMFQ>>>DLDGDGALSMFQ
132	EF1/EF2(x,y) ATAHDLLIDADD-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/EF2(x,y) ATAHDLL>>>F2(x,y) ATAHDLLIDADD
133	ALAGDGALSMFE-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	ALAGDGALSMFE>>>ALAGDGALSMFE
134	EF1/EF2(-z) DTDHDLLIDADN-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/EF2(-z) DTDHDLLI>>>EF2(-z) DTDHDLLIDADN
135	DLDGDGALSMFQ-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	DLDGDGALSMFQ>>>DLDGDGALSMFQ
136	45Ca2+-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	45Ca2+>>>45Ca2+
137	CBB-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	CBB>>>CBB
138	EF1/EF2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/EF2(-z)>>>EF1/EF2(-z)
139	EF1/EF2(x,y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/EF2(x,y)>>>EF1/EF2(x,y)
140	EF2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF2(-z)>>>EF2(-z)
141	EF2(x,y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF2(x,y)>>>EF2(x,y)
142	EF1(-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(>>>EF1(
143	x,y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	x,y)>>>x,y)
144	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
145	N-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	N>>>N
146	N-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	N>>>N
147	Ca+CP-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Ca+CP>>>Ca+CP
148	Ca-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Ca>>>Ca
149	E-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	E>>>E
150	N-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	N>>>N
151	Ca+CP-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Ca+CP>>>Ca+CP
152	Ca-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Ca>>>Ca
153	E-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	E>>>E
154	N-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	N>>>N
155	GST-A-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST-A>>>GST-A
156	GST-Cdc6-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST-Cdc6>>>GST-Cdc6
157	GST-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST>>>GST
158	FLAG-PR70-->id=3, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	FLAG-PR70>>>FLAG-PR70
159	C-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	C>>>C
160	GST-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST>>>GST
161	GST-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST>>>GST
162	A-->id=3228, page=4, size=24, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	A>>>A
163	EF1(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(-z)>>>EF1(-z)
164	A-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	A>>>A
165	B-->id=3228, page=4, size=24, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	B>>>B
166	C-->id=3228, page=4, size=24, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	C>>>C
167	ATAHDLLIDADD-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	ATAHDLLIDADD>>>ATAHDLLIDADD
168	X Y Z-X-Y -Z-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->['U']--->note	X Y Z-X-Y -Z>>>X Y Z-X-Y -Z
169	X Y Z-X-Y -Z-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->['U']--->note	X Y Z-X-Y -Z>>>X Y Z-X-Y -Z
170	12 3 4 5-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	12 3 4 5>>>12 3 4 5
171	6 7 8 9 10-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	6 7 8 9 10>>>6 7 8 9 10
172	FIGURE 2. Interaction of PR70 with PP2A is enhanced by calcium-binding. A, the figure shows a diagram of the location and sequences of wild-type PR70 (WT) and PR70 mutants containing substitutions of calcium-binding residues within the EF-hand motifs (EF mutants). The canonical EF-hand residues involved in coordination of calcium are indicated by the letters x, y, z, -x, -y, and -z using standard nomenclature (28). The x, y, and -z residues that were mutated are shown in bold type. B, GST fusions of wild-type PR70 (PR70) and the EF-hand mutant were analyzed for calcium binding by 45Ca2 overlay assay. The amounts of GST-PR70 in each lane were determined by staining the gel with Coomassie Brilliant Blue (CBB). C, calcium enhances binding of PR70 to the A-subunit of PP2A but not to Cdc6. FLAG-PR70 and the indicated EFhand mutants were transiently expressed in COS-7 cells, and the cells were lysed in standard buffer (N) or lysis buffer containing EGTA (E) or CaCl2 (Ca). Calpeptin (50 M) was included in some experiments (Ca CP). The lysates were incubated with GST alone (GST), GST-A, or GST-Cdc6, and bound proteins were detected by immunoblotting with anti-FLAG (FLAG-PR70), anti-Asubunit (A), anti-C-subunit (C), and anti-GST (GST) antibodies.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 2. Interactio>>>ST (GST) antibodies.
173	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
174	16108 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	16108 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
175	VOLUME 283 · NUMBER 23 · JUNE 6, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	VOLUME 283 · NUMBER >>>ER 23 · JUNE 6, 2008
176	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
177	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
178	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
179	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
180	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
181	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
182	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
183	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
184	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
185	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
186	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
187	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
188	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
189	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
190	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
191	PP2A and Cdc6 Bind to Distinct Regions of PR70--Comparison of the amino acid sequences of the PPP2R3 regulatory subunits identified a conserved domain in the central region of PR70 (supplemental Figs.  S1 and  S4A). The R3 domain is 66% identical and 82% conserved between human PR70 (PPP2R3B) and PR72 (PPP2R3A). A series of truncation mutants were constructed to identify regions within PR70 that were important for interaction with PP2A and Cdc6. FLAG-tagged mutants were expressed in COS-7 cells and immunoprecipitated with anti-FLAG antibody. The ability of the mutants to incorporate into endogenous PP2A heterotrimers was determined by immunoblotting for associated A- and C-subunits. The N1 mutant contains a deletion of the entire N-terminal PR70unique region and interacted with endogenous PP2A subunits to the same extent as full-length PR70 (Fig. 4B). Deletion of a C-terminal segment that included the PR70-unique region ( C) had little, if any, effect on the interaction with PP2A. However, deletions of N-terminal regions of the conserved R3 domain, N2 and N3, resulted in proteins that failed to interact with PP2A. These data indicated that the region between amino acids 125 and 136 of PR70 were necessary for interaction with the PP2A core dimer.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	PP2A and Cdc6 Bind t>>>the PP2A core dimer.
192	The sequence between residues 125 and 136 of PR70 contains a hydrophobic motif (FYF) that was conserved in PPP2R3 proteins from humans to flies (Fig. 5A). The role of the FYF motif was tested by mutating these residues to alanines (Fig. 5B) and determining the effects on interaction with the AC core dimer. Mutation of any one of these residues resulted in a significant loss of interaction with endogenous PP2A (Fig. 5C). A longer exposure of the immunoblot showed that small amounts of the A- and C-subunits could be detected in immunoprecipitates of each of the mutants (not shown). Although the interaction of the FYF mutants was severely compromised in intact cells, these mutants still bound to PP2A when assayed in vitro by GST pulldown experiments (not shown). These results indicate that the FYF motif contributes to the interaction of PR70 with the A-subunit.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The sequence between>>> with the A-subunit.
193	The N- and C-terminal truncation mutants of PR70 were also tested for their ability to interact with Cdc6. Pulldown experiments with GST-Cdc6 were performed with full-length PR70, the N3, and the C mutants in the presence and absence of calcium. As expected, full-length PR70 and the C mutant interacted with the A-subunit of PP2A, whereas the N3 mutant did not (Fig. 6A). Furthermore, there was an enhanced interaction of PR70 and the C mutant in the presence of calcium. As shown previously with full-length PR70 (Fig. 2C), the enhanced binding of PR70 and the C mutant was accompanied by a decrease in the amount of C-subunit associated with GST-A (Fig. 6A, lanes 3 and 9). This decrease in associated C-subunit was not observed with the N3 mutant, which did not bind to GST-A. These observations suggest that the decrease in C-subunit in the presence of calcium is due to displacement of endogenous regulatory and catalytic subunits from GST-A by excess free PR70.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The N- and C-termina>>>by excess free PR70.
194	GST PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST PR70>>>GST PR70
195	FL-PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	FL-PR70>>>FL-PR70
196	C-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	C>>>C
197	FL-PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	FL-PR70>>>FL-PR70
198	C-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	C>>>C
199	A-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	A>>>A
200	FL-PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	FL-PR70>>>FL-PR70
201	C OE-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	C OE>>>C OE
202	A OE-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	A OE>>>A OE
203	C-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	C>>>C
204	A-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	A>>>A
205	Em-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Em>>>Em
206	pVec-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	pVec>>>pVec
207	EF1/EF2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/EF2(-z)>>>EF1/EF2(-z)
208	EF1/EF2-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/EF2>>>EF1/EF2
209	(x,-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	(x,>>>(x,
210	y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	y)>>>y)
211	EF2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF2(-z)>>>EF2(-z)
212	EF2(x,-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF2(x,>>>EF2(x,
213	y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	y)>>>y)
214	EF1(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(-z)>>>EF1(-z)
215	EF1(x,-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(x,>>>EF1(x,
216	y)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	y)>>>y)
217	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
218	B-->id=4191, page=5, size=20, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	B>>>B
219	A-->id=4191, page=5, size=20, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	A>>>A
220	EF1/2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1/2(-z)>>>EF1/2(-z)
221	EF1(-z) EF2(-z)-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EF1(-z) EF2(-z)>>>EF1(-z) EF2(-z)
222	NE Ca-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	NE Ca>>>NE Ca
223	12 3-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	12 3>>>12 3
224	ECa-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	ECa>>>ECa
225	45-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	45>>>45
226	ECa-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	ECa>>>ECa
227	6 7-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	6 7>>>6 7
228	ECa-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	ECa>>>ECa
229	89-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	89>>>89
230	C-->id=4191, page=5, size=20, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	C>>>C
231	12 3 4 5 6 7 8-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	12 3 4 5 6 7 8>>>12 3 4 5 6 7 8
232	GST-A pulldown-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST-A pulldown>>>GST-A pulldown
233	GST-Cdc6 pulldown-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST-Cdc6 pulldown>>>GST-Cdc6 pulldown
234	Anti-FLAG IP-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Anti-FLAG IP>>>Anti-FLAG IP
235	FIGURE 3. The calcium-enhanced association of PP2A with PR70 requires EF2. FLAG-PR70 and the indicated EF-hand mutants were transiently expressed in COS-7 cells, and the cells were lysed in standard lysis buffer (N)or lysis buffer containing EGTA (E) or CaCl2 (Ca). A, GST pulldown assays were performed with the different lysates using immobilized GST-A. Bound proteins were detected by immunoblotting with anti-FLAG (FL-PR70), anti-A-subunit (A), and anti-C-subunit (C) antibodies. B, GST pulldown assays were performed using immobilized GST-Cdc6 as described for A. Lane 1 of panels A and B shows a control pulldown assay using GST alone. C, COS-7 cells were transiently transfected with FLAG-tagged wild-type PR70 or the indicated EFhand mutants, and lysates prepared with standard buffer were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted as described in A. Overexposures of the antiA-subunit (AOE) and anti-C-subunit (COE) immunoblots are also shown.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 3. The calciu>>>lots are also shown.
236	Em-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Em>>>Em
237	pVec-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	pVec>>>pVec
238	C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	C>>>C
239	N3-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	N3>>>N3
240	N2-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	N2>>>N2
241	N1-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	N1>>>N1
242	PR70-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PR70>>>PR70
243	FLAG-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	FLAG>>>FLAG
244	Conserved R3 domain-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	Conserved R3 domain>>>Conserved R3 domain
245	EF1 EF2-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	EF1 EF2>>>EF1 EF2
246	PR70-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	PR70>>>PR70
247	unique-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	unique>>>unique
248	PR70-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	PR70>>>PR70
249	unique-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	unique>>>unique
250	FYF motif-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	FYF motif>>>FYF motif
251	PR70-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	PR70>>>PR70
252	C-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	C>>>C
253	N1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	N1>>>N1
254	N2-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	N2>>>N2
255	N3-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	N3>>>N3
256	1-575-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	1-575>>>1-575
257	125-575 136-575 162-575 1-441-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	125-575 136-575 162->>>36-575 162-575 1-441
258	A-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	A>>>A
259	C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	C>>>C
260	A-->id=3228, page=4, size=24, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	A>>>A
261	B-->id=3228, page=4, size=24, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	B>>>B
262	Anti-FLAG IP-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Anti-FLAG IP>>>Anti-FLAG IP
263	FIGURE 4. Mapping of PP2A binding domains in PR70. A, a schematic diagram of PR70 showing the region containing the conserved R3 domain and PR70-unique regions. The truncation mutants used in binding assays are shown below with their corresponding designations on the left and amino acid numbers on the right. The conserved FYF (FYF motif) and EF-hand motifs (EF1 and EF2) are also depicted. B, interaction of PR70 truncation mutants with the A- and C-subunits of PP2A. FLAG-tagged PR70 (PR70) and the indicated truncation mutants were transiently expressed in COS-7 cells. The cells were lysed and the FLAG-tagged proteins immunoprecipitated with anti-FLAG antibody (Anti-FLAG IP). The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A), and anti-Csubunit (C) antibodies. A control immunoprecipitate using lysate from cells transfected with the empty expression vector (Emp Vec) is shown in the first lane.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 4. Mapping of>>>n in the first lane.
264	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
265	JUNE 6, 2008 · VOLUME 283 · NUMBER 23-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	JUNE 6, 2008 · VOLUM>>>LUME 283 · NUMBER 23
266	JOURNAL OF BIOLOGICAL CHEMISTRY 16109-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16109
267	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
268	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
269	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
270	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
271	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
272	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
273	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
274	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
275	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
276	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
277	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
278	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
279	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
280	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
281	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
282	Both full-length PR70 and the N3 mutant bound to GSTCdc6 (Fig. 6B). However, only full-length PR70 was able to recruit additional A- and C-subunits in the presence of calcium. In contrast, the C mutant bound very poorly to Cdc6 in the presence or absence of calcium. A low level of the A- and C-subunits was pulled down with GST-Cdc6 from lysates expressing the N3 or C mutants (Fig. 6B, lanes 5­ 6 and 8­9). Similar amounts of these subunits were also bound to GST-Cdc6 in lysates from non-transfected cells (not shown) suggesting that GST-Cdc6 can interact with endogenous A- and C-subunits in the absence of expressed PR70 (presumably by binding to endogenous PR70). The amounts of A- and C-subunits bound to GST-Cdc6 in experiments with the N3 or C mutants were increased in the presence of calcium. This observation provides additional support for the conclusion that calcium can regulate the association of PP2A with Cdc6 and shows that a C-terminal region of PR70, which includes the PR70-unique domain, is necessary for interaction with Cdc6.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Both full-length PR7>>>teraction with Cdc6.
283	PR70 Regulates Cdc6 Levels--Because phosphorylation of the N-terminal regulatory sites of Cdc6 inhibits degradation, loss of the phosphatase that dephosphorylates these sites should promote accumulation of Cdc6. Therefore, RNA interference was used to determine if knockdown of PR70 affected the levels of Cdc6. Knocking down the catalytic subunit of PP2A increased the levels of endogenous Cdc6 in HeLa cells (Fig. 7A). Treatment of cells with either a control siRNA or an siRNA that knocks down protein phosphatase 5 had no effect on Cdc6 levels. Knocking down the PR70 subunit also caused a substantial increase in the levels of Cdc6 (Fig. 7B). The increase in Cdc6 levels occurred with two PR70 siRNAs targeted to distinct regions of the mRNA. The accumulation of Cdc6 following knockdown with PR70-1 siRNA appeared to be greater than that with PR70-2 siRNA, which is consistent with the greater efficiency of the PR70-1 siRNA in reducing PR70 levels (supplemental Fig.  S2). Phosphorylation site mutants of Cdc6 were then used to test the role of phosphorylation in the accumulation of Cdc6 caused by knockdown of PR70. Knockdown of PR70 caused an increase in the levels of expressed wild-type GFP-Cdc6 compared with transfection with a control siRNA (Fig. 7B). Transfection with a mutant of Cdc6 in which all three N-terminal phosphorylation sites had been mutated to phospho-mimicking aspartic acid residues (DDD-Cdc6) resulted in substantially higher levels of expression than those observed with the wild-type protein as previously reported (15). PR70-1 siRNA had little or no effect on the levels of DDD-Cdc6. The ability of PR70 knockdown to cause accumulation of Cdc6 was also greatly diminished when the phosphorylation sites were mutated to non-phosphorylatable alanine residues (AAACdc6). Similar results were seen in U2OS cells. These data indicate that knockdown of PR70 results in an increase in the levels of endogenous and exogenous Cdc6 that is dependent on the presence of phosphorylatable residues at the N-terminal phosphorylation sites.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	PR70 Regulates Cdc6 >>>osphorylation sites.
284	C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	C>>>C
285	A-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	A>>>A
286	FLAG-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	FLAG>>>FLAG
287	Em-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Em>>>Em
288	pVec-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	pVec>>>pVec
289	AAA-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	AAA>>>AAA
290	AYA-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	AYA>>>AYA
291	FYA-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	FYA>>>FYA
292	FAF-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	FAF>>>FAF
293	AYF-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	AYF>>>AYF
294	PR70-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PR70>>>PR70
295	DN3-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	DN3>>>DN3
296	IPTFYFPRGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPTFYFPRGRP>>>IPTFYFPRGRP
297	IPRFYFGEGLP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPRFYFGEGLP>>>IPRFYFGEGLP
298	VPAFYFPCGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	VPAFYFPCGRP>>>VPAFYFPCGRP
299	IPKFYFPKGCP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPKFYFPKGCP>>>IPKFYFPKGCP
300	IPRFYFPHGKP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPRFYFPHGKP>>>IPRFYFPHGKP
301	Human PR70 Human PR72 Mouse PR59 Xenopus PR70 Drosophila PR72-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Human PR70 Human PR7>>>PR70 Drosophila PR72
302	EF1 EF2-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	EF1 EF2>>>EF1 EF2
303	FYF motif-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	FYF motif>>>FYF motif
304	PR70-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70>>>PR70
305	125-IPTFYFPRGRP-135-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	125-IPTFYFPRGRP-135>>>125-IPTFYFPRGRP-135
306	IPTAYFPRGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPTAYFPRGRP>>>IPTAYFPRGRP
307	IPTFAFPRGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPTFAFPRGRP>>>IPTFAFPRGRP
308	IPTFYAPRGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPTFYAPRGRP>>>IPTFYAPRGRP
309	IPTAYAPRGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPTAYAPRGRP>>>IPTAYAPRGRP
310	IPTAAAPRGRP-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	IPTAAAPRGRP>>>IPTAAAPRGRP
311	WT AYF FAF FYA AYA AAA-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	WT AYF FAF FYA AYA A>>> AYF FAF FYA AYA AAA
312	A-->id=5010, page=6, size=23, fam=Times, col=#100f0d, type=title, textLines=3--->[]--->note	A>>>A
313	B-->id=5010, page=6, size=23, fam=Times, col=#100f0d, type=title, textLines=3--->[]--->note	B>>>B
314	C-->id=5010, page=6, size=23, fam=Times, col=#100f0d, type=title, textLines=3--->[]--->note	C>>>C
315	Anti-FLAG IP-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Anti-FLAG IP>>>Anti-FLAG IP
316	FIGURE 5. A conserved hydrophobic motif is involved in the interaction of PR70 with PP2A. A, an alignment of the N-terminal region of the conserved R3 domains (residues 125­135 of PR70) of PPP2R3 subunits from various species. Conserved FYF residues are shown in bold. B, a diagram showing the residues within the PR70 that were changed in the mutant forms of PR70 listed on the left. Amino acid substitutions are shown in bold. C, FLAG-tagged PR70 (PR70), the N3 mutant ( N3), and the indicated FYF mutants were transiently expressed in COS-7 cells. The cells were lysed, and tagged proteins were immunoprecipitated with antiFLAG antibody (Anti-FLAG IP). The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A), and anti-C-subunit (C) antibodies. A control immunoprecipitate using lysate from cells transfected with the empty expression vector (Emp Vec)is shown in the first lane.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 5. A conserve>>>n in the first lane.
317	FLAG-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	FLAG>>>FLAG
318	FLAG-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	FLAG>>>FLAG
319	-A-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	-A>>>-A
320	GST-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	GST>>>GST
321	PR70-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PR70>>>PR70
322	GST-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	GST>>>GST
323	C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	C>>>C
324	N3-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	N3>>>N3
325	9-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	9>>>9
326	C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	C>>>C
327	--->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	->>>-
328	GST-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	GST>>>GST
329	12 3 4 56 78-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	12 3 4 56 78>>>12 3 4 56 78
330	C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	C>>>C
331	EE-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	EE>>>EE
332	E-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	E>>>E
333	Ca-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Ca>>>Ca
334	Ca-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Ca>>>Ca
335	Ca-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Ca>>>Ca
336	A-->id=4191, page=5, size=20, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	A>>>A
337	B-->id=4191, page=5, size=20, fam=Times, col=#100f0d, type=title, textLines=5--->[]--->note	B>>>B
338	GST-A pulldown-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST-A pulldown>>>GST-A pulldown
339	GST-Cdc6 pulldown-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	GST-Cdc6 pulldown>>>GST-Cdc6 pulldown
340	FIGURE 6. The C-terminal region of PR70 mediates interaction with Cdc6. FLAG-PR70 (lanes 1­3), the N3 mutant (lanes 4 ­ 6), and the C mutant (lanes 7­9) were transiently expressed in COS-7 cells, and the cells were lysed in the presence of EGTA (E) or CaCl2 (Ca). GST pulldown assays were performed using immobilized GST-A (panel A) or GST-Cdc6 (panel B), and bound proteins were detected by immunoblotting with anti-FLAG (FLAG), anti-A-subunit (A) or anti-C-subunit (C) antibodies. Control pulldowns with GST alone (GST) were carried out with all three expressed proteins using lysates prepared with standard buffer (lanes 1, 4, and 7).-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 6. The C-term>>>(lanes 1, 4, and 7).
341	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
342	16110 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	16110 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
343	VOLUME 283 · NUMBER 23 · JUNE 6, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	VOLUME 283 · NUMBER >>>ER 23 · JUNE 6, 2008
344	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
345	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
346	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
347	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
348	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
349	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
350	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
351	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
352	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
353	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
354	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
355	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
356	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
357	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
358	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
359	The effects of overexpressing PR70 on Cdc6 levels were also determined. When HeLa cells were transfected with expression plasmids containing the CMV promoter, FLAG-tagged PR70 was expressed at levels 5- to 10-fold higher than the endogenous protein (not shown). Co-expression of CDK2 and Cdc6 caused a substantial increase in Cdc6 levels as reported previously (15). Expression of wild-type PR70 caused an increase in the levels of both co-transfected and endogenous Cdc6 (Fig. 8A). Expression of the N3 or EF1/2 mutants, which cannot interact with the AC core dimer but bind to Cdc6, also increased the levels of Cdc6. In contrast, expression of the C mutant, which binds to the AC core dimer but not to Cdc6, had relatively little effect on the levels of exogenous or endogenous Cdc6. The potential role of phosphorylation in the effects of overexpressed PR70 was tested using the phosphorylation site mutants of Cdc6. Although co-expression of PR70 caused some increase in the levels of the AAA mutant of Cdc6, the effect was much less than its effect on wild-type Cdc6 (Fig. 8B). Similarly, co-expression of PR70 had little effect on the levels of the DDD mutant of Cdc6 even though endogenous Cdc6 was increased. Thus, the ability of overexpressed PR70 to cause accumulation of the protein was inhibited when the phosphorylation sites in Cdc6 were mutated. The effects of PR70 overexpression to cause accumulation of Cdc6 suggest it acts in a dominant-negative manner to block Cdc6 dephosphorylation (see "Discussion").-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The effects of overe>>> (see "Discussion").
360	Luc-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Luc>>>Luc
361	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
362	Luc-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Luc>>>Luc
363	Luc-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Luc>>>Luc
364	Luc-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	Luc>>>Luc
365	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
366	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
367	PR70-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	PR70>>>PR70
368	EV-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EV>>>EV
369	wtCdc6 DDD-Cdc6 AAA-Cdc6-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	wtCdc6 DDD-Cdc6 AAA->>>c6 DDD-Cdc6 AAA-Cdc6
370	GPDH-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	GPDH>>>GPDH
371	siRNA-->id=3, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	siRNA>>>siRNA
372	plasmid-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	plasmid>>>plasmid
373	GFP-Cdc6-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	GFP-Cdc6>>>GFP-Cdc6
374	B-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	B>>>B
375	EV-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	EV>>>EV
376	wtCdc6 DDD-Cdc6 AAA-Cdc6-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	wtCdc6 DDD-Cdc6 AAA->>>c6 DDD-Cdc6 AAA-Cdc6
377	PR70 siRNA-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70 siRNA>>>PR70 siRNA
378	C-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	C>>>C
379	Luc siRNA-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Luc siRNA>>>Luc siRNA
380	plasmid-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	plasmid>>>plasmid
381	D-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	D>>>D
382	A-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	A>>>A
383	Luc-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Luc>>>Luc
384	PP2A-C-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PP2A-C>>>PP2A-C
385	Mock-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Mock>>>Mock
386	PP5-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PP5>>>PP5
387	Cdc6-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Cdc6>>>Cdc6
388	PP2A-C-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PP2A-C>>>PP2A-C
389	PP5-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PP5>>>PP5
390	siRNA-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	siRNA>>>siRNA
391	PR70-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70>>>PR70
392	Cdc6-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Cdc6>>>Cdc6
393	PR70-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70>>>PR70
394	Actin-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Actin>>>Actin
395	Luc-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Luc>>>Luc
396	Mock-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Mock>>>Mock
397	PR70-1-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PR70-1>>>PR70-1
398	PR70-2-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	PR70-2>>>PR70-2
399	siRNA-->id=3224, page=4, size=9, fam=Times, col=#121213, type=parrnote, textLines=142--->[]--->note	siRNA>>>siRNA
400	FIGURE 7. Knockdown of PR70 increases the levels of Cdc6. A, HeLa cells were mock transfected (Mock) or transfected with control (Luc), PP2A catalytic subunit (PP2A-C), or PP5 (PP5) siRNA. Cells were harvested 48 h after transfection and immunoblotted with anti-Cdc6, PP2A-C, PR70, or PP5 antibodies. B, HeLa cells were mock transfected (Mock) or transfected with control (Luc), PR70-1, or PR70-2 siRNA. Cells were harvested 48 h after transfection and immunoblotted with antiCdc6, PR70, or actin (as a loading control) antibodies. C, HeLa cells were co-transfected with control or PR70-1 siRNA and plasmids encoding GFP-tagged versions of wild type Cdc6 (wtCdc6) or Cdc6 in which the N-terminal phosphorylation sites were mutated to aspartic acid (DDD-Cdc6) or alanine (AAA-Cdc6). Forty-eight hours later, the cells were harvested and lysates were analyzed by immunoblotting with antibodies against Cdc6 or glyceraldehyde-3 phosphate dehydrogenase (GPDH) as a loading control. D, duplicate samples of the lysates described in B, from cells co-transfected with Cdc6 and either luciferase control (Luc siRNA)or PR70-1 (PR70) siRNAs were immunoblotted with anti-PR70 antibodies to confirm knockdown of PR70.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 7. Knockdown >>>m knockdown of PR70.
401	Empty Vec-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Empty Vec>>>Empty Vec
402	tor-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	tor>>>tor
403	Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Cdc6>>>Cdc6
404	+ CDK2-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	+ CDK2>>>+ CDK2
405	Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Cdc6>>>Cdc6
406	Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Cdc6>>>Cdc6
407	+ PR70EF1/2-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	+ PR70EF1/2>>>+ PR70EF1/2
408	Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Cdc6>>>Cdc6
409	+ PR70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	+ PR70>>>+ PR70
410	C-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	C>>>C
411	Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Cdc6>>>Cdc6
412	+ PR70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	+ PR70>>>+ PR70
413	N3-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	N3>>>N3
414	Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Cdc6>>>Cdc6
415	+ PR70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	+ PR70>>>+ PR70
416	FLAG-PR70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	FLAG-PR70>>>FLAG-PR70
417	Myc-CDK2-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Myc-CDK2>>>Myc-CDK2
418	Actin-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Actin>>>Actin
419	Empty Vector-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Empty Vector>>>Empty Vector
420	DDD-Cdc-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	DDD-Cdc>>>DDD-Cdc
421	6 + PR7-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	6 + PR7>>>6 + PR7
422	0-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	0>>>0
423	DDD-Cdc-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	DDD-Cdc>>>DDD-Cdc
424	6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	6>>>6
425	AAA-Cdc-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	AAA-Cdc>>>AAA-Cdc
426	6 + PR70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	6 + PR70>>>6 + PR70
427	AAA-Cdc-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	AAA-Cdc>>>AAA-Cdc
428	6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	6>>>6
429	wtCdc-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	wtCdc>>>wtCdc
430	6 + PR-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	6 + PR>>>6 + PR
431	70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	70>>>70
432	wtCdc-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	wtCdc>>>wtCdc
433	6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	6>>>6
434	A-->id=5833, page=7, size=30, fam=Times, col=#100f0d, type=title, textLines=2--->[]--->note	A>>>A
435	B-->id=5833, page=7, size=30, fam=Times, col=#100f0d, type=title, textLines=2--->[]--->note	B>>>B
436	GFP-Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	GFP-Cdc6>>>GFP-Cdc6
437	Endo-Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Endo-Cdc6>>>Endo-Cdc6
438	GFP-Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	GFP-Cdc6>>>GFP-Cdc6
439	FLAG-PR70-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	FLAG-PR70>>>FLAG-PR70
440	Actin-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Actin>>>Actin
441	Endo-Cdc6-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->note	Endo-Cdc6>>>Endo-Cdc6
442	FIGURE 8. Overexpression of PR70 increases the levels of Cdc6. A, U2OS cells were transfected with empty vector, or plasmids encoding GFP-Cdc6 (Cdc6), myc-tagged CDK2 (CDK2), or FLAG-tagged constructs of wild-type PR70 (PR70) or the indicated PR70 mutants. Cells were harvested 24 h after transfection, and lysates were analyzed by immunoblotting with antibodies against Cdc6, actin, FLAG, or myc as indicated at the right. The Cdc6 antibodies detected both the expressed Cdc6 (GFP-Cdc6) and endogenous Cdc6 (Endo-Cdc6). B, U2OS cells were co-transfected with empty vector or plasmids expressing FLAG-PR70 and plasmids expressing GFP-tagged versions of wildtype Cdc6 (wtCdc6), or the AAA (AAA-Cdc6), or DDD (DDD-Cdc6) triple phosphorylation site mutants of Cdc6. Cells were harvested 24 h later, and lysates were analyzed by immunoblotting with antibodies against Cdc6, actin, or the FLAG epitope as indicated at the right.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 8. Overexpres>>>icated at the right.
443	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
444	JUNE 6, 2008 · VOLUME 283 · NUMBER 23-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	JUNE 6, 2008 · VOLUM>>>LUME 283 · NUMBER 23
445	JOURNAL OF BIOLOGICAL CHEMISTRY 16111-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16111
446	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
447	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
448	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
449	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
450	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
451	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
452	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
453	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
454	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
455	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
456	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
457	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
458	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
459	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
460	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
461	Knockdown of PR70 Causes G1 Arrest--The potential role of PR70 in progression through G1 was determined by determining the cell cycle distribution of cells in which PR70 was depleted by RNA interference. Knockdown of PR70 caused accumulation of cells in G0/G1 and depletion of cells in S and G2/M (Fig. 9). The apparent G1 arrest occurred with either of two siRNAs that target distinct regions of PR70. The level of G1 arrest correlated with the extent of PR70 knockdown. The lower levels of PR70 achieved with the PR70 siRNA-1 compared with PR70 siRNA-2 corresponded to a greater increase in the number of G1 cells (76% versus 65%). The G1 arrest caused by knockdown of PR70 supports a role for this PP2A regulatory subunit in progression through G1 phase.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Knockdown of PR70 Ca>>>on through G1 phase.
462	DISCUSSION-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=62--->[]--->title	DISCUSSION>>>DISCUSSION
463	The formation of pre-replicative complexes during the initiation of DNA replication is regulated, in part, by the availability of Cdc6. Cyclin-dependent kinases phosphorylate regulatory sites within the N-terminal domain of Cdc6 and block ubiquitination by APC/Ccdh1 and subsequent degradation by the proteasome (15). The results reported here help establish the form of PP2A complexed with the PR70 regulatory subunit as a physiological Cdc6 phosphatase and are consistent with a model in which PR70 targets PP2A to Cdc6 through direct protein-protein interactions. Knockdown of PR70 by RNA interference results in an increase in the levels of Cdc6 protein, consistent with a role for this subunit in regulating the stability of Cdc6. Overexpression of PR70 appeared to act in a dominant-negative manner to also increase the levels of Cdc6. The observations that increased protein levels did not occur with phosphorylation site mutants of Cdc6 are consistent with a role for PR70 in regulating Cdc6 phosphorylation and stability. A novel aspect of this model is the potential regulation of Cdc6 dephosphorylation by calcium. Calcium enhances the recruitment of the core dimer of PP2A to Cdc6 by binding to the EF-hand motifs of PR70, raising the possibility that changes in intracellular calcium can regulate the accumulation of Cdc6 and initiation of DNA replication. However, it remains to be determined if physiological changes in intracellular calcium-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The formation of pre>>>ntracellular calcium
464	No.-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->parr	No.>>>No.
465	of-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	of>>>of
466	Cells-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	Cells>>>Cells
467	PR70-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70>>>PR70
468	GPDH-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	GPDH>>>GPDH
469	Non-transfectedLuc-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Non-transfectedLuc>>>Non-transfectedLuc
470	control PR70 siRNA-2-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	control PR70 siRNA-2>>>control PR70 siRNA-2
471	PR70-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70>>>PR70
472	siRNA-1-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	siRNA-1>>>siRNA-1
473	Luc control-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Luc control>>>Luc control
474	0-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	0>>>0
475	100-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	100>>>100
476	200-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	200>>>200
477	300-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	300>>>300
478	400-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	400>>>400
479	G0/G1 55.3-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G0/G1 55.3>>>G0/G1 55.3
480	G2/M 20.9-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G2/M 20.9>>>G2/M 20.9
481	S 22.2-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	S 22.2>>>S 22.2
482	Non-transfected-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	Non-transfected>>>Non-transfected
483	0-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	0>>>0
484	100-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	100>>>100
485	200-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	200>>>200
486	300-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	300>>>300
487	400-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	400>>>400
488	500-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	500>>>500
489	G0/G1 52.1-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G0/G1 52.1>>>G0/G1 52.1
490	G2/M 24.4-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G2/M 24.4>>>G2/M 24.4
491	S 22.2-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	S 22.2>>>S 22.2
492	PR70 siRNA-2-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70 siRNA-2>>>PR70 siRNA-2
493	0-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	0>>>0
494	200-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	200>>>200
495	400-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	400>>>400
496	600-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	600>>>600
497	G0/G1 65-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G0/G1 65>>>G0/G1 65
498	G2/M 17.9-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G2/M 17.9>>>G2/M 17.9
499	S 16.3-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	S 16.3>>>S 16.3
500	PR70 siRNA-1-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	PR70 siRNA-1>>>PR70 siRNA-1
501	0-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	0>>>0
502	200-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	200>>>200
503	400-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	400>>>400
504	600-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	600>>>600
505	800-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	800>>>800
506	FL3 Area-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	FL3 Area>>>FL3 Area
507	0-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	0>>>0
508	200-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	200>>>200
509	400-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	400>>>400
510	600-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	600>>>600
511	800-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	800>>>800
512	G0/G1 76.5-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G0/G1 76.5>>>G0/G1 76.5
513	G2/M 10.7-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	G2/M 10.7>>>G2/M 10.7
514	S 12.1-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->note	S 12.1>>>S 12.1
515	A-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	A>>>A
516	B-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	B>>>B
517	C-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	C>>>C
518	D-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	D>>>D
519	E-->id=6335, page=8, size=15, fam=Times, col=#231f20, type=title, textLines=8--->[]--->note	E>>>E
520	FIGURE 9. Knockdown of PR70 causes G1 arrest. A­D, U2OS cells were left untreated (A), or transfected with control siRNA (B), PR70-2 siRNA (C), or PR70-1 siRNA (D). Forty-eight hours later, the cells were harvested and analyzed by flow cytometry. The data are plotted as the number of cells versus DNA content determined by FL3 area. The percentages of cells in G0/G1,S,and G2/M phases are indicated. E, duplicate transfections were harvested after 48 h and immunoblotted with anti-PR70 antibody to confirm knockdown.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->capfig	FIGURE 9. Knockdown >>>o confirm knockdown.
521	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
522	16112 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	16112 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
523	VOLUME 283 · NUMBER 23 · JUNE 6, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	VOLUME 283 · NUMBER >>>ER 23 · JUNE 6, 2008
524	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
525	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
526	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
527	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
528	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
529	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
530	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
531	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
532	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
533	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
534	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
535	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
536	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
537	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
538	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
539	concentrations are sufficient to regulate association of PP2A with Cdc6.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	concentrations are s>>>n of PP2A with Cdc6.
540	Biochemical analysis of the interaction of PR70 with the AC core dimer suggested an unanticipated mechanism for regulating PP2A activity. Experiments with N-terminal truncation mutants showed that PR70 can associate with Cdc6 independently of the A- and C-subunits. This observation contrasts with the prevailing view of PP2A in which the regulatory subunits have been thought to be constitutively associated with the core dimer (18 ­20). The importance of the existence of PP2A in heterotrimeric forms is supported by data showing that some PP2A regulatory subunits are only stable when incorporated into holoenzymes (29 ­31). Overexpression of the PPP2R2 family member, B , leads to proteasome-dependent degradation of the free protein but not protein incorporation into holoenzymes (31). In contrast, several lines of evidence indicate that members of the PPP2R3/PR72 family are stable regardless of whether or not they are incorporated into holoenzymes. As shown here, mutants of PR70 that cannot bind to PP2A accumulate to the same levels as the wild-type protein. The apparent stability of PR70 is also independent of interaction with Cdc6, because a mutant ( C) that interacts poorly with Cdc6 accumulates to similar levels. Similarly, mutations of the related PR72 subunit that block interaction with the AC core dimer have no effect on the levels of expressed protein (27). In addition, the PR59 subunit is not degraded following loss of the A-subunit (31). Thus, in contrast to the PPP2R2/B and PPP2R5/B56 families of PP2A regulatory subunits, members of the PPP2R3/PR72 family are stable proteins whose levels and functional interactions with substrates and other proteins may be independent of the core dimer of PP2A.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	Biochemical analysis>>> core dimer of PP2A.
541	The stability of expressed PR70 may also account for its ability to act in an apparent dominant-negative manner to increase the levels of Cdc6. Excess free PR70 would associate with Cdc6 and displace endogenous PR70-AC holoenzyme. Loss of the active AC core dimer from Cdc6 would inhibit dephosphorylation leading to decreased ubiquitination by APC/Ccdh1 and increase protein levels. A dominant-negative action of overexpressed PR70 is supported by observations that the effects on Cdc6 levels are not dependent on interaction of PR70 with the AC core dimer (e.g. the N3 and EF1/2 mutants) but are dependent on interaction with Cdc6 (e.g. the C mutant). Like knockdown of PR70, the dominant-negative actions of PR70 to increase Cdc6 levels appear to be dependent on intact phosphorylation sites, because the levels of co-expressed DDD and AAA mutants of Cdc6 were not significantly affected. Forced overexpression of the related PR72 subunit has also been reported to act in a dominant-negative manner. Expression of either wild-type PR72 or an EF-hand 2 mutant, which cannot bind the AC core dimer, both cause G1 arrest in U2OS cells (27).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The stability of exp>>> in U2OS cells (27).
542	The accumulation of cells with G0/G1 DNA content following knockdown by RNA interference is consistent with an important role for PR70 in progression of cells through into S phase. Similarly, overexpression of a fragment that contains the complete R3 domain and the C terminus of PR70 (termed PR48 or N1 in this study) also causes G1 arrest, presumably through a dominant-negative action (17). The G1 arrest in cells depleted of PR70 coincides with an increase in Cdc6 protein levels. A previous study showed that increases in phosphorylation and stability of Cdc6 enhance formation of pre-replicative complexes (15). The increase in pre-replicative complex formation would be expected to enhance entry into S phase. Consistent with this idea, expression of exogenous wild-type Cdc6 leads to accelerated entry into S phase (14). However, exogenous expression of a non-phosphorylatable (AAA) mutant of Cdc6 (5) or an N-terminally truncated version of Cdc6, missing the CDK phosphorylation sites and destruction motifs recognized by APC/Ccdh1 (14), inhibit initiation of DNA replication and entry into S phase. It is possible that, even though phosphorylation is required for stabilization of Cdc6 and assembly of prereplicative complexes during G1, an additional Cdc6 dephosphorylation or degradation step is needed to initiate DNA replication. Knockdown or overexpression of PR70 might inhibit this step and retard entry into S phase. Although the ability of PR70 knockdown to cause G1 arrest is consistent with regulation of Cdc6, an equally likely possibility is that PR70 plays other roles during G1. PR70 may regulate the activity of other proteins involved in cell cycle control, either through PP2A-mediated dephosphorylation or actions that are independent of PP2A.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The accumulation of >>>independent of PP2A.
543	The effects of calcium on the PR70-dependent association of PP2A with Cdc6 are consistent with a more general role for the PPP2R3/PR72 family in mediating calcium-regulated dephosphorylation. All four members of this family contain conserved EF-hand sequence motifs (supplemental Fig.  S1). The EF-hands of PR72 are also functional calcium binding sites, and, similar to PR70, calcium binding to the second EF-hand enhances interaction with the A-subunit (27). PR72 has been shown to mediate calcium-dependent dephosphorylation of threonine-75 in the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). This report showed that, in addition to the role of EF-hand 2 in interaction with the AC core dimer, calcium binding to EF-hand 1 increased the phosphatase activity of the PR72-holoenzyme toward DARPP-32 (32). Both PR72 and its alternative splice variant (PR130) have been reported to interact with the mammalian Naked cuticle protein and regulate Wnt signaling (33, 34). Calcium may therefore influence Wnt signaling through recruitment and/or regulation of PP2A associated with Naked cuticle. The other member of the PPP2R3 family, PR59, targets PP2A to the retinoblastoma-related p107 protein (35) and may provide a mechanism for calcium regulation of the cell cycle functions of p107.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The effects of calci>>>e functions of p107.
544	The sites involved in the interaction with PP2A and Cdc6 mapped to distinct regions of PR70, consistent with a role in bridging the two proteins. The N-terminal domain of PR70, which is not conserved with other members of the PPP2R3 family, is not required for interaction with either PP2A or Cdc6. Deletion of the C-terminal region, including the PR70-unique sequence and a portion of the conserved R3 domain, had no effect on interaction with the PP2A core dimer but severely inhibited binding to Cdc6. Conversely, deletion of N-terminal sequences within the conserved R3 domain blocked binding to the A-subunit but had no effect on interaction with Cdc6. The N-terminal region required for interaction with the A-subunit contains an FYF amino acid motif that plays a role in the interaction and is conserved between members of the PPP2R3 family. The A-subunit of PP2A is a HEAT repeat protein (36). The FYF motif in PR70 resembles the FG amino acid repeats (FXFG and GLFG) within the nucleoporin family of nuclear pore proteins. The nucleoporins interact with nuclear transport factors, including importin- , which are also HEAT repeat proteins. The FG repeats of nucleoporins bind to shallow hydrophobic pockets in importin- (37, 38). The A-subunit of PP2A contains exposed hydrophobic surfaces, predicted to play a role in interaction with the regulatory subunits (36), that are possible sites of interaction with the FYF motif of PR70.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The sites involved i>>>e FYF motif of PR70.
545	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
546	JUNE 6, 2008 · VOLUME 283 · NUMBER 23-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	JUNE 6, 2008 · VOLUM>>>LUME 283 · NUMBER 23
547	JOURNAL OF BIOLOGICAL CHEMISTRY 16113-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16113
548	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
549	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
550	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
551	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
552	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
553	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
554	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
555	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
556	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
557	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
558	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
559	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
560	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
561	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
562	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at:
563	The requirement for the N-terminal region of the R3 domain of PR70 for binding to the A-subunit is distinct from results observed with the PR72 protein. A fragment of PR72 consisting of amino acids 219 ­ 473 (corresponding to residues 257­509 of PR70) interacts with the A-subunit in the yeast two-hybrid assay (27). This fragment of PR72 is missing the N-terminal region of the R3 domain. Two fragments of PR72 containing putative A-subunit binding domains prepared by in vitro translation (corresponding to residues 234 ­339 and 378 ­ 436 of PR70) interacted with the A-subunit in vitro using GST pulldown assays (39). These PR72 fragments also do not contain the conserved N-terminal region of the R3 domain that was necessary for interaction of PR70 with the AC core dimer in our assays. These observations indicate that additional regions of PR70, beyond those required in PR72, are required for binding to the A-subunit, or that the differences observed are due to different assays employed.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	The requirement for >>>ent assays employed.
564	In summary, the present study shows that the PR70 regulatory subunit targets PP2A to Cdc6 and that PP2A is likely to be a physiological Cdc6 phosphatase. The targeting of PP2A to Cdc6 is enhanced by binding of calcium to PR70 raising the possibility that changes in intracellular calcium can influence formation of pre-replicative complexes through regulation of Cdc6 dephosphorylation.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=906--->[]--->parr	In summary, the pres>>>6 dephosphorylation.
565	REFERENCES-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->[]--->parr	REFERENCES>>>REFERENCES
566	1. Bell, S. P., and Dutta, A. (2002) Annu. Rev. Biochem. 71, 333­374 2. Diffley, J. F. (2004) Curr. Biol. 14, R778 ­R786-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	1. Bell, S. P., and >>>Biol. 14, R778 ­R786
567	3. Williams, R. S., Shohet, R. V., and Stillman, B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 142­147-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	3. Williams, R. S., >>>U. S. A. 94, 142­147
568	4. Yan, Z., DeGregori, J., Shohet, R., Leone, G., Stillman, B., Nevins, J. R., and Williams, R. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3603­3608-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	4. Yan, Z., DeGregor>>> S. A. 95, 3603­3608
569	5. Jiang, W., Wells, N. J., and Hunter, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6193­ 6198-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	5. Jiang, W., Wells,>>>S. A. 96, 6193­ 6198
570	6. Herbig, U., Griffith, J. W., and Fanning, E. (2000) Mol. Biol. Cell 11, 4117­ 4130-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	6. Herbig, U., Griff>>> Cell 11, 4117­ 4130
571	7. Petersen, B. O., Lukas, J., Sorensen, C. S., Bartek, J., and Helin, K. (1999) EMBO J. 18, 396 ­ 410-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	7. Petersen, B. O., >>>MBO J. 18, 396 ­ 410
572	8. Pelizon, C., Madine, M. A., Romanowski, P., and Laskey, R. A. (2000) Genes Dev. 14, 2526 ­2533-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	8. Pelizon, C., Madi>>> Dev. 14, 2526 ­2533
573	9. Delmolino, L. M., Saha, P., and Dutta, A. (2001) J. Biol. Chem. 276, 26947­26954 10. Coverley, D., Pelizon, C., Trewick, S., and Laskey, R. A. (2000) J. Cell Sci. 113, 1929 ­1938-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	9. Delmolino, L. M.,>>>Sci. 113, 1929 ­1938
574	11. Mendez, J., and Stillman, B. (2000) Mol. Cell Biol. 20, 8602­ 8612-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	11. Mendez, J., and >>>Biol. 20, 8602­ 8612
575	12. Alexandrow, M. G., and Hamlin, J. L. (2004) Mol. Cell Biol. 24, 1614 ­1627 13. Harper, J. W., Burton, J. L., and Solomon, M. J. (2002) Genes Dev. 16, 2179 ­2206-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	12. Alexandrow, M. G>>> Dev. 16, 2179 ­2206
576	14. Petersen, B. O., Wagener, C., Marinoni, F., Kramer, E. R., Melixetian, M., Denchi, E. L., Gieffers, C., Matteucci, C., Peters, J. M., and Helin, K. (2000) Genes Dev. 14, 2330 ­2343-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	14. Petersen, B. O.,>>> Dev. 14, 2330 ­2343
577	15. Mailand, N., and Diffley, J. F. (2005) Cell 122, 915­926-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	15. Mailand, N., and>>>5) Cell 122, 915­926
578	16. Duursma, A., and Agami, R. (2005) Mol. Cell Biol. 25, 6937­ 6947-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	16. Duursma, A., and>>>Biol. 25, 6937­ 6947
579	17. Yan, Z., Fedorov, S. A., Mumby, M. C., and Williams, R. S. (2000) Mol. Cell Biol. 20, 1021­1029-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	17. Yan, Z., Fedorov>>> Biol. 20, 1021­1029
580	18. Virshup, D. M. (2000) Curr. Opin. Cell Biol. 12, 180 ­185-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	18. Virshup, D. M. (>>>l Biol. 12, 180 ­185
581	19. Janssens, V., and Goris, J. (2001) Biochem. J. 353, 417­ 439-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	19. Janssens, V., an>>>em. J. 353, 417­ 439
582	20. Silverstein, A. M., Davis, A. J., Bielinski, V. A., Esplin, E. D., Mahmood, N. A., and Mumby, M. C. (2003) in Handbook of Cellular Signaling (Bradshaw, R. A., and Dennis, E. A., eds) pp. 405­ 415, Academic Press, San Diego, CA-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	20. Silverstein, A. >>>Press, San Diego, CA
583	21. Stevens, I., Janssens, V., Martens, E., Dilworth, S., Goris, J., and Van Hoof, C. (2003) Eur. J. Biochem. 270, 376 ­387-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	21. Stevens, I., Jan>>>ochem. 270, 376 ­387
584	22. Pearson, G. W., Earnest, S., and Cobb, M. H. (2006) Mol. Cell Biol. 26, 3039 ­3047-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	22. Pearson, G. W., >>>Biol. 26, 3039 ­3047
585	23. Ali, A., Zhang, J., Bao, S., Liu, I., Otterness, D., Dean, N. M., Abraham, R. T., and Wang, X. F. (2004) Genes Dev. 18, 249 ­254-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	23. Ali, A., Zhang, >>>es Dev. 18, 249 ­254
586	24. Mumby, M. C., Green, D. D., and Russell, K. R. (1985) J. Biol. Chem. 260, 13763­13770-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	24. Mumby, M. C., Gr>>>em. 260, 13763­13770
587	25. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. (Tokyo) 95, 511­519-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	25. Maruyama, K., Mi>>> (Tokyo) 95, 511­519
588	26. Sontag, E., Fedorov, S., Kamibayashi, C., Robbins, D., Cobb, M., and Mumby, M. (1993) Cell 75, 887­ 897-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	26. Sontag, E., Fedo>>>3) Cell 75, 887­ 897
589	27. Janssens, V., Jordens, J., Stevens, I., Van Hoof, C., Martens, E., De Smedt, H., Engelborghs, Y., Waelkens, E., and Goris, J. (2003) J. Biol. Chem. 278, 10697­10706-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	27. Janssens, V., Jo>>>em. 278, 10697­10706
590	28. Strynadka, N. C., and James, M. N. (1989) Annu. Rev. Biochem. 58, 951­998-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	28. Strynadka, N. C.>>>Biochem. 58, 951­998
591	29. Silverstein, A. M., Barrow, C. A., Davis, A. J., and Mumby, M. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 4221­ 4226-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	29. Silverstein, A. >>>S. A. 99, 4221­ 4226
592	30. Li, X., Scuderi, A., Letsou, A., and Virshup, D. M. (2002) Mol. Cell Biol. 22, 3674 ­3684-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	30. Li, X., Scuderi,>>>Biol. 22, 3674 ­3684
593	31. Strack, S., Ruediger, R., Walter, G., Dagda, R. K., Barwacz, C. A., and Cribbs, J. T. (2002) J. Biol. Chem. 277, 20750 ­20755-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	31. Strack, S., Rued>>>m. 277, 20750 ­20755
594	32. Ahn, J. H., Sung, J. Y., McAvoy, T., Nishi, A., Janssens, V., Goris, J., Greengard, P., and Nairn, A. C. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9876 ­9881-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	32. Ahn, J. H., Sung>>>. A. 104, 9876 ­9881
595	33. Creyghton, M. P., Roel, G., Eichhorn, P. J., Hijmans, E. M., Maurer, I., Destree, O., and Bernards, R. (2005) Genes Dev. 19, 376 ­386-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	33. Creyghton, M. P.>>>es Dev. 19, 376 ­386
596	34. Creyghton, M. P., Roel, G., Eichhorn, P. J., Vredeveld, L. C., Destree, O., and Bernards, R. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 5397­5402-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	34. Creyghton, M. P.>>>S. A. 103, 5397­5402
597	35. Voorhoeve, P. M., Hijmans, E. M., and Bernards, R. (1999) Oncogene 18, 515­524-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	35. Voorhoeve, P. M.>>>Oncogene 18, 515­524
598	36. Groves, M. R., Hanlon, N., Turowski, P., Hemmings, B. A., and Barford, D. (1999) Cell 96, 99 ­110-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	36. Groves, M. R., H>>>99) Cell 96, 99 ­110
599	37. Bayliss, R., Littlewood, T., and Stewart, M. (2000) Cell 102, 99 ­108-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	37. Bayliss, R., Lit>>>0) Cell 102, 99 ­108
600	38. Bayliss, R., Littlewood, T., Strawn, L. A., Wente, S. R., and Stewart, M. (2002) J. Biol. Chem. 277, 50597­50606-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	38. Bayliss, R., Lit>>>em. 277, 50597­50606
601	39. Li, X., and Virshup, D. M. (2002) Eur. J. Biochem. 269, 546 ­552-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->parr	39. Li, X., and Virs>>>ochem. 269, 546 ­552
602	PR70 Targets PP2A to Cdc6-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=14--->[]--->note	PR70 Targets PP2A to>>>Targets PP2A to Cdc6
603	16114 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	16114 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
604	VOLUME 283 · NUMBER 23 · JUNE 6, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=59--->['U']--->note	VOLUME 283 · NUMBER >>>ER 23 · JUNE 6, 2008
605	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[u'a']--->note	at>>>at
606	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Centro>>>Centro
607	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Nacional>>>Nacional
608	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	de>>>de
609	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Investigaciones>>>Investigaciones
610	Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Oncológicas,>>>Oncológicas,
611	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	on>>>on
612	May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	May>>>May
613	28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	28,>>>28,
614	2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	2010>>>2010
615	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	www.jbc.org>>>www.jbc.org
616	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	Downloaded>>>Downloaded
617	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=432--->[]--->note	from>>>from
618	http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	http://www.jbc.org/c>>>/M710313200.DC1.html
619	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=32--->[]--->note	Supplemental Materia>>>ial can be found at: