0	The Loss of PIN1 Deregulates Cyclin E and Sensitizes Mouse, otherFeat=[]-->, belongsTo=title
1	Embryo Fibroblasts to Genomic Instability*S, otherFeat=[]-->, belongsTo=parr
2	Received for publication, May 26, 2005, and in revised form, October 11, 2005 Published, JBC Papers in Press, October 13, 2005, DOI 10.1074/jbc.M505770200, otherFeat=[]-->, belongsTo=parrnote
3	Elizabeth S. Yeh1, Brian O. Lew, and Anthony R. Means2, otherFeat=[]-->, belongsTo=title
4	From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, otherFeat=[]-->, belongsTo=title
5	During the G0/G1-S phase transition, the timely synthesis and, otherFeat=[]-->, belongsTo=parr
6	degradation of key regulatory proteins is required for normal cell, otherFeat=[]-->, belongsTo=parr
7	cycle progression. Two of these proteins, c-Myc and cyclin E, are, otherFeat=[]-->, belongsTo=parr
8	recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) com-, otherFeat=[]-->, belongsTo=parr
9	plex. SCFCdc4 binds to a similar phosphodegron sequence in c-Myc, otherFeat=[]-->, belongsTo=parr
10	and cyclin E proteins resulting in ubiquitylation and degradation of, otherFeat=[]-->, belongsTo=parr
11	both proteins via the 26 S proteosome. Since the prolyl isomerase, otherFeat=[]-->, belongsTo=parr
12	Pin1 binds the c-Myc phosphodegron and participates in regulation, otherFeat=[]-->, belongsTo=parr
13	of c-Myc turnover, we hypothesized that Pin1 would bind to and, otherFeat=[]-->, belongsTo=parr
14	regulate cyclin E turnover in a similar manner. Here we show that, otherFeat=[]-->, belongsTo=parr
15	Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts., otherFeat=[]-->, belongsTo=parr
16	Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends, otherFeat=[]-->, belongsTo=parr
17	on Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence, otherFeat=[]-->, belongsTo=parr
18	of Pin1 results in an increased steady-state level of cyclin E and, otherFeat=[]-->, belongsTo=parr
19	stalling of the cells in the G1/S phase of the cell cycle. The cellular, otherFeat=[]-->, belongsTo=parr
20	changes that result from the loss of Pin1 predispose Pin1 null mouse, otherFeat=[]-->, belongsTo=parr
21	embryo fibroblasts to undergo more rapid genomic instability when, otherFeat=[]-->, belongsTo=parr
22	immortalized by conditional inactivation of p53 and sensitizes, otherFeat=[]-->, belongsTo=parr
23	these cells to more aggressive Ras-dependent transformation and, otherFeat=[]-->, belongsTo=parr
24	tumorigenesis., otherFeat=[]-->, belongsTo=parr
25	Mitogenic stimuli initiate a sequence of events that result in the entry of, otherFeat=[]-->, belongsTo=parr
26	quiescent cells into S phase (1). Critical for this important transition is the, otherFeat=[]-->, belongsTo=parr
27	ordered synthesis and degradation of transcription factors such as c-Jun, otherFeat=[]-->, belongsTo=parr
28	and c-Myc (Myc) and cyclins such as cyclin D and cyclin E (1). Deregulation, otherFeat=[]-->, belongsTo=parr
29	of the turnover of these proteins, such that they remain active at inappro-, otherFeat=[]-->, belongsTo=parr
30	priate times during cell cycle progression, is frequently found in human, otherFeat=[]-->, belongsTo=parr
31	cancer. Thus, an understanding of the molecular mechanisms that regulate, otherFeat=[]-->, belongsTo=parr
32	protein turnover is crucial to provide insight into the oncogenic process., otherFeat=[]-->, belongsTo=parr
33	Three proteins important for the progression of cells into S phase, c-Jun,, otherFeat=[]-->, belongsTo=parr
34	c-Myc, and cyclin E, are ubiquitylated by a common member of the Skp1/, otherFeat=[]-->, belongsTo=parr
35	Cul1/Rbx1 (SCF)3 group of ubiquitin enzymes in which the F-box compo-, otherFeat=[]-->, belongsTo=parr
36	nent, which serves as the ubiquitin E3 ligase, is Cdc4 (SEL-10, Fbw7, Ago), otherFeat=[]-->, belongsTo=parr
37	(2). SCFCdc4 binds to a component of each protein that has been termed the, otherFeat=[]-->, belongsTo=parr
38	"phosphodegron" (3) to promote the ubiquitylation and degradation of, otherFeat=[]-->, belongsTo=parr
39	these proteins via the 26 S proteosome. c-Jun and Myc have an additional, otherFeat=[]-->, belongsTo=parr
40	binding protein in common, the peptidyl prolyl cis/trans-isomerase Pin1,, otherFeat=[]-->, belongsTo=parr
41	which binds and isomerizes prolyl bonds in the context of phospho-Ser/, otherFeat=[]-->, belongsTo=parr
42	Thr-Pro motifs (4, 5)., otherFeat=[]-->, belongsTo=parr
43	Previously, we described the mechanism by which Pin1 promotes, otherFeat=[]-->, belongsTo=parr
44	Myc degradation (5). The Cdc4 phosphodegron of Myc is present in a, otherFeat=[]-->, belongsTo=parr
45	domain termed Myc box 1 (MB1), containing the sequence LPpTP-, otherFeat=[]-->, belongsTo=parr
46	PLpSP (where pT represents phosphothreonine and pS represents, otherFeat=[]-->, belongsTo=parr
47	phosphoserine), in which the two phosphorylation events occur, otherFeat=[]-->, belongsTo=parr
48	sequentially and are catalyzed by ERK (Ser62) and GSK3 (Thr58),, otherFeat=[]-->, belongsTo=parr
49	respectively (6, 7). Pin1 binds to the doubly phosphorylated motif in a, otherFeat=[]-->, belongsTo=parr
50	manner requiring phospho-Thr58 and promotes a conformational, otherFeat=[]-->, belongsTo=parr
51	change that presents phospho-Ser62 as a substrate for the protein phos-, otherFeat=[]-->, belongsTo=parr
52	phatase PP2A (5). Ubiquitylated Myc is only phosphoryated on Thr58, otherFeat=[]-->, belongsTo=parr
53	(5), suggesting that either dephosphorylation of Ser62 occurs before, otherFeat=[]-->, belongsTo=parr
54	Cdc4 can bind to Myc or before ubiquitylation can occur. Regardless of, otherFeat=[]-->, belongsTo=parr
55	the precise mechanism involved, inhibition of PP2A or the absence of, otherFeat=[]-->, belongsTo=parr
56	Pin1 results in a stabilization of Myc (5). Myc can also be stabilized by, otherFeat=[]-->, belongsTo=parr
57	mutating Thr58 to Ala, and this mutation renders Myc oncogenic in a, otherFeat=[]-->, belongsTo=parr
58	primary human cell transformation assay (5). Since residues in the Myc, otherFeat=[]-->, belongsTo=parr
59	phosphodegron, including Thr58 and Pro57, are frequently mutated in, otherFeat=[]-->, belongsTo=parr
60	lymphomas (8 ?11), deregulation of Myc due to mutations of this region, otherFeat=[]-->, belongsTo=parr
61	of the protein can play a role in human cancer (9)., otherFeat=[]-->, belongsTo=parr
62	The phosphodegron of cyclin E, LpTPPXpSG, is remarkably similar, otherFeat=[]-->, belongsTo=parr
63	to that of Myc (12). In the case of cyclin E, the phosphorylation events, otherFeat=[]-->, belongsTo=parr
64	are catalyzed by Cdk2 (Ser384) and GSK3 (Thr380) (11), respectively,, otherFeat=[]-->, belongsTo=parr
65	and x-ray structural studies have shown that phospho-Thr380 plays a, otherFeat=[]-->, belongsTo=parr
66	crucial role in the binding of cyclin E by Cdc4 (3, 12). As is the case for, otherFeat=[]-->, belongsTo=parr
67	Myc, deregulation of cyclin E can result in cell cycle defects (13?17) that, otherFeat=[]-->, belongsTo=parr
68	predispose cells to oncogenesis (18 ?23). Such defects include aberrant, otherFeat=[]-->, belongsTo=parr
69	DNA replication and the loss of genomic integrity. Indeed, cyclin E, otherFeat=[]-->, belongsTo=parr
70	deregulation is associated with many human cancers including breast, otherFeat=[]-->, belongsTo=parr
71	(18, 20, 24 ?26), ovarian (19), and bladder cancer (27), yet the precise, otherFeat=[]-->, belongsTo=parr
72	mechanism responsible for ensuring the timely cell cycle-dependent, otherFeat=[]-->, belongsTo=parr
73	turnover of cyclin E is incompletely understood. For these reasons and, otherFeat=[]-->, belongsTo=parr
74	because of the similarities in the phosphodegron motif and degradation, otherFeat=[]-->, belongsTo=parr
75	process between Myc and cyclin E, we investigated whether cyclin E was, otherFeat=[]-->, belongsTo=parr
76	also a Pin1-binding protein. Here we show that Pin1 binds cyclin E and, otherFeat=[]-->, belongsTo=parr
77	modulates cyclin E levels. In the absence of Pin1, cyclin E is deregulated, otherFeat=[]-->, belongsTo=parr
78	in a way that leads to its stabilization, which, in combination with other, otherFeat=[]-->, belongsTo=parr
79	protein alterations in Pin1 null cells, leads to cell cycle defects. We also, otherFeat=[]-->, belongsTo=parr
80	demonstrate a correlation between the cell cycle defects that occur in, otherFeat=[]-->, belongsTo=parr
81	mouse embryo fibroblasts (MEFs) null for Pin1 and increased rate in the, otherFeat=[]-->, belongsTo=parr
82	progression of genomic instability when these cells are immortalized by, otherFeat=[]-->, belongsTo=parr
83	inactivating p53 function. Finally, we show that the cell cycle defects, otherFeat=[]-->, belongsTo=parr
84	resulting from the loss of Pin1 sensitize immortalized Pin1 null cells to, otherFeat=[]-->, belongsTo=parr
85	more extensive and aggressive transformation and tumorigenesis, otherFeat=[]-->, belongsTo=parr
86	induced by the Ras oncogene., otherFeat=[]-->, belongsTo=parr
87	MATERIALS AND METHODS, otherFeat=['U']-->, belongsTo=parr
88	Cells and Cell Culture--HEK 293 cells were acquired from the Amer-, otherFeat=[]-->, belongsTo=parr
89	ican Type Culture Collection. 293 cells were grown in Dulbecco's mod-, otherFeat=[]-->, belongsTo=parr
90	* This work was supported by National Institutes of Health (NIH) Grant CA082845 (to, otherFeat=[]-->, belongsTo=parrnote
91	A. R. M.). The costs of publication of this article were defrayed in part by the payment, otherFeat=[]-->, belongsTo=parrnote
92	of page charges. This article must therefore be hereby marked "advertisement"in, otherFeat=[]-->, belongsTo=parrnote
93	accordance with 18 U.S.C. Section 1734 solely to indicate this fact., otherFeat=[]-->, belongsTo=parrnote
94	S The on-line version of this article (available at http://www.jbc.org) contains supple-, otherFeat=[]-->, belongsTo=parrnote
95	mental Figs. 1 and 2., otherFeat=[]-->, belongsTo=parrnote
96	1 Supported in part by NCI, NIH, T32 Training Grant CA-059365 and a grant from the, otherFeat=[]-->, belongsTo=parrnote
97	Milheim Foundation for Cancer Research., otherFeat=[]-->, belongsTo=parrnote
98	2 To whom correspondence should be addressed: Dept. of Pharmacology and Cancer, otherFeat=[]-->, belongsTo=parrnote
99	Biology, Duke University Medical Center, Box 3813, Durham, NC 27710-3813. Tel.:, otherFeat=[]-->, belongsTo=parrnote
100	919-681-6209; Fax: 919-681-7767; E-mail: means001@mc.duke.edu., otherFeat=[]-->, belongsTo=parrnote
101	3 The abbreviations used are: SCF, Skp1/Cul1/Rbx1; MEF, mouse embryo fibroblast; GST,, otherFeat=[]-->, belongsTo=parrnote
102	glutathione S-transferase; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuri-, otherFeat=[]-->, belongsTo=parrnote
103	dine; WT, wild type; MMTV, murine mammary tumor virus., otherFeat=[]-->, belongsTo=parrnote
104	THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 1, pp. 241?251, January 6, 2006, otherFeat=[]-->, belongsTo=nota_cab_pie
105	? 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A., otherFeat=[]-->, belongsTo=nota_cab_pie
106	JANUARY 6, 2006 ? VOLUME 281 ? NUMBER 1, otherFeat=['U']-->, belongsTo=nota_cab_pie
107	JOURNAL OF BIOLOGICAL CHEMISTRY 241, otherFeat=[]-->, belongsTo=nota_cab_pie
108	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
109	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
110	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
111	de, otherFeat=[]-->, belongsTo=nota_cab_pie
112	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
113	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
114	on, otherFeat=[]-->, belongsTo=nota_cab_pie
115	September, otherFeat=[]-->, belongsTo=nota_cab_pie
116	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
117	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
118	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
119	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
120	from, otherFeat=[]-->, belongsTo=nota_cab_pie
121	http://www.jbc.org/cgi/content/full/M505770200/DC1, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
122	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
123	ified Eagle's medium, containing 10% fetal bovine serum. Phoenix cells, otherFeat=[]-->, belongsTo=parr
124	(293 derivative) were maintained in Dulbecco's modified Eagle's, otherFeat=[]-->, belongsTo=parr
125	medium containing 10% heat-inactivated fetal bovine serum. Pin1 null, otherFeat=[]-->, belongsTo=parr
126	mice, originally generated by Fujimori et al. (28), were obtained from, otherFeat=[]-->, belongsTo=parr
127	Hoffmann-LaRoche. The pin1 gene deletion was transferred into an, otherFeat=[]-->, belongsTo=parr
128	isogenic C57BL6 background using marker-assisted speed congenic, otherFeat=[]-->, belongsTo=parr
129	breeding by Jackson Laboratory. pin1 / and pin1 / embryonic fibro-, otherFeat=[]-->, belongsTo=parr
130	blasts were isolated from the isogenic C57BL6 strain as previously, otherFeat=[]-->, belongsTo=parr
131	described (29). MEFs were grown in Dulbecco's modified Eagle's, otherFeat=[]-->, belongsTo=parr
132	medium containing sodium pyruvate and 10% heat-inactivated fetal, otherFeat=[]-->, belongsTo=parr
133	bovine serum., otherFeat=[]-->, belongsTo=parr
134	293 cells were transfected using Lipofectamine or Lipofectamine, otherFeat=[]-->, belongsTo=parr
135	2000 (Invitrogen) and Opti-MEM (Invitrogen) serum-reduced media, otherFeat=[]-->, belongsTo=parr
136	per the manufacturer's suggestion. Cells were plated 1 day prior to, otherFeat=[]-->, belongsTo=parr
137	transfection at a density of 1 106 cells/100-mm plate. Briefly, 2.5 g, otherFeat=[]-->, belongsTo=parr
138	of DNA was mixed with 15?30 l of Lipofectamine 2000 or Lipo-, otherFeat=[]-->, belongsTo=parr
139	fectamine, respectively, in 0.5 ml of Opti-MEM and incubated for 15, otherFeat=[]-->, belongsTo=parr
140	min at room temperature. The mixture was added to the cells for 12?18, otherFeat=[]-->, belongsTo=parr
141	h prior to treatment with drug or lysis., otherFeat=[]-->, belongsTo=parr
142	The following plasmids were used for transfections. pCS2-Myc-cy-, otherFeat=[]-->, belongsTo=parr
143	clin E, cyclin ET62A, cyclin ET62A/T380A, cyclin ES372A, and cyclin ES384A, otherFeat=[]-->, belongsTo=parr
144	were generous gifts from J. Wade Harper (Harvard Medical School,, otherFeat=[]-->, belongsTo=parr
145	Boston, MA). pBabe-Puro-Ras was kindly provided by Chris Counter, otherFeat=[]-->, belongsTo=parr
146	(Duke University, Durham, NC), and pBabe-Hygro-p53DD was from, otherFeat=[]-->, belongsTo=parr
147	William Hahn (Dana Farber Research Institute, Boston, MA). pWZL-, otherFeat=[]-->, belongsTo=parr
148	Blast-Myc was generated as previously described (5)., otherFeat=[]-->, belongsTo=parr
149	Protein Binding Assays--GST pull-down assays were performed as, otherFeat=[]-->, belongsTo=parr
150	previously described (5). Briefly, GST and GST-Pin1 fusion proteins, otherFeat=[]-->, belongsTo=parr
151	with either wild type Xenopus Pin1, an xPin1 WW domain mutant with, otherFeat=[]-->, belongsTo=parr
152	substitutions at W11A and W34A, or a xPin1 catalysis-deficient mutant, otherFeat=[]-->, belongsTo=parr
153	with a substitution at C109A was prepared as described (30). The GST, otherFeat=[]-->, belongsTo=parr
154	and GST-Pin1 recombinant proteins were maintained on GSH-agarose, otherFeat=[]-->, belongsTo=parr
155	beads and stored at 4 ?C. Cells were lysed in buffer containing PBS, 10, otherFeat=[]-->, belongsTo=parr
156	mM EDTA, 1 mM dithiothreitol, and 1% Triton X-100 supplemented, otherFeat=[]-->, belongsTo=parr
157	with Pefabloc (Roche Applied Science) and NaF. Lysates were clarified, otherFeat=[]-->, belongsTo=parr
158	by centrifugation prior to binding assays. Binding was performed at 4 ?C, otherFeat=[]-->, belongsTo=parr
159	for 1 h using 10 g of GST proteins incubated with 500 g of total lysate, otherFeat=[]-->, belongsTo=parr
160	protein. Beads were washed four times in 1 ml of lysis buffer per wash., otherFeat=[]-->, belongsTo=parr
161	Subsequently, proteins were solubilized in 2 SDS sample buffer and, otherFeat=[]-->, belongsTo=parr
162	separated by SDS-PAGE. Bound proteins were analyzed by Western, otherFeat=[]-->, belongsTo=parr
163	blotting., otherFeat=[]-->, belongsTo=parr
164	For immunoprecipitations, cells were lysed in buffer containing 50, otherFeat=[]-->, belongsTo=parr
165	mM Tris, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 1% Triton X-100, otherFeat=[]-->, belongsTo=parr
166	supplemented with Pefabloc (Roche Applied Science) and NaF. Lysates, otherFeat=[]-->, belongsTo=parr
167	were clarified by centrifugation. 500 g of total lysate protein was used, otherFeat=[]-->, belongsTo=parr
168	per sample. Prior to binding, the lysate samples were precleared with, otherFeat=[]-->, belongsTo=parr
169	Protein A/G-Sepharose for 10 min at 4 ?C. Binding was performed using, otherFeat=[]-->, belongsTo=parr
170	2 g (per sample) of anti-9E10 (Santa Cruz Biotechnology, Inc., Santa, otherFeat=[]-->, belongsTo=parr
171	Cruz, CA) for Myc-tagged protein. For experiments using Xenopus, otherFeat=[]-->, belongsTo=parr
172	interphase extracts, extracts were prepared as previously described (30)., otherFeat=[]-->, belongsTo=parr
173	Binding was performed by preincubating anti-Pin1 to Protein A beads, otherFeat=[]-->, belongsTo=parr
174	(Amersham Biosciences) in the presence of bovine serum albumin. The, otherFeat=[]-->, belongsTo=parr
175	beads were washed three times in PBS prior to incubation with 400 gof, otherFeat=[]-->, belongsTo=parr
176	total extract protein. Beads were washed four times in a modified radio-, otherFeat=[]-->, belongsTo=parr
177	immune precipitation buffer containing 10 mM Tris, pH 7.5, 150 mM, otherFeat=[]-->, belongsTo=parr
178	NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS. Bound proteins, otherFeat=[]-->, belongsTo=parr
179	were separated and analyzed as described above., otherFeat=[]-->, belongsTo=parr
180	Western Blotting--The following antibodies were used for the detec-, otherFeat=[]-->, belongsTo=parr
181	tion of proteins by Western blotting: anti-Cdk2 M2 (Santa Cruz Bio-, otherFeat=[]-->, belongsTo=parr
182	technology), anti-cyclin E M20 (Santa Cruz Biotechnology), anti-cyclin, otherFeat=[]-->, belongsTo=parr
183	E HE12 (Upstate Biotechnology), anti-Myc 9E10 (Santa Cruz Biotech-, otherFeat=[]-->, belongsTo=parr
184	nology), anti-Pin1 (30), anti-p27 C-19 (Santa Cruz Biotechnology), anti-, otherFeat=[]-->, belongsTo=parr
185	p53 FL-393 (Santa Cruz Biotechnology), anti-phosphocyclin E Thr380, otherFeat=[]-->, belongsTo=parr
186	(Santa Cruz Biotechnology), and anti- -actin (Sigma). Equal amounts, otherFeat=[]-->, belongsTo=parr
187	of protein for each sample were separated by SDS-PAGE and trans-, otherFeat=[]-->, belongsTo=parr
188	ferred to Immobilon-P membrane (Millipore Corp.). Membranes were, otherFeat=[]-->, belongsTo=parr
189	blocked in TBS-0.2% Tween 20 containing 5% milk for 1 h prior to, otherFeat=[]-->, belongsTo=parr
190	incubation with primary antibody. Primary antibodies were diluted, otherFeat=[]-->, belongsTo=parr
191	1:1000 in the blocking solution with the exception of anti-Pin1, which, otherFeat=[]-->, belongsTo=parr
192	was diluted 1:10,000 unless otherwise stated. Membranes were washed, otherFeat=[]-->, belongsTo=parr
193	a minimum of three times in Tris-buffered saline, 0.2% Tween 20 for 15, otherFeat=[]-->, belongsTo=parr
194	min each wash. Horseradish peroxidase-conjugated secondary antibod-, otherFeat=[]-->, belongsTo=parr
195	ies were diluted into the blocking buffer and incubated for2hat room, otherFeat=[]-->, belongsTo=parr
196	temperature. Primary antibodies were detected with horseradish perox-, otherFeat=[]-->, belongsTo=parr
197	idase-conjugated secondary antibodies using the ECL reagent (Amer-, otherFeat=[]-->, belongsTo=parr
198	sham Biosciences) per the manufacturer's instructions., otherFeat=[]-->, belongsTo=parr
199	Retroviral Transduction--The pin1 / and pin1 / cells containing, otherFeat=[]-->, belongsTo=parr
200	p53DD and Ras were generated by amphotrophic retroviral transduction, otherFeat=[]-->, belongsTo=parr
201	and polyclonal selection using 10 g/ml hygromycin B (Calbiochem) or, otherFeat=[]-->, belongsTo=parr
202	0.5 g/ml puromycin (Calbiochem). Viral medium was obtained by, otherFeat=[]-->, belongsTo=parr
203	Lipofectamine transfection of the Phoenix packaging cell line with 10, otherFeat=[]-->, belongsTo=parr
204	g of DNA. Cells were allowed to recover for 24 h, after which time the, otherFeat=[]-->, belongsTo=parr
205	medium was collected and filtered through a 0.45- m filter (16) to, otherFeat=[]-->, belongsTo=parr
206	remove any cellular debris. Filtered viral medium was supplemented, otherFeat=[]-->, belongsTo=parr
207	with 4 g/ml Polybrene (Sigma) and added to primary MEFs at passage, otherFeat=[]-->, belongsTo=parr
208	3 for 24 h. Cells were allowed to recover in complete medium for 24 h at, otherFeat=[]-->, belongsTo=parr
209	37 ?C prior to drug selection., otherFeat=[]-->, belongsTo=parr
210	Cellular Transformation--The ability of cells to undergo anchorage, otherFeat=[]-->, belongsTo=parr
211	independent growth was tested as previously described (32). 5 104, otherFeat=[]-->, belongsTo=parr
212	cells were plated in 0.3% Nobel agar, Dulbecco's modified Eagle's, otherFeat=[]-->, belongsTo=parr
213	medium and heat-inactivated fetal bovine serum on 35-mm plates con-, otherFeat=[]-->, belongsTo=parr
214	taining a 2-mm grid. Plates were incubated at 37 ?C for 21 days and, otherFeat=[]-->, belongsTo=parr
215	assessed for colony growth in soft agar. Cells were fed one time weekly, otherFeat=[]-->, belongsTo=parr
216	with 1 ml of agar/medium mixture. Colonies were counted by light, otherFeat=[]-->, belongsTo=parr
217	microscopy. The mean S.D. represents two independent experi-, otherFeat=[]-->, belongsTo=parr
218	ments, each performed in triplicate., otherFeat=[]-->, belongsTo=parr
219	Tumor Formation in Immunodeficient (SCID) Mice--SCID mice, otherFeat=[]-->, belongsTo=parr
220	were obtained from Charles River and housed in the Comprehensive, otherFeat=[]-->, belongsTo=parr
221	Cancer Center Isolation Facility under a 12-h light, 12-h dark cycle., otherFeat=[]-->, belongsTo=parr
222	Food and water were provided ad libitum, and all care was given in, otherFeat=[]-->, belongsTo=parr
223	compliance with National Institutes of Health and institutional guide-, otherFeat=[]-->, belongsTo=parr
224	lines on the use of laboratory and experimental animals. Prior to injec-, otherFeat=[]-->, belongsTo=parr
225	tion, cells were free of micoplasma, drugs, and antibiotics. Five mice, otherFeat=[]-->, belongsTo=parr
226	were injected per cell type with a single subcutaneous injection of 1, otherFeat=[]-->, belongsTo=parr
227	107 cells/animal. Data are represented as number of mice that developed, otherFeat=[]-->, belongsTo=parr
228	tumors over total number of animals., otherFeat=[]-->, belongsTo=parr
229	Real Time PCR--Quantitation of cellular mRNA levels was deter-, otherFeat=[]-->, belongsTo=parr
230	mined by real time PCR using the SyberGreen Supermix (Bio-Rad) rea-, otherFeat=[]-->, belongsTo=parr
231	gent per the manufacturer's suggestions. Briefly primers to the NH2-, otherFeat=[]-->, belongsTo=parr
232	terminal region of cyclin E were created to contain the following, otherFeat=[]-->, belongsTo=parr
233	sequences: 5 -ACG GAC CAC AGC AAC ATG AA-3 and 5 -AAA, otherFeat=[]-->, belongsTo=parr
234	CAC GGC CAC ATT TGC CT-3 (IDT). Total cellular RNA was iso-, otherFeat=[]-->, belongsTo=parr
235	lated by harvesting confluent 100-mm plates of pin1 / and pin1 /, otherFeat=[]-->, belongsTo=parr
236	MEFs using the TRIzol (Invitrogen) reagent. Cells were harvested in 1, otherFeat=[]-->, belongsTo=parr
237	ml of TRIzol and cleaned with chloroform. Resulting material was puri-, otherFeat=[]-->, belongsTo=parr
238	fied by ethanol precipitation. First strand cDNA was generated using, otherFeat=[]-->, belongsTo=parr
239	Superscript III reverse transcriptase (Invitrogen). Reaction mixtures, otherFeat=[]-->, belongsTo=parr
240	were denatured at 65 ?C for 6 min followed by cDNA synthesis at 50 ?C, otherFeat=[]-->, belongsTo=parr
241	for 50 min, and then the reactions were terminated by incubation at, otherFeat=[]-->, belongsTo=parr
242	85 ?C for 5 min. Resultant cDNA was mixed with designated primers, otherFeat=[]-->, belongsTo=parr
243	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
244	242 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
245	VOLUME 281 ? NUMBER 1 ? JANUARY 6, 2006, otherFeat=['U']-->, belongsTo=nota_cab_pie
246	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
247	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
248	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
249	de, otherFeat=[]-->, belongsTo=nota_cab_pie
250	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
251	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
252	on, otherFeat=[]-->, belongsTo=nota_cab_pie
253	September, otherFeat=[]-->, belongsTo=nota_cab_pie
254	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
255	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
256	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
257	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
258	from, otherFeat=[]-->, belongsTo=nota_cab_pie
259	and SyberGreen dye. The mixture was analyzed by real time PCR (Bio-, otherFeat=[]-->, belongsTo=parr
260	Rad) under the following conditions: step 1, 95 ?C for 3 min; step 2,, otherFeat=[]-->, belongsTo=parr
261	60.8 ?C for 0 ?15 min; step 3, 72 ?C for 0 ?15 min; repeat steps 2 and 3 for, otherFeat=[]-->, belongsTo=parr
262	45 cycles; step 5, 72 ?C for 2 min; step 6, 45 ?C for 0 ?10 min. Samples, otherFeat=[]-->, belongsTo=parr
263	were analyzed two times in duplicate per trial., otherFeat=[]-->, belongsTo=parr
264	Cell Proliferation--2.5 105 cells were seeded into 60-mm plates in, otherFeat=[]-->, belongsTo=parr
265	duplicate. Every 24 h, cells were trypsinized and counted by Coulter, otherFeat=[]-->, belongsTo=parr
266	counter. Data are represented as the average of the two samples for both, otherFeat=[]-->, belongsTo=parr
267	cell types at each time point. These data are representative of five indi-, otherFeat=[]-->, belongsTo=parr
268	vidual experiments., otherFeat=[]-->, belongsTo=parr
269	BrdUrd Incorporation--1 105 cells were seeded onto coverslips in, otherFeat=[]-->, belongsTo=parr
270	6-well plates and incubated overnight or until 70 ? 80% confluent. Cells, otherFeat=[]-->, belongsTo=parr
271	were pulsed with BrdUrd for 30 min at a final concentration of 10 M., otherFeat=[]-->, belongsTo=note
272	Following incubation with BrdUrd, cells were fixed in cold MeOH for 5, otherFeat=[]-->, belongsTo=parr
273	min at 20 ?C and then rehydrated in PBS. 1 ml of 2 M HCl/Triton, otherFeat=[]-->, belongsTo=note
274	X-100 was added to each well, followed by incubation at room temper-, otherFeat=[]-->, belongsTo=parr
275	ature for 30 min. Cells were washed once for 5 min in 1 ml of 0.1 M, otherFeat=[]-->, belongsTo=parr
276	sodium tetraborate (pH 8.5) and twice for 5 min in PBS followed by, otherFeat=[]-->, belongsTo=parr
277	blocking in 1% bovine serum albumin/PBS for 30 min. To perform, otherFeat=[]-->, belongsTo=parr
278	staining, anti-BrdUrd, diluted 1:100 in 0.1% bovine serum albumin/PBS,, otherFeat=[]-->, belongsTo=parr
279	was added to each coverslip and incubated at 4 ?C overnight. Coverslips, otherFeat=[]-->, belongsTo=parr
280	were washed three times in PBS for 5 min and then incubated with, otherFeat=[]-->, belongsTo=parr
281	anti-mouse fluorescein isothiocyanate diluted 1:200 in 0.1% bovine, otherFeat=[]-->, belongsTo=parr
282	serum albumin/PBS and incubated at room temperature for 60 min, otherFeat=[]-->, belongsTo=parr
283	followed by three washes in PBS. Coverslips were mounted on slides, otherFeat=[]-->, belongsTo=parr
284	with fluor antioxidant containing 1:5000 4 ,6-diamidino-2-phenylin-, otherFeat=[]-->, belongsTo=parr
285	dole and analyzed by fluorescence microscopy (Zeiss-Axiscop). Each of, otherFeat=[]-->, belongsTo=parr
286	three individual experiments were performed in triplicate, and 300, otherFeat=[]-->, belongsTo=parr
287	cells were counted per replicate., otherFeat=[]-->, belongsTo=parr
288	Flow Cytometry--MEFs were grown until 70 ? 80% confluent in, otherFeat=[]-->, belongsTo=parr
289	60-mm plates. Cells were harvested by washing once in Hanks' balanced, otherFeat=[]-->, belongsTo=parr
290	salt solution followed by trypsinization. Cells were pelleted by centrifu-, otherFeat=[]-->, belongsTo=parr
291	gation at 500 g for 10 min and washed once in PBS, followed by, otherFeat=[]-->, belongsTo=parr
292	fixation with cold 70% ethanol. At this time, cells were either stored at, otherFeat=[]-->, belongsTo=parr
293	20 ?C until used or kept on ice for 2 h prior to staining. DNA staining, otherFeat=[]-->, belongsTo=parr
294	was performed by resuspending cells in PBS containing propidium, otherFeat=[]-->, belongsTo=parr
295	iodide (5 g/ml) and RNase A (50 units) and then analyzed by fluores-, otherFeat=[]-->, belongsTo=parr
296	cence-activated cell sorting (Duke University Comprehensive Cancer, otherFeat=[]-->, belongsTo=parr
297	Center Flow Cytometry Shared Resource, Durham, NC). Three sepa-, otherFeat=[]-->, belongsTo=parr
298	rate experiments were performed with each cell type analyzed in tripli-, otherFeat=[]-->, belongsTo=parr
299	cate in each experiment., otherFeat=[]-->, belongsTo=parr
300	RESULTS, otherFeat=[]-->, belongsTo=parr
301	Pin1 Binds to the Cyclin E-Cdk2 Complex and Influences the Stability, otherFeat=[]-->, belongsTo=parr
302	of Cyclin E Protein--Cyclin E has been reported to undergo phospho-, otherFeat=[]-->, belongsTo=parr
303	rylation on several Ser and Thr residues in cells (12, 26, 33?36). At least, otherFeat=[]-->, belongsTo=parr
304	four of these sites are implicated in its degradation (12, 26, 33?36) (Fig., otherFeat=[]-->, belongsTo=parr
305	1A). Three sites, Thr62, Thr380, and Ser384 (shown in black), influence, otherFeat=[]-->, belongsTo=parr
306	the Cdc4-dependent turnover of cyclin E, whereas Ser372 (shown in, otherFeat=[]-->, belongsTo=parr
307	gray) may be involved in cyclin E degradation but by an ill defined, otherFeat=[]-->, belongsTo=parr
308	pathway (12, 26, 33). To determine whether endogenous Pin1 and cyclin, otherFeat=[]-->, belongsTo=parr
309	E interact, we used Xenopus interphase extracts and immunoprecipi-, otherFeat=[]-->, belongsTo=parr
310	tated Pin1 using our previously described anti-Xenopus Pin1 antibody, otherFeat=[]-->, belongsTo=parr
311	(29). As shown in Fig. 1B, endogenous cyclin E and Pin1 do interact in, otherFeat=[]-->, belongsTo=parr
312	this assay. Next, we transfected 293 cells with wild type (WT), Myc-, otherFeat=[]-->, belongsTo=parr
313	tagged cyclin E and subjected the extracts to GST pull-down assays, otherFeat=[]-->, belongsTo=parr
314	using GST alone, as a negative control, or GST-Pin1. As shown in the, otherFeat=[]-->, belongsTo=parr
315	first two lanes of Fig. 1C, GST-Pin1 also binds Myc-tagged cyclin E. We, otherFeat=[]-->, belongsTo=parr
316	next evaluated the importance of the three Cdc4-specific phosphoryla-, otherFeat=[]-->, belongsTo=parr
317	tion sites, Thr62, Thr380, and Ser384, in Pin1 binding by transfecting, otherFeat=[]-->, belongsTo=parr
318	Myc-tagged cyclin E cDNA constructs that were mutated in one or, otherFeat=[]-->, belongsTo=parr
319	more of the phosphorylation sites illustrated in Fig. 1A. Surprisingly,, otherFeat=[]-->, belongsTo=parr
320	mutation of Thr62, Thr380, or the combination of Thr62 and Thr380 does, otherFeat=[]-->, belongsTo=parrnote
321	not affect Pin1 binding (Fig. 1C, lanes 3? 8). On the other hand, muta-, otherFeat=[]-->, belongsTo=parr
322	tion of Ser384 nearly abolishes Pin1 binding (Fig. 1C, lanes 9 and 10)., otherFeat=[]-->, belongsTo=parr
323	Since Ser384 is phosphorylated by the Cdk2 component of the cyclin, otherFeat=[]-->, belongsTo=parr
324	E-Cdk2 heterodimer (12), this result suggested that Pin1 might bind to, otherFeat=[]-->, belongsTo=parr
325	the heterodimer in a manner that requires an active Cdk2 protein to, otherFeat=[]-->, belongsTo=parr
326	catalyze the phosphorylation of Ser384 on cyclin E. This hypothesis is, otherFeat=[]-->, belongsTo=parr
327	supported by the data in Fig. 1, D?F. First, when Pin1 is associated with, otherFeat=[]-->, belongsTo=parr
328	WT endogenous or Myc-tagged cyclin E, immunoblot analysis reveals, otherFeat=[]-->, belongsTo=parr
329	the presence of both cyclin E and Cdk2 (Fig. 1, D and E, lanes 1 and 2,, otherFeat=[]-->, belongsTo=parr
330	and F, lanes 1 and 2, respectively). Second, preincubation of the cells, otherFeat=[]-->, belongsTo=parr
331	containing Myc-cyclin E with the selective Cdk2 inhibitor roscovitine, otherFeat=[]-->, belongsTo=parr
332	prior to lysis and the addition of GST-Pin1 markedly attenuated the, otherFeat=[]-->, belongsTo=parr
333	binding of both cyclin E and Cdk2 (Fig. 1E, lanes 3 and 4). This result was, otherFeat=[]-->, belongsTo=parr
334	repeated three times with similar results, and quantitation of these, otherFeat=[]-->, belongsTo=parr
335	results can be found in supplemental Fig. 1. Although roscovitine is an, otherFeat=[]-->, belongsTo=parr
336	inhibitor of Cdk2 activity, it may also affect Cdk2 and/or cyclin E protein, otherFeat=[]-->, belongsTo=parr
337	levels (37). Thus, we evaluated Cdk2 and cyclin E levels by immunoblot-, otherFeat=[]-->, belongsTo=parr
338	ting extracts derived from cells that had been treated with either vehicle, otherFeat=[]-->, belongsTo=parr
339	(Me2SO)or30 M roscovitine. As shown in Fig. 1E (right panel), treat-, otherFeat=[]-->, belongsTo=parr
340	ment with 30 M roscovitine resulted in minimal changes in cyclin E or, otherFeat=[]-->, belongsTo=parr
341	Cdk2 levels. Collectively, these results suggest that the efficient binding, otherFeat=[]-->, belongsTo=parr
342	of Pin1 to cyclin E depends on Cdk2 activity and an intact Ser384 residue,, otherFeat=[]-->, belongsTo=parr
343	which is the site on cyclin E that is phosphorylated by Cdk2., otherFeat=[]-->, belongsTo=parr
344	To confirm the observation that phosphorylation of Ser384 is impor-, otherFeat=[]-->, belongsTo=parr
345	tant for Pin1 binding to cyclin E, we expressed the Myc-tagged cyclin E, otherFeat=[]-->, belongsTo=parr
346	constructs in 293 cells and examined Pin1 binding by immunoprecipi-, otherFeat=[]-->, belongsTo=parr
347	tation using antibody to the Myc tag. We then probed the immunopre-, otherFeat=[]-->, belongsTo=parr
348	cipitates with antibodies to Cdk2, Pin1, or Myc (to detect the immuno-, otherFeat=[]-->, belongsTo=parr
349	precipitated cyclin E proteins). In addition to WT, the T62A/T380A, otherFeat=[]-->, belongsTo=parr
350	double mutant, and the S384A mutant cyclin E proteins, we also, otherFeat=[]-->, belongsTo=parr
351	expressed a S372A mutant, since this site has also been implicated in, otherFeat=[]-->, belongsTo=parr
352	cyclin E protein turnover (12) and is a potential Ser(P)-Pro binding site, otherFeat=[]-->, belongsTo=parr
353	for Pin1. As shown in the left panel of Fig. 1F, Pin1 was present in the, otherFeat=[]-->, belongsTo=parr
354	immunoprecipitates of WT, T62A/T380A and S372A cyclin E but was, otherFeat=[]-->, belongsTo=parr
355	considerably less abundant in the immunoprecipitate containing the, otherFeat=[]-->, belongsTo=parr
356	S384A mutant protein. This was true, although a similar amount of, otherFeat=[]-->, belongsTo=parr
357	Cdk2 (as shown by Western blot) and of each cyclin E protein (based on, otherFeat=[]-->, belongsTo=parr
358	Coomassie staining) was immunoprecipitated (Fig. 1F, left). In addition,, otherFeat=[]-->, belongsTo=parr
359	each of the whole cell extracts contained similar amounts of Cdk2, Pin1,, otherFeat=[]-->, belongsTo=parr
360	and the appropriate cyclin E protein (Fig. 1F, right). Collectively, these, otherFeat=[]-->, belongsTo=parr
361	results show that Ser384 phosphorylation is important for the associa-, otherFeat=[]-->, belongsTo=parr
362	tion of Pin1 with cyclin E-Cdk2 but not for the assembly of the cyclin, otherFeat=[]-->, belongsTo=parr
363	E-Cdk2 complex., otherFeat=[]-->, belongsTo=parr
364	Since the phosphorylation of Thr380 and Ser384 on cyclin E are impor-, otherFeat=[]-->, belongsTo=parr
365	tant for regulating the Cdc4-mediated turnover of cyclin E protein (12,, otherFeat=[]-->, belongsTo=parr
366	26, 33), we next asked whether, in the absence of Pin1, cyclin E levels, otherFeat=[]-->, belongsTo=parr
367	were elevated. We subjected equal amounts of extracts from MEFs,, otherFeat=[]-->, belongsTo=parr
368	either WT or null for Pin1, to Western blotting and found that steady-, otherFeat=[]-->, belongsTo=parr
369	state levels of cyclin E were up-regulated 2-fold in the absence of Pin1, otherFeat=[]-->, belongsTo=parr
370	(Fig. 2A and supplemental Fig. 2). Interestingly, this up-regulation of, otherFeat=[]-->, belongsTo=parr
371	cyclin E in Pin1 null cells was accompanied by a considerable decrease in, otherFeat=[]-->, belongsTo=parr
372	the level of phosphorylated Thr380. Since phosphorylation of Thr380, otherFeat=[]-->, belongsTo=parr
373	promotes the degradation of cyclin E and this process is impaired in the, otherFeat=[]-->, belongsTo=parr
374	absence of Pin1, these results are compatible with a role for Pin1 in, otherFeat=[]-->, belongsTo=parr
375	regulating the turnover of cyclin E. Because the cyclin E gene is activated, otherFeat=[]-->, belongsTo=parr
376	during the progression from G0/G1 to S phase, we also questioned, otherFeat=[]-->, belongsTo=parr
377	whether the changes in cyclin E protein levels in the absence of Pin1, otherFeat=[]-->, belongsTo=parr
378	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
379	JANUARY 6, 2006 ? VOLUME 281 ? NUMBER 1, otherFeat=['U']-->, belongsTo=nota_cab_pie
380	JOURNAL OF BIOLOGICAL CHEMISTRY 243, otherFeat=[]-->, belongsTo=nota_cab_pie
381	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
382	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
383	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
384	de, otherFeat=[]-->, belongsTo=nota_cab_pie
385	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
386	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
387	on, otherFeat=[]-->, belongsTo=nota_cab_pie
388	September, otherFeat=[]-->, belongsTo=nota_cab_pie
389	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
390	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
391	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
392	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
393	from, otherFeat=[]-->, belongsTo=nota_cab_pie
394	FIGURE 1. Cyclin E binds to Pin1. A, phosphorylation sites on cyclin E protein that are involved in cyclin E turnover. Thr62, Thr380, and Ser384 have been described to influence cyclin E turnover, otherFeat=[]-->, belongsTo=fig_caption
395	promoted by Cdc4. Ser372 is also involved in cyclin E turnover by an unknown mechanism. B, endogenous Pin1 interacts with endogenous cyclin E. Xenopus Pin1 was immunoprecipitated,, otherFeat=[]-->, belongsTo=fig_caption
396	using anti-xPin1 antibody or rabbit IgG as a negative control, from Xenopus interphase extracts. Cyclin E was found only with the xPin1 co-immunoprecipitate. C, mutation of Ser384 impairs, otherFeat=[]-->, belongsTo=fig_caption
397	binding of cyclin E to Pin1. 293T cells were transfected with either Myc-tagged wild type cyclin E, cyclin ET62A, cyclin ET380A, cyclin ET62A/T380A, or cyclin ES384A prior to lysis. Lysates were, otherFeat=[]-->, belongsTo=fig_caption
398	subjected to GST pull-down with either GST beads as a negative control or GST-Pin1. Bound wild type and mutant cyclin E proteins were detected by Western blot, using anti-9E10 antibody, otherFeat=[]-->, belongsTo=fig_caption
399	for the Myc tag. Wild type cyclin E, cyclin ET62A, cyclin ET380A, and cyclin ET62A/T380A protein associated with GST-Pin1, whereas cyclin ES384A binding to Pin1 was attenuated. D, endogenous, otherFeat=[]-->, belongsTo=fig_caption
400	cyclin E-Cdk2 associates with Pin1. Nontransfected 293T cells were lysed and subjected to GST pull-down using GST alone or GST-Pin1. Both endogenous cyclin E and endogenous Cdk2 were, otherFeat=[]-->, belongsTo=fig_caption
401	found to be associated with GST-Pin1 but not with GST alone. E, Pin1 binds to the cyclin E-Cdk2 complex, and inhibition of Cdk2 with roscovitine impairs cyclin E-Cdk2 complex association, otherFeat=[]-->, belongsTo=fig_caption
402	with Pin1. Left, 293T cells were transfected with wild type cyclin E and treated with either Me2SO or 30 M roscovitine. 500 g of total protein from lysates were used to perform GST pull-down, otherFeat=[]-->, belongsTo=fig_caption
403	as described in A. Binding of cyclin E-Cdk2 to Pin1 was decreased in the presence of roscovitine. Right, 293T cells were transfected with wild type cyclin E prior to treatment with Me2SO vehicle, otherFeat=[]-->, belongsTo=fig_caption
404	or 30 M roscovitine. Whole cell lysates containing 35 g of total protein were subjected to Western blotting using the anti-HE12 antibody to detect cyclin E. As determined by densitometry,, otherFeat=[]-->, belongsTo=fig_caption
405	the ratio of cyclin E to actin in the Me2SO treatment is 1.1, whereas the ratio of cyclin E to actin under roscovitine treatment is 1.3. Similarly, the ratio of Cdk2 to actin in the Me2SO-treated lane, otherFeat=[]-->, belongsTo=fig_caption
406	is 2, whereas the ratio or Cdk2 to actin under roscovitine treatment is 2.1. No significant changes in the amounts of cyclin E and Cdk2 proteins were observed with 30 M roscovitine treatment, otherFeat=[]-->, belongsTo=fig_caption
407	relative to Me2SO treatment. F, Cdk2 remains associated with cyclin ES384A when Pin1 binding is impaired. Left, 293T cells were mock-transfected (lane 1) or transfected with Myc-tagged wild, otherFeat=[]-->, belongsTo=fig_caption
408	type cyclin E, cyclin ET62A/T380A, cyclin ET372A, or cyclin ES384A (lanes 2?5) prior to immunoprecipitation using the Myc tag with the anti-9E10 antibody. Bound proteins were resolved using, otherFeat=[]-->, belongsTo=fig_caption
409	anti-Cdk2 antibody and anti-Pin1 antibody. Cdk2 was bound to cyclin E and to each cyclin E mutant protein, whereas Pin1 binding was decreased with cyclin ES384A immunoprecipitate,, otherFeat=[]-->, belongsTo=fig_caption
410	confirming the result seen in B. Right,25 g of whole cell lysate for samples containing mock-transfected cells or cells transfected with Myc-tagged wild type cyclin E, cyclin ET62A/T380A, cyclin, otherFeat=[]-->, belongsTo=fig_caption
411	ET372A, or cyclin ES384A were subjected to Western blotting using the anti-Myc (9E10). Cyclin E was expressed in the appropriate lanes (lanes 2?5 but not lane 1) at comparable levels between, otherFeat=[]-->, belongsTo=fig_caption
412	samples. IB, immunoblotting; IP, immunoprecipitation., otherFeat=[]-->, belongsTo=fig_caption
413	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
414	244 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
415	VOLUME 281 ? NUMBER 1 ? JANUARY 6, 2006, otherFeat=['U']-->, belongsTo=nota_cab_pie
416	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
417	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
418	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
419	de, otherFeat=[]-->, belongsTo=nota_cab_pie
420	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
421	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
422	on, otherFeat=[]-->, belongsTo=nota_cab_pie
423	September, otherFeat=[]-->, belongsTo=nota_cab_pie
424	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
425	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
426	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
427	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
428	from, otherFeat=[]-->, belongsTo=nota_cab_pie
429	might be attributed to increased cyclin E mRNA. We performed real, otherFeat=[]-->, belongsTo=parr
430	time PCR on total RNA isolated from either pin1 / or pin1 / MEFs, otherFeat=[]-->, belongsTo=parr
431	and found that, although there appeared to be a slight increase in cyclin, otherFeat=[]-->, belongsTo=parr
432	EmRNAin pin1 / cells, the increase was not statistically significant, otherFeat=[]-->, belongsTo=parr
433	(Fig. 2B). We next probed the immunoblots for two components that, otherFeat=[]-->, belongsTo=parr
434	are known to interact with cyclin E to regulate its level and/or activity in, otherFeat=[]-->, belongsTo=parr
435	the cell. As shown in Fig. 2A, neither the level of Cdk2 nor the cyclin-, otherFeat=[]-->, belongsTo=parr
436	dependent kinase inhibitor p27 was changed in pin1 / cells. Finally, we, otherFeat=[]-->, belongsTo=parr
437	tested whether cyclin E is appropriately degraded in the absence of Pin1., otherFeat=[]-->, belongsTo=parr
438	Either Pin1 WT or Pin1 KO cells were treated with cycloheximide for, otherFeat=[]-->, belongsTo=parr
439	6 h. Fig. 2C shows that whereas inhibition of translation led to a 70%, otherFeat=[]-->, belongsTo=parr
440	decrease in cyclin E protein in the wild type cells, cyclin E turnover was, otherFeat=[]-->, belongsTo=parr
441	impaired in the pin1 / cells and showed only a small decrease ( 20%), otherFeat=[]-->, belongsTo=parr
442	6 h after the addition of cycloheximide (Fig. 2C). Based on these results,, otherFeat=[]-->, belongsTo=parr
443	we conclude that the primary way by which Pin1 regulates cyclin E is, otherFeat=[]-->, belongsTo=parr
444	likely to be at the protein rather than the mRNA level., otherFeat=[]-->, belongsTo=parr
445	The Absence of Pin1 in MEFs Results in Cell Cycle Defects--pin1 /, otherFeat=[]-->, belongsTo=parr
446	MEFs contain increased amounts of cyclin E (this study) and c-Myc (5),, otherFeat=[]-->, belongsTo=parr
447	and deregulation of either cyclin E or c-Myc results in defects in the cell, otherFeat=[]-->, belongsTo=parr
448	cycle (1). Cyclin E protein overexpression leads to an accelerated pro-, otherFeat=[]-->, belongsTo=parr
449	gression of G0/G1 to S phase coupled with an overall increase in the, otherFeat=[]-->, belongsTo=parr
450	length of the cell cycle, possibly due to an increase in the duration of S, otherFeat=[]-->, belongsTo=parr
451	phase (13, 14, 16, 38). Since cyclin E and c-Myc are deregulated in, otherFeat=[]-->, belongsTo=parr
452	pin1 / MEFs, we questioned whether cell cycle progression through S, otherFeat=[]-->, belongsTo=parr
453	phase might be compromised. First, as shown in Fig. 3A, the doubling, otherFeat=[]-->, belongsTo=parr
454	time of pin1 / MEFs is considerably slower than that of WT MEFs., otherFeat=[]-->, belongsTo=parr
455	This result is similar to that reported by others using MEFs isolated, otherFeat=[]-->, belongsTo=parr
456	from Pin1 null mice of a different genetic background than ours (28, 40)., otherFeat=[]-->, belongsTo=parr
457	Second, to evaluate whether this slower cell cycle progression might, otherFeat=[]-->, belongsTo=parr
458	reflect changes in S phase entry or progression, we pulse-labeled asyn-, otherFeat=[]-->, belongsTo=parr
459	chronously growing populations of MEFs with BrdUrd and quantified, otherFeat=[]-->, belongsTo=parr
460	its presence by immunocytochemical analysis of both pin1 / and, otherFeat=[]-->, belongsTo=parr
461	pin1 / MEFs. As shown in Fig. 3B (which represents three collective, otherFeat=[]-->, belongsTo=parr
462	experiments with each experiment containing n 300/cell type), the, otherFeat=[]-->, belongsTo=parr
463	absence of Pin1 in MEFs results in a statistically significant (p 0.0037, otherFeat=[]-->, belongsTo=parr
464	evaluated by Student's t test) decrease in the percentage of cells that, otherFeat=[]-->, belongsTo=parr
465	incorporate BrdUrd. This outcome could be explained by either a, otherFeat=[]-->, belongsTo=parr
466	decrease in the number of cells entering S phase during the time of the, otherFeat=[]-->, belongsTo=parr
467	BrdUrd pulse or an inability of cells that enter S phase to synthesize, otherFeat=[]-->, belongsTo=parr
468	DNA efficiently. To distinguish between these possibilities, we sub-, otherFeat=[]-->, belongsTo=parr
469	jected asynchronously growing cells to single parameter DNA profiling, otherFeat=[]-->, belongsTo=parr
470	to determine the percentage of cells in G1, S, and G2/M phases. Fig. 3C, otherFeat=[]-->, belongsTo=parr
471	(left and middle bars) shows that a higher percentage of pin1 / cells, otherFeat=[]-->, belongsTo=parr
472	are in G1 and S phases relative to WT cells. On the other hand, we found, otherFeat=[]-->, belongsTo=parr
473	a concomitant decrease in the percentage of pin1 / cells in G2/M as, otherFeat=[]-->, belongsTo=parr
474	seen in Fig. 3C (right bars). Thus, progression through the G1/S phases, otherFeat=[]-->, belongsTo=parr
475	FIGURE 2. Pin1 influences the stability of cyclin E protein. A, steady-state levels of cyclin E protein are elevated in Pin1 / MEFs. Equal amounts of protein (50 g) from pin1 / and, otherFeat=[]-->, belongsTo=fig_caption
476	pin1 / MEFs were analyzed for protein content by Western blotting (IB). Protein levels were detected using anti-cyclin E (M20), anti-phospho-cyclin E T380, anti-Cdk2, anti-p27,, otherFeat=[]-->, belongsTo=fig_caption
477	anti-Pin1, and anti- -actin as a loading control. Cyclin E levels were elevated 2-fold, and Thr(P)380 levels were markedly decreased in the absence of Pin1 as determined by, otherFeat=[]-->, belongsTo=fig_caption
478	densitometry and normalized to actin. Cdk2 and p27 levels were unchanged. Results are representative of five individual experiments. B, levels of cyclin E mRNA in the presence and, otherFeat=[]-->, belongsTo=fig_caption
479	absence of Pin1. Levels of cyclin E mRNA present in MEFs were quantified using real time PCR. Pin1 / MEFs displayed a slight increase in mRNA quantities, but this was not, otherFeat=[]-->, belongsTo=fig_caption
480	statistically significant. C, loss of Pin1 affects cyclin E protein stability. pin1 / and pin1 / MEFs were treated with 100 g/ml cycloheximide and harvested at 0 and 6 h. The middle, otherFeat=[]-->, belongsTo=fig_caption
481	panel represents a lower exposure of the right panel and was used for quantitation. Treatment with cycloheximide for 6 h led to a 67% decrease in cyclin E protein in pin1 / cells (left), otherFeat=[]-->, belongsTo=fig_caption
482	but to only a slight decrease in cyclin E protein in pin1 / cells (middle) as determined by densitometry and normalized to actin. KO, knock-out., otherFeat=[]-->, belongsTo=fig_caption
483	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
484	JANUARY 6, 2006 ? VOLUME 281 ? NUMBER 1, otherFeat=['U']-->, belongsTo=nota_cab_pie
485	JOURNAL OF BIOLOGICAL CHEMISTRY 245, otherFeat=[]-->, belongsTo=nota_cab_pie
486	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
487	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
488	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
489	de, otherFeat=[]-->, belongsTo=nota_cab_pie
490	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
491	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
492	on, otherFeat=[]-->, belongsTo=nota_cab_pie
493	September, otherFeat=[]-->, belongsTo=nota_cab_pie
494	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
495	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
496	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
497	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
498	from, otherFeat=[]-->, belongsTo=nota_cab_pie
499	of the cell cycle is impaired in pin1 / cells. These data are represent-, otherFeat=[]-->, belongsTo=parr
500	ative of three individual experiments, which each used 3 replicate, otherFeat=[]-->, belongsTo=parr
501	samples/cell type, and are highly statistically significant (see Fig. 3C)., otherFeat=[]-->, belongsTo=parr
502	The results in Fig. 3, B and C, are entirely consistent with data obtained, otherFeat=[]-->, belongsTo=parr
503	by Ekholm-Reed et al. (24), who overexpressed cyclin E in human cells, otherFeat=[]-->, belongsTo=parr
504	and support the concept that the absence of Pin1, at least in part due to, otherFeat=[]-->, belongsTo=parr
505	the deregulation of cyclin E, compromises the ability of cells to synthe-, otherFeat=[]-->, belongsTo=parr
506	size DNA and to progress through S phase., otherFeat=[]-->, belongsTo=parr
507	Immortilization of Pin1 / MEFs with a Dominant Negative p53, otherFeat=[]-->, belongsTo=parr
508	Results in a More Rapid Progression toward Genomic Instability--In, otherFeat=[]-->, belongsTo=parr
509	addition to cell cycle defects involving G1/S progression, cyclin E and, otherFeat=[]-->, belongsTo=parr
510	c-Myc protein deregulation is involved in the generation of chromo-, otherFeat=[]-->, belongsTo=parr
511	somal instability (16, 20, 22, 25, 27, 41?50) and correlates with tumor, otherFeat=[]-->, belongsTo=parr
512	formation (18 ?22, 25, 41, 51? 64). Because c-Myc and cyclin E levels are, otherFeat=[]-->, belongsTo=parr
513	increased in pin1 / cells, we evaluated the pin1 / cells for markers of, otherFeat=[]-->, belongsTo=parr
514	genomic instability. We first quantified the number of micronucleated, otherFeat=[]-->, belongsTo=parr
515	cells in early passage (passage 3) primary MEFs of the pin1 / and, otherFeat=[]-->, belongsTo=parr
516	pin1 / genotypes. As shown in the left pair of bars in Fig. 4B, micro-, otherFeat=[]-->, belongsTo=parr
517	nuclei occur in about 5% of the WT MEFs, and, although the Pin1 null, otherFeat=[]-->, belongsTo=parr
518	cells show only about a 2% increase in this percentage (to about 7%), the, otherFeat=[]-->, belongsTo=parr
519	difference is highly statistically significant (p 0.00000158, Student's t, otherFeat=[]-->, belongsTo=parr
520	test, n 1000). These data show that even at passage 3, primary MEFs, otherFeat=[]-->, belongsTo=parr
521	form micronuclei and that the absence of Pin1 exacerbates this defect., otherFeat=[]-->, belongsTo=parr
522	To determine whether these DNA changes result in aneuploidy, we, otherFeat=[]-->, belongsTo=parr
523	subjected asynchronously cycling populations of passage 3 primary, otherFeat=[]-->, belongsTo=parr
524	pin1 / and pin1 / cells to single parameter DNA profiling but, otherFeat=[]-->, belongsTo=parr
525	found that both populations of primary cells displayed normal distribu-, otherFeat=[]-->, belongsTo=parr
526	tion in each phase of the cell cycle as measured by fluorescence-acti-, otherFeat=[]-->, belongsTo=parr
527	vated cell sorting (Fig. 4C, top panels; data represent three individual, otherFeat=[]-->, belongsTo=parr
528	experiments performed with 3 replicates/cell type in each experiment)., otherFeat=[]-->, belongsTo=parr
529	Inactivation of p53 will immortalize mouse cells and lead to progres-, otherFeat=[]-->, belongsTo=parr
530	sive genomic instability (27, 65, 66). To determine whether this p53, otherFeat=[]-->, belongsTo=parr
531	inactivation-dependent process would also be sensitized by the absence, otherFeat=[]-->, belongsTo=parr
532	of Pin1, we infected the passage 3 pin1 / and pin1 / MEFs with a, otherFeat=[]-->, belongsTo=parr
533	retrovirus encoding a dominant-negative p53 that expresses a truncated, otherFeat=[]-->, belongsTo=parr
534	version of the p53 protein termed p53DD (Fig. 4A). p53DD is the C-ter-, otherFeat=[]-->, belongsTo=parr
535	minal portion of p53 containing amino acids 302?390 and functions to, otherFeat=[]-->, belongsTo=parr
536	prevent transcription by inhibiting p53 tetramer formation (67). Upon, otherFeat=[]-->, belongsTo=parr
537	stable expression of p53DD, we obtained cell lines that continue to grow, otherFeat=[]-->, belongsTo=parr
538	for greater than 50 passages (data not shown), which is many more than, otherFeat=[]-->, belongsTo=parr
539	the 4 ? 6 passages number that is typical of primary MEFs. Since Pin1, otherFeat=[]-->, belongsTo=parr
540	generally functions in the timing of events that occur during cell prolif-, otherFeat=[]-->, belongsTo=parr
541	eration (30, 68, 69) and we found an increase in micronuclei formation, otherFeat=[]-->, belongsTo=parr
542	even in passage 3 primary MEFs, we wanted to evaluate the effect of Pin1, otherFeat=[]-->, belongsTo=parr
543	FIGURE 3. The absence of Pin1 in MEFs results in cell cycle defects. A, Pin1 null MEFs have longer doubling times compared with wild type. Equal numbers of pin1 / and pin1 /, otherFeat=[]-->, belongsTo=fig_caption
544	MEFs were plated into 6-well dishes. Cells from duplicate wells per each cell type were trypsinized and counted every 24 h for 6 days. The graph is representative of five similar, otherFeat=[]-->, belongsTo=fig_caption
545	independent experiments. pin1 / cells display a growth deficiency. B, Pin1 null cells undergo DNA synthesis more slowly as measured by BrdUrd incorporation. Cells were plated, otherFeat=[]-->, belongsTo=fig_caption
546	on glass coverslips in equal numbers. Cells were pulsed with BrdUrd prior to fixation and staining with anti-BrdUrd antibody to assess the number of cells which have incorporated, otherFeat=[]-->, belongsTo=fig_caption
547	BrdUrd. Cells lacking Pin1 incorporated less BrdUrd. This experiment was performed three times, counting n 300 per cell type per experiment, and is statistically significant as, otherFeat=[]-->, belongsTo=fig_caption
548	analyzed by the Student's t test (p 0.0037). C, pin1 / MEFs progress more slowly through G1/S phases. Asynchronous populations of pin1, otherFeat=[]-->, belongsTo=fig_caption
549	/ and pin1 / MEFs were trypsinized, otherFeat=[]-->, belongsTo=fig_caption
550	and fixed for single parameter fluorescence-activated cell sorting analysis. Cells were resuspended in PBS containing RNase and stained with propidium iodide to analyze DNA, otherFeat=[]-->, belongsTo=fig_caption
551	content. This experiment represents three individual experiments using 3 replicates/cell type/experiment and is statistically significant as analyzed by Student's t test (see right, otherFeat=[]-->, belongsTo=fig_caption
552	panel for p value). The graph represents the total percentage of cells in G1, S, and G2/M phases. The Pin1, otherFeat=[]-->, belongsTo=fig_caption
553	/ MEFs show a greater percentage of cells in G1 and S phases than pin1, otherFeat=[]-->, belongsTo=??
554	/, otherFeat=[]-->, belongsTo=??
555	MEFs, and a concomitant decrease in G2/M occurs in the pin1, otherFeat=[]-->, belongsTo=??
556	/ population. ko, knock-out., otherFeat=[]-->, belongsTo=parrnote
557	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
558	246 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
559	VOLUME 281 ? NUMBER 1 ? JANUARY 6, 2006, otherFeat=['U']-->, belongsTo=nota_cab_pie
560	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
561	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
562	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
563	de, otherFeat=[]-->, belongsTo=nota_cab_pie
564	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
565	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
566	on, otherFeat=[]-->, belongsTo=nota_cab_pie
567	September, otherFeat=[]-->, belongsTo=nota_cab_pie
568	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
569	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
570	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
571	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
572	from, otherFeat=[]-->, belongsTo=nota_cab_pie
573	absence as early as possible after the stable integration of p53DD. This, otherFeat=[]-->, belongsTo=parr
574	cell selection and verification of protein expression required three cell, otherFeat=[]-->, belongsTo=parr
575	passages, so MEFs at passage 3 after infection of the retrovirus express-, otherFeat=[]-->, belongsTo=parr
576	ing p53DD (or a total of six passages altogether) were used in all subse-, otherFeat=[]-->, belongsTo=parr
577	quent experiments. Fig. 4A shows that p53DD is expressed to a similar, otherFeat=[]-->, belongsTo=parr
578	extent in both pin1 / and pin1 / MEFs and that this does not alter, otherFeat=[]-->, belongsTo=parr
579	the expression of endogenous p53. Expression of p53DD caused a, otherFeat=[]-->, belongsTo=parr
580	marked increase in micronuclei formation in both Pin1-replete and, otherFeat=[]-->, belongsTo=parr
581	-null cells, although the absence of Pin1 increased this from about 17.5, otherFeat=[]-->, belongsTo=parr
582	to 28% of the cells (p 0.000000287, Student's t test, n 1000) (Fig. 4B,, otherFeat=[]-->, belongsTo=parr
583	right pair of bars). This increase in the percentage of micronucleated, otherFeat=[]-->, belongsTo=parr
584	cells is mirrored by a change in the DNA profile in both pin1 / and, otherFeat=[]-->, belongsTo=parr
585	pin1 / cells expressing p53DD (Fig. 4C, bottom panels; data represent, otherFeat=[]-->, belongsTo=parr
586	three individual experiments performed with 3 replicates/cell type in, otherFeat=[]-->, belongsTo=parr
587	each experiment). Aneuploidy is evident in both cell populations but is, otherFeat=[]-->, belongsTo=parr
588	considerably more advanced in the cells null for Pin1 (e.g. 47% versus, otherFeat=[]-->, belongsTo=parr
589	62% of cells with 4 N DNA content in pin1 / and pin1 / cells,, otherFeat=[]-->, belongsTo=parr
590	respectively; Fig. 4C, lanes M5 and M6, compare rows B and D). These, otherFeat=[]-->, belongsTo=parr
591	results support our conjecture that the absence of Pin1 promotes, otherFeat=[]-->, belongsTo=parr
592	genomic instability in cells in a more accelerated fashion than in the, otherFeat=[]-->, belongsTo=parr
593	presence of Pin1 and that the loss of Pin1 acts in concert with the loss of, otherFeat=[]-->, belongsTo=parr
594	p53 function to promote this effect., otherFeat=[]-->, belongsTo=parr
595	The Absence of Pin1 Sensitizes Cells to More Aggressive Transforma-, otherFeat=[]-->, belongsTo=parr
596	tion and Tumorigenesis in Vivo--Studies from the cyclin E knock-out, otherFeat=[]-->, belongsTo=parr
597	mouse indicate that the loss of cyclin E protects mouse cells from Ras-, otherFeat=[]-->, belongsTo=parr
598	mediated transformation (70). Because cyclin E is up-regulated in, otherFeat=[]-->, belongsTo=parr
599	pin1 / cells and these cells show increased genomic instability at early, otherFeat=[]-->, belongsTo=parr
600	passage after immortalization, we hypothesized that deletion of Pin1, otherFeat=[]-->, belongsTo=parr
601	might lead to changes in the genome that are sufficient to promote more, otherFeat=[]-->, belongsTo=parr
602	advanced cellular transformation of MEFs. In support of this hypothe-, otherFeat=[]-->, belongsTo=parr
603	sis, we have previously reported that c-Myc is stabilized in the absence, otherFeat=[]-->, belongsTo=parr
604	of Pin1 (5). Since Myc and Ras collaborate to transform rodent cells (47),, otherFeat=[]-->, belongsTo=parr
605	it seemed plausible that the addition of oncogenic Ras alone to Pin1 /, otherFeat=[]-->, belongsTo=parr
606	primary MEFs might induce spontaneous transformation. However,, otherFeat=[]-->, belongsTo=parr
607	expression of RasG12V alone in pin1 / cells caused the early passage, otherFeat=[]-->, belongsTo=parr
608	primary MEFs to senesce, similar to what we found to occur in wild type, otherFeat=[]-->, belongsTo=parr
609	MEFs (data not shown)., otherFeat=[]-->, belongsTo=parr
610	Previous reports indicate that whereas cooperating oncogenes such, otherFeat=[]-->, belongsTo=parr
611	as Myc and Ras can spontaneously transform rodent cells, several lines, otherFeat=[]-->, belongsTo=parr
612	of evidence suggest that the expression of these oncogenes co-selects for, otherFeat=[]-->, belongsTo=parr
613	the loss of p53 and INK4a (71, 72). Boehm et al. (73) recently demon-, otherFeat=[]-->, belongsTo=parr
614	strated that p53DD collaborates with Myc and Ras to efficiently trans-, otherFeat=[]-->, belongsTo=parr
615	form MEFs. These authors demonstrated that whereas p53DD and Ras, otherFeat=[]-->, belongsTo=parr
616	alone can result in transformation of wild type MEFs, the transforma-, otherFeat=[]-->, belongsTo=parr
617	FIGURE 4. Immortalization of Pin1 / MEFs with a dominant negative p53 results in a more rapid progression toward genomic instability. A, creation of stable cell line, otherFeat=[]-->, belongsTo=fig_caption
618	expressing p53DD. Primary MEFs were infected with retrovirus containing the dominant negative form of p53, p53DD. Cell lysates were analyzed by Western blotting (IB) and probed, otherFeat=[]-->, belongsTo=fig_caption
619	for the expression of p53DD using anti-p53 antibody. p53DD is expressed as a truncated form of p53 protein and runs at a lower Mr (middle) than native p53 protein (top). B, Pin1 null, otherFeat=[]-->, belongsTo=fig_caption
620	cells show increased formation of micronuclei. Cells were plated on coverslips prior to methanol fixation and staining with 4 ,6-diamidino-2-phenylindole. Cells were visualized by, otherFeat=[]-->, belongsTo=fig_caption
621	fluorescence microscopy and scored by counting total number of cells ( 300 cells counted per cell type in three individual experiments; see figure for p value) compared with the, otherFeat=[]-->, belongsTo=fig_caption
622	number of cells containing micronuclei. C, immortalization of Pin1 / MEFs with a dominant negative p53 results in a more rapid progression toward aneuploidy. Primary MEFs, otherFeat=[]-->, belongsTo=fig_caption
623	(passage 3) and p53DD MEFs (passage 3 after the addition of p53DD) were fixed and stained with propidium iodide prior to analysis by single parameter flow cytometry to determine, otherFeat=[]-->, belongsTo=fig_caption
624	DNA content. Primary pin1 / and pin1 / MEFs did not display aneuploidy, whereas immortalized cells of both genotypes resulted in a higher number of cells containing4NDNA, otherFeat=[]-->, belongsTo=fig_caption
625	content indicating aneuploidy. pin1 / cells contained the highest percentage of cells with 4 N DNA content (compare rows B and D, M5 and M6). KO, knock-out., otherFeat=[]-->, belongsTo=fig_caption
626	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
627	JANUARY 6, 2006 ? VOLUME 281 ? NUMBER 1, otherFeat=['U']-->, belongsTo=nota_cab_pie
628	JOURNAL OF BIOLOGICAL CHEMISTRY 247, otherFeat=[]-->, belongsTo=nota_cab_pie
629	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
630	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
631	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
632	de, otherFeat=[]-->, belongsTo=nota_cab_pie
633	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
634	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
635	on, otherFeat=[]-->, belongsTo=nota_cab_pie
636	September, otherFeat=[]-->, belongsTo=nota_cab_pie
637	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
638	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
639	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
640	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
641	from, otherFeat=[]-->, belongsTo=nota_cab_pie
642	tion of these cells can be accelerated by the addition of another onco-, otherFeat=[]-->, belongsTo=parr
643	protein, such as Myc. Thus, MEFs expressing p53DD and Ras were less, otherFeat=[]-->, belongsTo=parr
644	efficiently transformed compared with MEFs expressing p53DD, Ras,, otherFeat=[]-->, belongsTo=parr
645	and Myc, and the MEFs only containing p53DD and Ras formed signifi-, otherFeat=[]-->, belongsTo=parr
646	cantly fewer colonies in soft agar than MEFs containing p53DD, Ras, and, otherFeat=[]-->, belongsTo=parr
647	Myc (73). These results demonstrate that the serial introduction of, otherFeat=[]-->, belongsTo=parr
648	oncogenic components (p53DD, Ras, Myc) can result in an additive, otherFeat=[]-->, belongsTo=parr
649	effect on transformation. In Fig. 5A, we demonstrate that expression of, otherFeat=[]-->, belongsTo=parr
650	oncogenic Ras results in the ability of p53DD, pin1, otherFeat=[]-->, belongsTo=parr
651	/ MEFs to form, otherFeat=[]-->, belongsTo=parr
652	colonies in soft agar to a higher extent than pin1 / MEFs expressing, otherFeat=[]-->, belongsTo=parr
653	the same components (Fig. 5A). The fact that these latter cells would, otherFeat=[]-->, belongsTo=parr
654	form colonies to a markedly lower extent is consistent with the conclu-, otherFeat=[]-->, belongsTo=parr
655	sions of the study by Boehm et al. (73). These results show that deletion, otherFeat=[]-->, belongsTo=parr
656	of Pin1 can sensitize cells to a more aggressive Ras-induced transforma-, otherFeat=[]-->, belongsTo=parr
657	tion relative to pin1 / cells. We postulate that the deregulation of cell, otherFeat=[]-->, belongsTo=parr
658	cycle proteins, such as cyclin E and c-Myc, in pin1 / cells may be at, otherFeat=[]-->, belongsTo=parr
659	least in part responsible for this difference., otherFeat=[]-->, belongsTo=parr
660	In order to test the in vivo relevance of Pin1 depletion in tumor, otherFeat=[]-->, belongsTo=parr
661	formation, we injected immunocompromised mice with four cell types:, otherFeat=[]-->, belongsTo=parr
662	pin1 / MEFs expressing p53DD alone, pin1 / MEFs expressing, otherFeat=[]-->, belongsTo=parr
663	p53DD and Ras, pin1, otherFeat=[]-->, belongsTo=parr
664	/ MEFs expressing p53DD alone, and pin1, otherFeat=[]-->, belongsTo=parr
665	/, otherFeat=[]-->, belongsTo=parrnote
666	MEFs expressing p53DD and Ras. Consistent with our demonstration, otherFeat=[]-->, belongsTo=parr
667	that loss of Pin1 promotes a more aggressive type of transformation in, otherFeat=[]-->, belongsTo=parr
668	conjunction with loss of p53 function and oncogenic Ras when com-, otherFeat=[]-->, belongsTo=parr
669	pared with WT cells, we found the same to be true in our tumorigenesis, otherFeat=[]-->, belongsTo=parr
670	assay. Whereas both pin1 / MEFs expressing p53DD and Ras and, otherFeat=[]-->, belongsTo=parr
671	pin1 / MEFs expressing p53DD and Ras supported tumor formation in, otherFeat=[]-->, belongsTo=parr
672	immunocompromised mice (Fig. 5B), the tumors that developed from, otherFeat=[]-->, belongsTo=parr
673	the pin1 / MEFs expressing p53DD and Ras were significantly larger in, otherFeat=[]-->, belongsTo=parr
674	volume (Fig. 5C) and size (Fig. 5D) than their WT counterparts just 1, otherFeat=[]-->, belongsTo=parr
675	week postinjection. In fact, two of the five SCID mice injected with, otherFeat=[]-->, belongsTo=parr
676	pin1 / MEFs expressing p53DD alone formed tumors (Fig. 5B). These, otherFeat=[]-->, belongsTo=parr
677	data support the idea that cells can exhibit varying levels of oncogenic, otherFeat=[]-->, belongsTo=parr
678	potency based on their ability to select for specific mutations, such as, otherFeat=[]-->, belongsTo=parr
679	loss of p53 or Myc overexpression. Thus, Ras is more efficient in causing, otherFeat=[]-->, belongsTo=parr
680	tumorigenesis when Pin1 null MEFs are immortalized by expression of, otherFeat=[]-->, belongsTo=parr
681	p53DD but not when cells contain normal levels of Pin1. Our study, otherFeat=[]-->, belongsTo=parr
682	demonstrates that the loss of Pin1, perhaps due to a deregulation of Pin1, otherFeat=[]-->, belongsTo=parr
683	protein substrates such as cyclin E and Myc, has a profound effect on, otherFeat=[]-->, belongsTo=parr
684	proliferation, genomic instability, and tumorigenesis of MEFs., otherFeat=[]-->, belongsTo=parr
685	DISCUSSION, otherFeat=[]-->, belongsTo=parr
686	Here we show that cyclin E is a Pin1-binding protein and that the, otherFeat=[]-->, belongsTo=parr
687	steady-state level of cyclin E protein is increased in Pin1-null MEFs, otherFeat=[]-->, belongsTo=parr
688	isolated from mice in which the pin1 gene deletion is maintained in an, otherFeat=[]-->, belongsTo=parr
689	isogenic C57BL6 background. This increased protein level seems to be, otherFeat=[]-->, belongsTo=parr
690	FIGURE 5. Pin1 depletion sensitizes cells to transformation and can initiate tumorigenesis in vivo. A, Pin1-null cells are more readily transformed in the presence of Ras. pin1 /, otherFeat=[]-->, belongsTo=fig_caption
691	or pin1 / cells were stably infected with p53DD alone or p53DD and RasG12V retrovirus as indicated. 50,000 cells were plated in 0.3% agar, incubated for 21 days, and assessed for, otherFeat=[]-->, belongsTo=fig_caption
692	colony growth in soft agar. The mean and S.D. represent two independent experiments performed in triplicate (p 0.0001, Student's t test). The pin1 / p53DD Ras cells formed, otherFeat=[]-->, belongsTo=fig_caption
693	colonies in soft agar to a much greater extent than pin1 / p53DD Ras cells. B, summary of in vivo tumor formation. Mice were injected with pin1, otherFeat=[]-->, belongsTo=fig_caption
694	/ and pin1 / MEF expressing, otherFeat=[]-->, belongsTo=??
695	p53DD alone and pin1, otherFeat=[]-->, belongsTo=??
696	/ and pin1 / MEF expressing p53 DD and RasG12V (n 5 mice for each cell type). Injections were performed subcutaneously at the shoulder. C, mice injected, otherFeat=[]-->, belongsTo=parrnote
697	with pin1 / MEF expressing p53DD and RasG12V formed more aggressive tumors than pin1 / MEF expressing p53DD and RasG12V. Tumor volume analysis represents the average, otherFeat=[]-->, belongsTo=parrnote
698	tumor volume calculated from the five mice injected per cell type (p 0.0019; compare pin1 / and pin1 / MEF expressing p53DD and Ras G12V by Student's t test). The volumes (in, otherFeat=[]-->, belongsTo=parrnote
699	mm3) of tumors derived from pin1 / MEFs are significantly larger than tumors derived from pin1 / MEFs. D, the tumors derived from pin1 / MEFs were markedly larger than the, otherFeat=[]-->, belongsTo=parrnote
700	tumors derived from pin1 / MEFs. Images are representative of tumor size (indicated with average diameter; top) prior to and after excision (bottom)., otherFeat=[]-->, belongsTo=parrnote
701	Loss of Pin1 Deregulates Cyclin E, otherFeat=[]-->, belongsTo=nota_cab_pie
702	248 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
703	VOLUME 281 ? NUMBER 1 ? JANUARY 6, 2006, otherFeat=['U']-->, belongsTo=nota_cab_pie
704	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
705	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
706	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
707	de, otherFeat=[]-->, belongsTo=nota_cab_pie
708	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
709	Oncol?gicas, otherFeat=[]-->, belongsTo=nota_cab_pie
710	on, otherFeat=[]-->, belongsTo=nota_cab_pie
711	September, otherFeat=[]-->, belongsTo=nota_cab_pie
712	11,, otherFeat=[]-->, belongsTo=nota_cab_pie
713	2006, otherFeat=[]-->, belongsTo=nota_cab_pie
714	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
715	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
716	from, otherFeat=[]-->, belongsTo=nota_cab_pie
717	predominantly due to a decreased rate of cyclin E turnover. Addition-, otherFeat=[]-->, belongsTo=parr
718	ally, we show that Pin1 binds to the cyclin E-Cdk2 complex in a phos-, otherFeat=[]-->, belongsTo=parr
719	phorylation-dependent manner; the interaction requires an intact, otherFeat=[]-->, belongsTo=parr
720	Ser384 and is decreased by inhibition of Cdk2 activity. Although anti-, otherFeat=[]-->, belongsTo=parr
721	bodies that specifically recognize Ser(P)384 are not available, these data, otherFeat=[]-->, belongsTo=parr
722	argue that Ser384 may need to be phosphorylated in order for Pin1 to, otherFeat=[]-->, belongsTo=parr
723	efficiently bind the cyclin E-Cdk2 complex. The fact that Ser384 is phos-, otherFeat=[]-->, belongsTo=parr
724	phorylated by the Cdk2 component of the cyclin E-Cdk2 complex (12), otherFeat=[]-->, belongsTo=parr
725	and this phosphorylation is required for SCFCdc4-dependent degrada-, otherFeat=[]-->, belongsTo=parr
726	tion of cyclin E (12, 33, 35) but not for formation of the cyclin E-Cdk2, otherFeat=[]-->, belongsTo=parr
727	complex suggests the possibility that Pin1 binds to cyclin E and facili-, otherFeat=[]-->, belongsTo=parr
728	tates its ubiquitylation in preparation for its degradation via the 26 S, otherFeat=[]-->, belongsTo=parr
729	proteosome. This hypothesis is consistent with our previous study,, otherFeat=[]-->, belongsTo=parr
730	which demonstrated that Myc is a phosphorylation-dependent Pin1-, otherFeat=[]-->, belongsTo=parr
731	binding protein, the loss of Pin1 increased the steady state level of Myc, otherFeat=[]-->, belongsTo=parr
732	in MEFs, and the deregulation of Myc protein was due to a decreased, otherFeat=[]-->, belongsTo=parr
733	rate of its ubiquitylation and degradation (5). Since both cyclin E and, otherFeat=[]-->, belongsTo=parr
734	Myc are degraded by SCFCdc4 (6, 7, 33, 36, 74) and the "phosphode-, otherFeat=[]-->, belongsTo=parr
735	grons" recognized by Cdc4 and Pin1 are remarkably similar, our data, otherFeat=[]-->, belongsTo=parr
736	support the idea that a mechanism with many common features con-, otherFeat=[]-->, belongsTo=parr
737	trols the degradation of these two important cell regulatory proteins., otherFeat=[]-->, belongsTo=parr
738	On the other hand the precise way in which Pin1 participates in the, otherFeat=[]-->, belongsTo=parr
739	Cdc4-dependent degradation of cyclin E and Myc may be different. In, otherFeat=[]-->, belongsTo=parr
740	the case of Myc, the binding of Pin1 required phosphorylation of Thr58,, otherFeat=[]-->, belongsTo=parr
741	which has been proposed to constitute the primary Cdc4 recognition, otherFeat=[]-->, belongsTo=parr
742	site (6, 7, 74). Pin1 binding resulted in a conformational change in the, otherFeat=[]-->, belongsTo=parr
743	phosphodegron that presented Ser(P)62 as a substrate for PP2A (5)., otherFeat=[]-->, belongsTo=parr
744	Since ubiquitylated Myc contained only Thr(P)58, we concluded that, otherFeat=[]-->, belongsTo=parr
745	Pin1/PP2A-mediated dephosphorylation of Ser62 was important for, otherFeat=[]-->, belongsTo=parr
746	ubiquitylation and/or degradation (5). We show in the current study, otherFeat=[]-->, belongsTo=parr
747	that Pin1 binding does not require phosphorylation of Thr380 in cyclin E,, otherFeat=[]-->, belongsTo=parr
748	which is equivalent to Thr58 in Myc and also constitutes the primary, otherFeat=[]-->, belongsTo=parr
749	binding site for Cdc4 based on crystallographic analysis of Cdc4 in com-, otherFeat=[]-->, belongsTo=parr
750	plex with a peptide mimic of the cyclin E phosphodegron (3). Rather,, otherFeat=[]-->, belongsTo=parr
751	effective Pin1 binding to cyclin E is dependent on phosphorylation of, otherFeat=[]-->, belongsTo=parr
752	Ser384, which is in a location in the phosphodegron equivalent to Ser62 in, otherFeat=[]-->, belongsTo=parrnote
753	Myc. Regardless of these differences, the outcome of Pin1 binding to a, otherFeat=[]-->, belongsTo=parr
754	phosphorylated form of cyclin E and Myc is the same, since this inter-, otherFeat=[]-->, belongsTo=parr
755	action promotes Cdc4-dependent degradation in each case. Clarifying, otherFeat=[]-->, belongsTo=parr
756	the precise mechanism by which Pin1 facilitates the turnover of cyclin E, otherFeat=[]-->, belongsTo=parr
757	will be an important future endeavor., otherFeat=[]-->, belongsTo=parr
758	There is considerable information demonstrating a role for Pin1 in, otherFeat=[]-->, belongsTo=parr
759	the G0/G1 to S phase transition, and many of its proposed targets are, otherFeat=[]-->, belongsTo=parr
760	deregulated in human cancer (4, 5, 75?91). Because cyclin E and Myc, otherFeat=[]-->, belongsTo=parr
761	levels (5) are increased in MEFs null for Pin1 and deregulation of cyclin, otherFeat=[]-->, belongsTo=parr
762	E and Myc have been linked to processes involved in tumorigenesis (16,, otherFeat=[]-->, belongsTo=parr
763	20, 22, 25, 27, 41? 44, 46, 92), such as hyperproliferation and genomic, otherFeat=[]-->, belongsTo=parr
764	instability, we evaluated whether Pin1 null MEFs would be more sus-, otherFeat=[]-->, belongsTo=parr
765	ceptible to Ras-dependent transformation after they were immortalized, otherFeat=[]-->, belongsTo=parr
766	by conditional inactivation of p53. Indeed, we found that the absence of, otherFeat=[]-->, belongsTo=parr
767	Pin1 led to increased genomic instability, and in the presence of p53DD, otherFeat=[]-->, belongsTo=parr
768	these cells could be more aggressively transformed by oncogenic Ras, otherFeat=[]-->, belongsTo=parr
769	than wild type cells. Thus, at least in a C57BL6 genetic background, loss, otherFeat=[]-->, belongsTo=parr
770	of function of Pin1 sensitizes MEFs to more extensive and aggressive, otherFeat=[]-->, belongsTo=parr
771	transformation and tumorigenesis., otherFeat=[]-->, belongsTo=parr
772	Contrary to our results, loss of Pin1 has been suggested to decrease, otherFeat=[]-->, belongsTo=parr
773	sensitivity to cancer (81). It would follow that mice null for Pin1 might, otherFeat=[]-->, belongsTo=parr
774	be "protected" from oncogene-mediated tumorigenesis. Wulf et al. (89), otherFeat=[]-->, belongsTo=parr
775	tested this hypothesis by breeding FVB mice containing a transgene, otherFeat=[]-->, belongsTo=parr
776	expressing MMTV-Neu or MMTV-H-Ras with Pin1-null mice main-, otherFeat=[]-->, belongsTo=parr
777	tained in SvJae/129/C57BL6 mixed genetic background. MMTV-Neu, otherFeat=[]-->, belongsTo=parr
778	and MMTV-Ras have previously been shown to form spontaneous, otherFeat=[]-->, belongsTo=parr
779	breast tumors, resulting in decreased survival (93, 94). Wulf et al. (67), otherFeat=[]-->, belongsTo=parr
780	hypothesized that the resulting pin1 / /ras or pin1 / /neu transgenic, otherFeat=[]-->, belongsTo=parr
781	populations would be less susceptible to tumor formation and have an, otherFeat=[]-->, belongsTo=parr
782	increased chance of survival. These authors found that the absence of, otherFeat=[]-->, belongsTo=parr
783	Pin1 prevented oncogenic Neu or Ras from inducing breast cancer and, otherFeat=[]-->, belongsTo=parr
784	attributed these effects to cyclin D deregulation due to the loss of Pin1., otherFeat=[]-->, belongsTo=parr
785	Correlating with these observations, MEFs from the Pin1 null mice, otherFeat=[]-->, belongsTo=parr
786	maintained in the SVJae/129/C57BL6 background demonstrated, otherFeat=[]-->, belongsTo=parr
787	decreased protein levels of cyclin D1, c-Jun, and -catenin (4, 79, 88)., otherFeat=[]-->, belongsTo=parr
788	Therefore, it is difficult to contend that depletion of Pin1 cannot also, otherFeat=[]-->, belongsTo=parr
789	decrease susceptibility to mammary cancer, at least in a FBV/129/SvJae/, otherFeat=[]-->, belongsTo=parr
790	C57BL6 mixed genetic background., otherFeat=[]-->, belongsTo=parr
791	How can one reconcile these apparently opposite outcomes of the, otherFeat=[]-->, belongsTo=parr
792	role for Pin1 in cancer? The differences in protein expression in the, otherFeat=[]-->, belongsTo=parr
793	SVJae/129/C57BL6 mixed background (e.g. c-Jun, cyclin D1, and, otherFeat=[]-->, belongsTo=parr
794	-catenin) versus the isogenic C57BL6 (e.g. c-Myc and cyclin E) predict, otherFeat=[]-->, belongsTo=parr
795	differential sensitivity of the two lines of mice to oncogenesis, and it is, otherFeat=[]-->, belongsTo=parr
796	highly unlikely that these are the only differences that exist between cells, otherFeat=[]-->, belongsTo=parr
797	derived from these two genetic backgrounds. It is certainly not surpris-, otherFeat=[]-->, belongsTo=parr
798	ing that identical mutations can have different biological effects when, otherFeat=[]-->, belongsTo=parr
799	studied in distinct genetic backgrounds, which probably reflect differ-, otherFeat=[]-->, belongsTo=parr
800	ential expression of potential modifier genes, and this could explain the, otherFeat=[]-->, belongsTo=parr
801	conundrum of the role of Pin1 in tumorigenesis. This idea was clearly, otherFeat=[]-->, belongsTo=parr
802	demonstrated by Reilly et al. (95), who proposed that the susceptibility, otherFeat=[]-->, belongsTo=parr
803	of mice to development of astrocytoma is intimately linked to the, otherFeat=[]-->, belongsTo=parr
804	genetic strain of mice in which the experiments are conducted. These, otherFeat=[]-->, belongsTo=parr
805	authors engineered different strains of mice (namely 129, 129/C57BL6,, otherFeat=[]-->, belongsTo=parr
806	129/SvJae, or C57BL6) to express mutant Nf1 or TP53 (95). The mutant, otherFeat=[]-->, belongsTo=parr
807	mice maintained in the isogenic C57BL6 genetic background always, otherFeat=[]-->, belongsTo=parr
808	formed astrocytomas. Conversely, the same mutations in mice main-, otherFeat=[]-->, belongsTo=parr
809	tained in 129, 129/C57BL6, or 129/SvJae backgrounds did not result in, otherFeat=[]-->, belongsTo=parr
810	astrocytoma formation. These data provoked the authors to suggest, otherFeat=[]-->, belongsTo=parr
811	that the study of specific gene mutations in mixed genetic backgrounds,, otherFeat=[]-->, belongsTo=parr
812	especially those including 129, might conceal the functions of certain, otherFeat=[]-->, belongsTo=parr
813	genes. Moreover, proteins other than Pin1 have also been reported to, otherFeat=[]-->, belongsTo=parr
814	have conflicting roles in tumorigenesis. For example, transforming, otherFeat=[]-->, belongsTo=parr
815	growth factor has been described as functioning either to suppress or, otherFeat=[]-->, belongsTo=parr
816	exacerbate invasive and/or metastatic behavior of tumor cells (39, 96,, otherFeat=[]-->, belongsTo=parr
817	97). Similarly, studies on the protein DCC demonstrate that the loss of, otherFeat=[]-->, belongsTo=parr
818	DCC occurs frequently in colorectal cancers, whereas forced expression, otherFeat=[]-->, belongsTo=parr
819	of the DCC ligand, netrin-1, promotes intestinal tumor development, otherFeat=[]-->, belongsTo=parr
820	(31). As a result of these observations, it was suggested that DCC and, otherFeat=[]-->, belongsTo=parr
821	netrin-1 be classified as "conditional" tumor suppressors to distinguish, otherFeat=[]-->, belongsTo=parr
822	them from "classical" tumor suppressors, such as p53 or Rb, which, otherFeat=[]-->, belongsTo=parr
823	always inhibit tumor formation due to their role in the cell cycle. Based, otherFeat=[]-->, belongsTo=parr
824	on this analysis, it is tempting to speculate that, at least in the context of, otherFeat=[]-->, belongsTo=parr
825	a C57BL6 genetic background, Pin1 can also function as a conditional, otherFeat=[]-->, belongsTo=parr
826	tumor suppressor due in part to its role in regulating the timely Cdc4-, otherFeat=[]-->, belongsTo=parr
827	dependent degradation of cyclin E and c-Myc., otherFeat=[]-->, belongsTo=parr
828	Acknowledgments--We thank Chris Counter for providing housing for the, otherFeat=[]-->, belongsTo=parrnote
829	mice used to perform the in vivo tumor analysis. In addition, we thank Tom, otherFeat=[]-->, belongsTo=parrnote
830	Ribar for performing the animal injections required to carry out these, otherFeat=[]-->, belongsTo=parrnote
831	experiments., otherFeat=[]-->, belongsTo=parrnote
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==============================
0	The Loss of PIN1 Deregulates Cyclin E and Sensitizes Mouse-->id=0, page=0, size=27, fam=Times, col=#231f20, type=title, textLines=1--->[]--->title	The Loss of PIN1 Der>>>and Sensitizes Mouse
1	Embryo Fibroblasts to Genomic Instability*S-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->note	Embryo Fibroblasts t>>>enomic Instability*S
2	Received for publication, May 26, 2005, and in revised form, October 11, 2005 Published, JBC Papers in Press, October 13, 2005, DOI 10.1074/jbc.M505770200-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Received for publica>>>.1074/jbc.M505770200
3	Elizabeth S. Yeh1, Brian O. Lew, and Anthony R. Means2-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=3--->[]--->note	Elizabeth S. Yeh1, B>>>nd Anthony R. Means2
4	From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=3--->[]--->note	From the Department >>>North Carolina 27710
5	During the G0/G1-S phase transition, the timely synthesis and degradation of key regulatory proteins is required for normal cell cycle progression. Two of these proteins, c-Myc and cyclin E, are recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) complex. SCFCdc4 binds to a similar phosphodegron sequence in c-Myc and cyclin E proteins resulting in ubiquitylation and degradation of both proteins via the 26 S proteosome. Since the prolyl isomerase Pin1 binds the c-Myc phosphodegron and participates in regulation of c-Myc turnover, we hypothesized that Pin1 would bind to and regulate cyclin E turnover in a similar manner. Here we show that Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts. Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends on Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 results in an increased steady-state level of cyclin E and stalling of the cells in the G1/S phase of the cell cycle. The cellular changes that result from the loss of Pin1 predispose Pin1 null mouse embryo fibroblasts to undergo more rapid genomic instability when immortalized by conditional inactivation of p53 and sensitizes these cells to more aggressive Ras-dependent transformation and tumorigenesis.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	During the G0/G1-S p>>>n and tumorigenesis.
6	Mitogenic stimuli initiate a sequence of events that result in the entry of quiescent cells into S phase (1). Critical for this important transition is the ordered synthesis and degradation of transcription factors such as c-Jun and c-Myc (Myc) and cyclins such as cyclin D and cyclin E (1). Deregulation of the turnover of these proteins, such that they remain active at inappropriate times during cell cycle progression, is frequently found in human cancer. Thus, an understanding of the molecular mechanisms that regulate protein turnover is crucial to provide insight into the oncogenic process. Three proteins important for the progression of cells into S phase, c-Jun, c-Myc, and cyclin E, are ubiquitylated by a common member of the Skp1/ Cul1/Rbx1 (SCF)3 group of ubiquitin enzymes in which the F-box component, which serves as the ubiquitin E3 ligase, is Cdc4 (SEL-10, Fbw7, Ago) (2). SCFCdc4 binds to a component of each protein that has been termed the "phosphodegron" (3) to promote the ubiquitylation and degradation of these proteins via the 26 S proteosome. c-Jun and Myc have an additional binding protein in common, the peptidyl prolyl cis/trans-isomerase Pin1, which binds and isomerizes prolyl bonds in the context of phospho-Ser/ Thr-Pro motifs (4, 5).-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Mitogenic stimuli in>>>r-Pro motifs (4, 5).
7	Previously, we described the mechanism by which Pin1 promotes Myc degradation (5). The Cdc4 phosphodegron of Myc is present in a domain termed Myc box 1 (MB1), containing the sequence LPpTPPLpSP (where pT represents phosphothreonine and pS represents phosphoserine), in which the two phosphorylation events occur sequentially and are catalyzed by ERK (Ser62) and GSK3 (Thr58), respectively (6, 7). Pin1 binds to the doubly phosphorylated motif in a manner requiring phospho-Thr58 and promotes a conformational change that presents phospho-Ser62 as a substrate for the protein phosphatase PP2A (5). Ubiquitylated Myc is only phosphoryated on Thr58 (5), suggesting that either dephosphorylation of Ser62 occurs before Cdc4 can bind to Myc or before ubiquitylation can occur. Regardless of the precise mechanism involved, inhibition of PP2A or the absence of Pin1 results in a stabilization of Myc (5). Myc can also be stabilized by mutating Thr58 to Ala, and this mutation renders Myc oncogenic in a primary human cell transformation assay (5). Since residues in the Myc phosphodegron, including Thr58 and Pro57, are frequently mutated in lymphomas (8 ­11), deregulation of Myc due to mutations of this region of the protein can play a role in human cancer (9).-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Previously, we descr>>>in human cancer (9).
8	The phosphodegron of cyclin E, LpTPPXpSG, is remarkably similar to that of Myc (12). In the case of cyclin E, the phosphorylation events are catalyzed by Cdk2 (Ser384) and GSK3 (Thr380) (11), respectively, and x-ray structural studies have shown that phospho-Thr380 plays a crucial role in the binding of cyclin E by Cdc4 (3, 12). As is the case for Myc, deregulation of cyclin E can result in cell cycle defects (13­17) that predispose cells to oncogenesis (18 ­23). Such defects include aberrant DNA replication and the loss of genomic integrity. Indeed, cyclin E deregulation is associated with many human cancers including breast (18, 20, 24 ­26), ovarian (19), and bladder cancer (27), yet the precise mechanism responsible for ensuring the timely cell cycle-dependent turnover of cyclin E is incompletely understood. For these reasons and because of the similarities in the phosphodegron motif and degradation process between Myc and cyclin E, we investigated whether cyclin E was also a Pin1-binding protein. Here we show that Pin1 binds cyclin E and modulates cyclin E levels. In the absence of Pin1, cyclin E is deregulated in a way that leads to its stabilization, which, in combination with other protein alterations in Pin1 null cells, leads to cell cycle defects. We also demonstrate a correlation between the cell cycle defects that occur in mouse embryo fibroblasts (MEFs) null for Pin1 and increased rate in the progression of genomic instability when these cells are immortalized by inactivating p53 function. Finally, we show that the cell cycle defects resulting from the loss of Pin1 sensitize immortalized Pin1 null cells to more extensive and aggressive transformation and tumorigenesis induced by the Ras oncogene.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	The phosphodegron of>>>by the Ras oncogene.
9	MATERIALS AND METHODS-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->title	MATERIALS AND METHOD>>>ATERIALS AND METHODS
10	Cells and Cell Culture--HEK 293 cells were acquired from the American Type Culture Collection. 293 cells were grown in Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum. Phoenix cells (293 derivative) were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum. Pin1 null mice, originally generated by Fujimori et al. (28), were obtained from Hoffmann-LaRoche. The pin1 gene deletion was transferred into an isogenic C57BL6 background using marker-assisted speed congenic breeding by Jackson Laboratory. pin1 / and pin1 / embryonic fibroblasts were isolated from the isogenic C57BL6 strain as previously described (29). MEFs were grown in Dulbecco's modified Eagle's medium containing sodium pyruvate and 10% heat-inactivated fetal bovine serum.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Cells and Cell Cultu>>> fetal bovine serum.
11	* This work was supported by National Institutes of Health (NIH) Grant CA082845 (to A. R. M.). 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=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	* This work was supp>>> indicate this fact.
12	S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	S The on-line versio>>>ental Figs. 1 and 2.
13	1 Supported in part by NCI, NIH, T32 Training Grant CA-059365 and a grant from the Milheim Foundation for Cancer Research.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	1 Supported in part >>>for Cancer Research.
14	2 To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710-3813. Tel.: 919-681-6209; Fax: 919-681-7767; E-mail: means001@mc.duke.edu.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	2 To whom correspond>>>eans001@mc.duke.edu.
15	3 The abbreviations used are: SCF, Skp1/Cul1/Rbx1; MEF, mouse embryo fibroblast; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; WT, wild type; MMTV, murine mammary tumor virus.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	3 The abbreviations >>>mammary tumor virus.
16	THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 1, pp. 241­251, January 6, 2006-->id=6, page=0, size=7, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	THE JOURNAL OF BIOLO>>>251, January 6, 2006
17	© 2006 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=note, textLines=8--->[]--->note	© 2006 by The Americ>>>rinted in the U.S.A.
18	JANUARY 6, 2006 · VOLUME 281 · NUMBER 1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	JANUARY 6, 2006 · VO>>>OLUME 281 · NUMBER 1
19	JOURNAL OF BIOLOGICAL CHEMISTRY 241-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->note	JOURNAL OF BIOLOGICA>>>OGICAL CHEMISTRY 241
20	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
21	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
22	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
23	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
24	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
25	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
26	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
27	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
28	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
29	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
30	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
31	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
32	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	from>>>from
33	http://www.jbc.org/cgi/content/full/M505770200/DC1-->id=6, page=0, size=7, fam=Times, col=#231f20, type=note, textLines=8--->[u'a']--->note	http://www.jbc.org/c>>>/full/M505770200/DC1
34	Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	Supplemental Materia>>>ial can be found at:
35	293 cells were transfected using Lipofectamine or Lipofectamine 2000 (Invitrogen) and Opti-MEM (Invitrogen) serum-reduced media per the manufacturer's suggestion. Cells were plated 1 day prior to transfection at a density of 1 106 cells/100-mm plate. Briefly, 2.5 g of DNA was mixed with 15­30 l of Lipofectamine 2000 or Lipofectamine, respectively, in 0.5 ml of Opti-MEM and incubated for 15 min at room temperature. The mixture was added to the cells for 12­18 h prior to treatment with drug or lysis.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	293 cells were trans>>> with drug or lysis.
36	The following plasmids were used for transfections. pCS2-Myc-cyclin E, cyclin ET62A, cyclin ET62A/T380A, cyclin ES372A, and cyclin ES384A were generous gifts from J. Wade Harper (Harvard Medical School, Boston, MA). pBabe-Puro-Ras was kindly provided by Chris Counter (Duke University, Durham, NC), and pBabe-Hygro-p53DD was from William Hahn (Dana Farber Research Institute, Boston, MA). pWZLBlast-Myc was generated as previously described (5).-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	The following plasmi>>>ously described (5).
37	Protein Binding Assays--GST pull-down assays were performed as previously described (5). Briefly, GST and GST-Pin1 fusion proteins with either wild type Xenopus Pin1, an xPin1 WW domain mutant with substitutions at W11A and W34A, or a xPin1 catalysis-deficient mutant with a substitution at C109A was prepared as described (30). The GST and GST-Pin1 recombinant proteins were maintained on GSH-agarose beads and stored at 4 °C. Cells were lysed in buffer containing PBS, 10 mM EDTA, 1 mM dithiothreitol, and 1% Triton X-100 supplemented with Pefabloc (Roche Applied Science) and NaF. Lysates were clarified by centrifugation prior to binding assays. Binding was performed at 4 °C for 1 h using 10 g of GST proteins incubated with 500 g of total lysate protein. Beads were washed four times in 1 ml of lysis buffer per wash. Subsequently, proteins were solubilized in 2 SDS sample buffer and separated by SDS-PAGE. Bound proteins were analyzed by Western blotting.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Protein Binding Assa>>>by Western blotting.
38	For immunoprecipitations, cells were lysed in buffer containing 50 mM Tris, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 1% Triton X-100 supplemented with Pefabloc (Roche Applied Science) and NaF. Lysates were clarified by centrifugation. 500 g of total lysate protein was used per sample. Prior to binding, the lysate samples were precleared with Protein A/G-Sepharose for 10 min at 4 °C. Binding was performed using 2 g (per sample) of anti-9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for Myc-tagged protein. For experiments using Xenopus interphase extracts, extracts were prepared as previously described (30). Binding was performed by preincubating anti-Pin1 to Protein A beads (Amersham Biosciences) in the presence of bovine serum albumin. The beads were washed three times in PBS prior to incubation with 400 gof total extract protein. Beads were washed four times in a modified radioimmune precipitation buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS. Bound proteins were separated and analyzed as described above.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	For immunoprecipitat>>> as described above.
39	Western Blotting--The following antibodies were used for the detection of proteins by Western blotting: anti-Cdk2 M2 (Santa Cruz Biotechnology), anti-cyclin E M20 (Santa Cruz Biotechnology), anti-cyclin E HE12 (Upstate Biotechnology), anti-Myc 9E10 (Santa Cruz Biotechnology), anti-Pin1 (30), anti-p27 C-19 (Santa Cruz Biotechnology), antip53 FL-393 (Santa Cruz Biotechnology), anti-phosphocyclin E Thr380 (Santa Cruz Biotechnology), and anti- -actin (Sigma). Equal amounts of protein for each sample were separated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore Corp.). Membranes were blocked in TBS-0.2% Tween 20 containing 5% milk for 1 h prior to incubation with primary antibody. Primary antibodies were diluted 1:1000 in the blocking solution with the exception of anti-Pin1, which was diluted 1:10,000 unless otherwise stated. Membranes were washed a minimum of three times in Tris-buffered saline, 0.2% Tween 20 for 15 min each wash. Horseradish peroxidase-conjugated secondary antibodies were diluted into the blocking buffer and incubated for2hat room temperature. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies using the ECL reagent (Amersham Biosciences) per the manufacturer's instructions.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Western Blotting--Th>>>urer's instructions.
40	Retroviral Transduction--The pin1 / and pin1 / cells containing p53DD and Ras were generated by amphotrophic retroviral transduction and polyclonal selection using 10 g/ml hygromycin B (Calbiochem) or 0.5 g/ml puromycin (Calbiochem). Viral medium was obtained by Lipofectamine transfection of the Phoenix packaging cell line with 10 g of DNA. Cells were allowed to recover for 24 h, after which time the medium was collected and filtered through a 0.45- m filter (16) to remove any cellular debris. Filtered viral medium was supplemented with 4 g/ml Polybrene (Sigma) and added to primary MEFs at passage 3 for 24 h. Cells were allowed to recover in complete medium for 24 h at 37 °C prior to drug selection.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Retroviral Transduct>>>r to drug selection.
41	Cellular Transformation--The ability of cells to undergo anchorage independent growth was tested as previously described (32). 5 104 cells were plated in 0.3% Nobel agar, Dulbecco's modified Eagle's medium and heat-inactivated fetal bovine serum on 35-mm plates containing a 2-mm grid. Plates were incubated at 37 °C for 21 days and assessed for colony growth in soft agar. Cells were fed one time weekly with 1 ml of agar/medium mixture. Colonies were counted by light microscopy. The mean S.D. represents two independent experiments, each performed in triplicate.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Cellular Transformat>>>ormed in triplicate.
42	Tumor Formation in Immunodeficient (SCID) Mice--SCID mice were obtained from Charles River and housed in the Comprehensive Cancer Center Isolation Facility under a 12-h light, 12-h dark cycle. Food and water were provided ad libitum, and all care was given in compliance with National Institutes of Health and institutional guidelines on the use of laboratory and experimental animals. Prior to injection, cells were free of micoplasma, drugs, and antibiotics. Five mice were injected per cell type with a single subcutaneous injection of 1 and SyberGreen dye. The mixture was analyzed by real time PCR (BioRad) under the following conditions: step 1, 95 °C for 3 min; step 2, 60.8 °C for 0 ­15 min; step 3, 72 °C for 0 ­15 min; repeat steps 2 and 3 for 45 cycles; step 5, 72 °C for 2 min; step 6, 45 °C for 0 ­10 min. Samples were analyzed two times in duplicate per trial.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Tumor Formation in I>>>duplicate per trial.
43	107 cells/animal. Data are represented as number of mice that developed tumors over total number of animals.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	107 cells/animal. Da>>>l number of animals.
44	Real Time PCR--Quantitation of cellular mRNA levels was determined by real time PCR using the SyberGreen Supermix (Bio-Rad) reagent per the manufacturer's suggestions. Briefly primers to the NH2terminal region of cyclin E were created to contain the following sequences: 5 -ACG GAC CAC AGC AAC ATG AA-3 and 5 -AAA CAC GGC CAC ATT TGC CT-3 (IDT). Total cellular RNA was isolated by harvesting confluent 100-mm plates of pin1 / and pin1 /-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Real Time PCR--Quant>>>of pin1 / and pin1 /
45	MEFs using the TRIzol (Invitrogen) reagent. Cells were harvested in 1 ml of TRIzol and cleaned with chloroform. Resulting material was purified by ethanol precipitation. First strand cDNA was generated using Superscript III reverse transcriptase (Invitrogen). Reaction mixtures were denatured at 65 °C for 6 min followed by cDNA synthesis at 50 °C for 50 min, and then the reactions were terminated by incubation at 85 °C for 5 min. Resultant cDNA was mixed with designated primers-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	MEFs using the TRIzo>>>h designated primers
46	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
47	242 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	242 JOURNAL OF BIOLO>>>BIOLOGICAL CHEMISTRY
48	VOLUME 281 · NUMBER 1 · JANUARY 6, 2006-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	VOLUME 281 · NUMBER >>> 1 · JANUARY 6, 2006
49	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
50	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
51	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
52	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
53	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
54	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
55	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
56	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
57	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
58	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
59	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
60	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
61	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	from>>>from
62	Cell Proliferation--2.5 105 cells were seeded into 60-mm plates in duplicate. Every 24 h, cells were trypsinized and counted by Coulter counter. Data are represented as the average of the two samples for both cell types at each time point. These data are representative of five individual experiments.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Cell Proliferation-->>>ividual experiments.
63	BrdUrd Incorporation--1 105 cells were seeded onto coverslips in 6-well plates and incubated overnight or until 70 ­ 80% confluent. Cells-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	BrdUrd Incorporation>>>80% confluent. Cells
64	were pulsed with BrdUrd for 30 min at a final concentration of 10 M.-->id=6, page=0, size=7, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	were pulsed with Brd>>>ncentration of 10 M.
65	Following incubation with BrdUrd, cells were fixed in cold MeOH for 5-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Following incubation>>>d in cold MeOH for 5
66	min at 20 °C and then rehydrated in PBS. 1 ml of 2 M HCl/Triton-->id=6, page=0, size=7, fam=Times, col=#231f20, type=note, textLines=8--->[]--->note	min at 20 °C and the>>>ml of 2 M HCl/Triton
67	X-100 was added to each well, followed by incubation at room temperature for 30 min. Cells were washed once for 5 min in 1 ml of 0.1 M sodium tetraborate (pH 8.5) and twice for 5 min in PBS followed by blocking in 1% bovine serum albumin/PBS for 30 min. To perform staining, anti-BrdUrd, diluted 1:100 in 0.1% bovine serum albumin/PBS, was added to each coverslip and incubated at 4 °C overnight. Coverslips were washed three times in PBS for 5 min and then incubated with anti-mouse fluorescein isothiocyanate diluted 1:200 in 0.1% bovine serum albumin/PBS and incubated at room temperature for 60 min followed by three washes in PBS. Coverslips were mounted on slides with fluor antioxidant containing 1:5000 4 ,6-diamidino-2-phenylindole and analyzed by fluorescence microscopy (Zeiss-Axiscop). Each of three individual experiments were performed in triplicate, and 300 cells were counted per replicate.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	X-100 was added to e>>>unted per replicate.
68	Flow Cytometry--MEFs were grown until 70 ­ 80% confluent in 60-mm plates. Cells were harvested by washing once in Hanks' balanced salt solution followed by trypsinization. Cells were pelleted by centrifugation at 500 g for 10 min and washed once in PBS, followed by fixation with cold 70% ethanol. At this time, cells were either stored at 20 °C until used or kept on ice for 2 h prior to staining. DNA staining was performed by resuspending cells in PBS containing propidium iodide (5 g/ml) and RNase A (50 units) and then analyzed by fluorescence-activated cell sorting (Duke University Comprehensive Cancer Center Flow Cytometry Shared Resource, Durham, NC). Three separate experiments were performed with each cell type analyzed in triplicate in each experiment.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Flow Cytometry--MEFs>>> in each experiment.
69	RESULTS-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	RESULTS>>>RESULTS
70	Pin1 Binds to the Cyclin E-Cdk2 Complex and Influences the Stability of Cyclin E Protein--Cyclin E has been reported to undergo phosphorylation on several Ser and Thr residues in cells (12, 26, 33­36). At least four of these sites are implicated in its degradation (12, 26, 33­36) (Fig. 1A). Three sites, Thr62, Thr380, and Ser384 (shown in black), influence the Cdc4-dependent turnover of cyclin E, whereas Ser372 (shown in gray) may be involved in cyclin E degradation but by an ill defined pathway (12, 26, 33). To determine whether endogenous Pin1 and cyclin E interact, we used Xenopus interphase extracts and immunoprecipitated Pin1 using our previously described anti-Xenopus Pin1 antibody (29). As shown in Fig. 1B, endogenous cyclin E and Pin1 do interact in this assay. Next, we transfected 293 cells with wild type (WT), Myctagged cyclin E and subjected the extracts to GST pull-down assays using GST alone, as a negative control, or GST-Pin1. As shown in the first two lanes of Fig. 1C, GST-Pin1 also binds Myc-tagged cyclin E. We next evaluated the importance of the three Cdc4-specific phosphorylation sites, Thr62, Thr380, and Ser384, in Pin1 binding by transfecting Myc-tagged cyclin E cDNA constructs that were mutated in one or more of the phosphorylation sites illustrated in Fig. 1A. Surprisingly,-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Pin1 Binds to the Cy>>>g. 1A. Surprisingly,
71	mutation of Thr62, Thr380, or the combination of Thr62 and Thr380 does-->id=9, page=0, size=6, fam=Times, col=#231f20, type=parrnote, textLines=61--->[]--->parr	mutation of Thr62, T>>>hr62 and Thr380 does
72	not affect Pin1 binding (Fig. 1C, lanes 3­ 8). On the other hand, mutation of Ser384 nearly abolishes Pin1 binding (Fig. 1C, lanes 9 and 10). Since Ser384 is phosphorylated by the Cdk2 component of the cyclin E-Cdk2 heterodimer (12), this result suggested that Pin1 might bind to the heterodimer in a manner that requires an active Cdk2 protein to catalyze the phosphorylation of Ser384 on cyclin E. This hypothesis is supported by the data in Fig. 1, D­F. First, when Pin1 is associated with WT endogenous or Myc-tagged cyclin E, immunoblot analysis reveals the presence of both cyclin E and Cdk2 (Fig. 1, D and E, lanes 1 and 2, and F, lanes 1 and 2, respectively). Second, preincubation of the cells containing Myc-cyclin E with the selective Cdk2 inhibitor roscovitine prior to lysis and the addition of GST-Pin1 markedly attenuated the binding of both cyclin E and Cdk2 (Fig. 1E, lanes 3 and 4). This result was repeated three times with similar results, and quantitation of these results can be found in supplemental Fig. 1. Although roscovitine is an inhibitor of Cdk2 activity, it may also affect Cdk2 and/or cyclin E protein levels (37). Thus, we evaluated Cdk2 and cyclin E levels by immunoblotting extracts derived from cells that had been treated with either vehicle (Me2SO)or30 M roscovitine. As shown in Fig. 1E (right panel), treatment with 30 M roscovitine resulted in minimal changes in cyclin E or Cdk2 levels. Collectively, these results suggest that the efficient binding of Pin1 to cyclin E depends on Cdk2 activity and an intact Ser384 residue, which is the site on cyclin E that is phosphorylated by Cdk2.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	not affect Pin1 bind>>>sphorylated by Cdk2.
73	To confirm the observation that phosphorylation of Ser384 is important for Pin1 binding to cyclin E, we expressed the Myc-tagged cyclin E constructs in 293 cells and examined Pin1 binding by immunoprecipitation using antibody to the Myc tag. We then probed the immunoprecipitates with antibodies to Cdk2, Pin1, or Myc (to detect the immunoprecipitated cyclin E proteins). In addition to WT, the T62A/T380A double mutant, and the S384A mutant cyclin E proteins, we also expressed a S372A mutant, since this site has also been implicated in cyclin E protein turnover (12) and is a potential Ser(P)-Pro binding site for Pin1. As shown in the left panel of Fig. 1F, Pin1 was present in the immunoprecipitates of WT, T62A/T380A and S372A cyclin E but was considerably less abundant in the immunoprecipitate containing the S384A mutant protein. This was true, although a similar amount of Cdk2 (as shown by Western blot) and of each cyclin E protein (based on Coomassie staining) was immunoprecipitated (Fig. 1F, left). In addition, each of the whole cell extracts contained similar amounts of Cdk2, Pin1, and the appropriate cyclin E protein (Fig. 1F, right). Collectively, these results show that Ser384 phosphorylation is important for the association of Pin1 with cyclin E-Cdk2 but not for the assembly of the cyclin E-Cdk2 complex.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	To confirm the obser>>>clin E-Cdk2 complex.
74	Since the phosphorylation of Thr380 and Ser384 on cyclin E are important for regulating the Cdc4-mediated turnover of cyclin E protein (12, 26, 33), we next asked whether, in the absence of Pin1, cyclin E levels were elevated. We subjected equal amounts of extracts from MEFs, either WT or null for Pin1, to Western blotting and found that steadystate levels of cyclin E were up-regulated 2-fold in the absence of Pin1 (Fig. 2A and supplemental Fig. 2). Interestingly, this up-regulation of cyclin E in Pin1 null cells was accompanied by a considerable decrease in the level of phosphorylated Thr380. Since phosphorylation of Thr380 promotes the degradation of cyclin E and this process is impaired in the absence of Pin1, these results are compatible with a role for Pin1 in regulating the turnover of cyclin E. Because the cyclin E gene is activated during the progression from G0/G1 to S phase, we also questioned whether the changes in cyclin E protein levels in the absence of Pin1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Since the phosphoryl>>> the absence of Pin1
75	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
76	JANUARY 6, 2006 · VOLUME 281 · NUMBER 1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	JANUARY 6, 2006 · VO>>>OLUME 281 · NUMBER 1
77	JOURNAL OF BIOLOGICAL CHEMISTRY 243-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->note	JOURNAL OF BIOLOGICA>>>OGICAL CHEMISTRY 243
78	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
79	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
80	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
81	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
82	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
83	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
84	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
85	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
86	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
87	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
88	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
89	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
90	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	from>>>from
91	FIGURE 1. Cyclin E binds to Pin1. A, phosphorylation sites on cyclin E protein that are involved in cyclin E turnover. Thr62, Thr380, and Ser384 have been described to influence cyclin E turnover promoted by Cdc4. Ser372 is also involved in cyclin E turnover by an unknown mechanism. B, endogenous Pin1 interacts with endogenous cyclin E. Xenopus Pin1 was immunoprecipitated, using anti-xPin1 antibody or rabbit IgG as a negative control, from Xenopus interphase extracts. Cyclin E was found only with the xPin1 co-immunoprecipitate. C, mutation of Ser384 impairs binding of cyclin E to Pin1. 293T cells were transfected with either Myc-tagged wild type cyclin E, cyclin ET62A, cyclin ET380A, cyclin ET62A/T380A, or cyclin ES384A prior to lysis. Lysates were subjected to GST pull-down with either GST beads as a negative control or GST-Pin1. Bound wild type and mutant cyclin E proteins were detected by Western blot, using anti-9E10 antibody for the Myc tag. Wild type cyclin E, cyclin ET62A, cyclin ET380A, and cyclin ET62A/T380A protein associated with GST-Pin1, whereas cyclin ES384A binding to Pin1 was attenuated. D, endogenous cyclin E-Cdk2 associates with Pin1. Nontransfected 293T cells were lysed and subjected to GST pull-down using GST alone or GST-Pin1. Both endogenous cyclin E and endogenous Cdk2 were found to be associated with GST-Pin1 but not with GST alone. E, Pin1 binds to the cyclin E-Cdk2 complex, and inhibition of Cdk2 with roscovitine impairs cyclin E-Cdk2 complex association with Pin1. Left, 293T cells were transfected with wild type cyclin E and treated with either Me2SO or 30 M roscovitine. 500 g of total protein from lysates were used to perform GST pull-down as described in A. Binding of cyclin E-Cdk2 to Pin1 was decreased in the presence of roscovitine. Right, 293T cells were transfected with wild type cyclin E prior to treatment with Me2SO vehicle or 30 M roscovitine. Whole cell lysates containing 35 g of total protein were subjected to Western blotting using the anti-HE12 antibody to detect cyclin E. As determined by densitometry, the ratio of cyclin E to actin in the Me2SO treatment is 1.1, whereas the ratio of cyclin E to actin under roscovitine treatment is 1.3. Similarly, the ratio of Cdk2 to actin in the Me2SO-treated lane is 2, whereas the ratio or Cdk2 to actin under roscovitine treatment is 2.1. No significant changes in the amounts of cyclin E and Cdk2 proteins were observed with 30 M roscovitine treatment relative to Me2SO treatment. F, Cdk2 remains associated with cyclin ES384A when Pin1 binding is impaired. Left, 293T cells were mock-transfected (lane 1) or transfected with Myc-tagged wild type cyclin E, cyclin ET62A/T380A, cyclin ET372A, or cyclin ES384A (lanes 2­5) prior to immunoprecipitation using the Myc tag with the anti-9E10 antibody. Bound proteins were resolved using anti-Cdk2 antibody and anti-Pin1 antibody. Cdk2 was bound to cyclin E and to each cyclin E mutant protein, whereas Pin1 binding was decreased with cyclin ES384A immunoprecipitate, confirming the result seen in B. Right,25 g of whole cell lysate for samples containing mock-transfected cells or cells transfected with Myc-tagged wild type cyclin E, cyclin ET62A/T380A, cyclin ET372A, or cyclin ES384A were subjected to Western blotting using the anti-Myc (9E10). Cyclin E was expressed in the appropriate lanes (lanes 2­5 but not lane 1) at comparable levels between samples. IB, immunoblotting; IP, immunoprecipitation.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	FIGURE 1. Cyclin E b>>>immunoprecipitation.
92	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
93	244 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	244 JOURNAL OF BIOLO>>>BIOLOGICAL CHEMISTRY
94	VOLUME 281 · NUMBER 1 · JANUARY 6, 2006-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	VOLUME 281 · NUMBER >>> 1 · JANUARY 6, 2006
95	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
96	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
97	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
98	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
99	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
100	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
101	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
102	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
103	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
104	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
105	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
106	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
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108	might be attributed to increased cyclin E mRNA. We performed real time PCR on total RNA isolated from either pin1 / or pin1 / MEFs and found that, although there appeared to be a slight increase in cyclin EmRNAin pin1 / cells, the increase was not statistically significant (Fig. 2B). We next probed the immunoblots for two components that are known to interact with cyclin E to regulate its level and/or activity in the cell. As shown in Fig. 2A, neither the level of Cdk2 nor the cyclindependent kinase inhibitor p27 was changed in pin1 / cells. Finally, we tested whether cyclin E is appropriately degraded in the absence of Pin1. Either Pin1 WT or Pin1 KO cells were treated with cycloheximide for 6 h. Fig. 2C shows that whereas inhibition of translation led to a 70% decrease in cyclin E protein in the wild type cells, cyclin E turnover was impaired in the pin1 / cells and showed only a small decrease ( 20%) 6 h after the addition of cycloheximide (Fig. 2C). Based on these results, we conclude that the primary way by which Pin1 regulates cyclin E is likely to be at the protein rather than the mRNA level.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	might be attributed >>>than the mRNA level.
109	The Absence of Pin1 in MEFs Results in Cell Cycle Defects--pin1 /-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	The Absence of Pin1 >>>ycle Defects--pin1 /
110	MEFs contain increased amounts of cyclin E (this study) and c-Myc (5), and deregulation of either cyclin E or c-Myc results in defects in the cell cycle (1). Cyclin E protein overexpression leads to an accelerated progression of G0/G1 to S phase coupled with an overall increase in the length of the cell cycle, possibly due to an increase in the duration of S phase (13, 14, 16, 38). Since cyclin E and c-Myc are deregulated in pin1 / MEFs, we questioned whether cell cycle progression through S phase might be compromised. First, as shown in Fig. 3A, the doubling time of pin1 / MEFs is considerably slower than that of WT MEFs. This result is similar to that reported by others using MEFs isolated from Pin1 null mice of a different genetic background than ours (28, 40). Second, to evaluate whether this slower cell cycle progression might reflect changes in S phase entry or progression, we pulse-labeled asynchronously growing populations of MEFs with BrdUrd and quantified its presence by immunocytochemical analysis of both pin1 / and pin1 / MEFs. As shown in Fig. 3B (which represents three collective experiments with each experiment containing n 300/cell type), the absence of Pin1 in MEFs results in a statistically significant (p 0.0037 evaluated by Student's t test) decrease in the percentage of cells that incorporate BrdUrd. This outcome could be explained by either a decrease in the number of cells entering S phase during the time of the BrdUrd pulse or an inability of cells that enter S phase to synthesize DNA efficiently. To distinguish between these possibilities, we subjected asynchronously growing cells to single parameter DNA profiling to determine the percentage of cells in G1, S, and G2/M phases. Fig. 3C (left and middle bars) shows that a higher percentage of pin1 / cells are in G1 and S phases relative to WT cells. On the other hand, we found a concomitant decrease in the percentage of pin1 / cells in G2/M as seen in Fig. 3C (right bars). Thus, progression through the G1/S phases-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	MEFs contain increas>>>ough the G1/S phases
111	FIGURE 2. Pin1 influences the stability of cyclin E protein. A, steady-state levels of cyclin E protein are elevated in Pin1 / MEFs. Equal amounts of protein (50 g) from pin1 / and pin1 / MEFs were analyzed for protein content by Western blotting (IB). Protein levels were detected using anti-cyclin E (M20), anti-phospho-cyclin E T380, anti-Cdk2, anti-p27, anti-Pin1, and anti- -actin as a loading control. Cyclin E levels were elevated 2-fold, and Thr(P)380 levels were markedly decreased in the absence of Pin1 as determined by densitometry and normalized to actin. Cdk2 and p27 levels were unchanged. Results are representative of five individual experiments. B, levels of cyclin E mRNA in the presence and absence of Pin1. Levels of cyclin E mRNA present in MEFs were quantified using real time PCR. Pin1 / MEFs displayed a slight increase in mRNA quantities, but this was not statistically significant. C, loss of Pin1 affects cyclin E protein stability. pin1 / and pin1 / MEFs were treated with 100 g/ml cycloheximide and harvested at 0 and 6 h. The middle panel represents a lower exposure of the right panel and was used for quantitation. Treatment with cycloheximide for 6 h led to a 67% decrease in cyclin E protein in pin1 / cells (left) but to only a slight decrease in cyclin E protein in pin1 / cells (middle) as determined by densitometry and normalized to actin. KO, knock-out.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	FIGURE 2. Pin1 influ>>>ctin. KO, knock-out.
112	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
113	JANUARY 6, 2006 · VOLUME 281 · NUMBER 1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	JANUARY 6, 2006 · VO>>>OLUME 281 · NUMBER 1
114	JOURNAL OF BIOLOGICAL CHEMISTRY 245-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->note	JOURNAL OF BIOLOGICA>>>OGICAL CHEMISTRY 245
115	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
116	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
117	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
118	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
119	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
120	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
121	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
122	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
123	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
124	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
125	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
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128	of the cell cycle is impaired in pin1 / cells. These data are representative of three individual experiments, which each used 3 replicate samples/cell type, and are highly statistically significant (see Fig. 3C). The results in Fig. 3, B and C, are entirely consistent with data obtained by Ekholm-Reed et al. (24), who overexpressed cyclin E in human cells and support the concept that the absence of Pin1, at least in part due to the deregulation of cyclin E, compromises the ability of cells to synthesize DNA and to progress through S phase.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	of the cell cycle is>>>ess through S phase.
129	Immortilization of Pin1 / MEFs with a Dominant Negative p53 Results in a More Rapid Progression toward Genomic Instability--In addition to cell cycle defects involving G1/S progression, cyclin E and c-Myc protein deregulation is involved in the generation of chromosomal instability (16, 20, 22, 25, 27, 41­50) and correlates with tumor formation (18 ­22, 25, 41, 51­ 64). Because c-Myc and cyclin E levels are increased in pin1 / cells, we evaluated the pin1 / cells for markers of genomic instability. We first quantified the number of micronucleated cells in early passage (passage 3) primary MEFs of the pin1 / and pin1 / genotypes. As shown in the left pair of bars in Fig. 4B, micronuclei occur in about 5% of the WT MEFs, and, although the Pin1 null cells show only about a 2% increase in this percentage (to about 7%), the difference is highly statistically significant (p 0.00000158, Student's t test, n 1000). These data show that even at passage 3, primary MEFs form micronuclei and that the absence of Pin1 exacerbates this defect. To determine whether these DNA changes result in aneuploidy, we subjected asynchronously cycling populations of passage 3 primary pin1 / and pin1 / cells to single parameter DNA profiling but found that both populations of primary cells displayed normal distribution in each phase of the cell cycle as measured by fluorescence-activated cell sorting (Fig. 4C, top panels; data represent three individual experiments performed with 3 replicates/cell type in each experiment).-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Immortilization of P>>>in each experiment).
130	Inactivation of p53 will immortalize mouse cells and lead to progressive genomic instability (27, 65, 66). To determine whether this p53 inactivation-dependent process would also be sensitized by the absence of Pin1, we infected the passage 3 pin1 / and pin1 / MEFs with a retrovirus encoding a dominant-negative p53 that expresses a truncated version of the p53 protein termed p53DD (Fig. 4A). p53DD is the C-terminal portion of p53 containing amino acids 302­390 and functions to prevent transcription by inhibiting p53 tetramer formation (67). Upon stable expression of p53DD, we obtained cell lines that continue to grow for greater than 50 passages (data not shown), which is many more than the 4 ­ 6 passages number that is typical of primary MEFs. Since Pin1 generally functions in the timing of events that occur during cell proliferation (30, 68, 69) and we found an increase in micronuclei formation even in passage 3 primary MEFs, we wanted to evaluate the effect of Pin1 absence as early as possible after the stable integration of p53DD. This cell selection and verification of protein expression required three cell passages, so MEFs at passage 3 after infection of the retrovirus expressing p53DD (or a total of six passages altogether) were used in all subsequent experiments. Fig. 4A shows that p53DD is expressed to a similar extent in both pin1 / and pin1 / MEFs and that this does not alter the expression of endogenous p53. Expression of p53DD caused a marked increase in micronuclei formation in both Pin1-replete and -null cells, although the absence of Pin1 increased this from about 17.5 to 28% of the cells (p 0.000000287, Student's t test, n 1000) (Fig. 4B, right pair of bars). This increase in the percentage of micronucleated cells is mirrored by a change in the DNA profile in both pin1 / and pin1 / cells expressing p53DD (Fig. 4C, bottom panels; data represent three individual experiments performed with 3 replicates/cell type in each experiment). Aneuploidy is evident in both cell populations but is considerably more advanced in the cells null for Pin1 (e.g. 47% versus 62% of cells with 4 N DNA content in pin1 / and pin1 / cells, respectively; Fig. 4C, lanes M5 and M6, compare rows B and D). These results support our conjecture that the absence of Pin1 promotes genomic instability in cells in a more accelerated fashion than in the presence of Pin1 and that the loss of Pin1 acts in concert with the loss of p53 function to promote this effect.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Inactivation of p53 >>>promote this effect.
131	FIGURE 3. The absence of Pin1 in MEFs results in cell cycle defects. A, Pin1 null MEFs have longer doubling times compared with wild type. Equal numbers of pin1 / and pin1 /-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	FIGURE 3. The absenc>>>of pin1 / and pin1 /
132	MEFs were plated into 6-well dishes. Cells from duplicate wells per each cell type were trypsinized and counted every 24 h for 6 days. The graph is representative of five similar independent experiments. pin1 / cells display a growth deficiency. B, Pin1 null cells undergo DNA synthesis more slowly as measured by BrdUrd incorporation. Cells were plated on glass coverslips in equal numbers. Cells were pulsed with BrdUrd prior to fixation and staining with anti-BrdUrd antibody to assess the number of cells which have incorporated BrdUrd. Cells lacking Pin1 incorporated less BrdUrd. This experiment was performed three times, counting n 300 per cell type per experiment, and is statistically significant as analyzed by the Student's t test (p 0.0037). C, pin1 / MEFs progress more slowly through G1/S phases. Asynchronous populations of pin1 / and pin1 / MEFs were trypsinized and fixed for single parameter fluorescence-activated cell sorting analysis. Cells were resuspended in PBS containing RNase and stained with propidium iodide to analyze DNA content. This experiment represents three individual experiments using 3 replicates/cell type/experiment and is statistically significant as analyzed by Student's t test (see right panel for p value). The graph represents the total percentage of cells in G1, S, and G2/M phases. The Pin1-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	MEFs were plated int>>>2/M phases. The Pin1
133	/ MEFs show a greater percentage of cells in G1 and S phases than pin1-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->note	/ MEFs show a greate>>>d S phases than pin1
134	/-->id=14, page=0, size=4, fam=Times, col=#231f20, type=parrnote, textLines=36--->[]--->note	/>>>/
135	MEFs, and a concomitant decrease in G2/M occurs in the pin1-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->note	MEFs, and a concomit>>>M occurs in the pin1
136	/ population. ko, knock-out.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	/ population. ko, kn>>>tion. ko, knock-out.
137	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
138	246 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	246 JOURNAL OF BIOLO>>>BIOLOGICAL CHEMISTRY
139	VOLUME 281 · NUMBER 1 · JANUARY 6, 2006-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	VOLUME 281 · NUMBER >>> 1 · JANUARY 6, 2006
140	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
141	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
142	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
143	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
144	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
145	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
146	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
147	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
148	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
149	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
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153	The Absence of Pin1 Sensitizes Cells to More Aggressive Transformation and Tumorigenesis in Vivo--Studies from the cyclin E knock-out mouse indicate that the loss of cyclin E protects mouse cells from Rasmediated transformation (70). Because cyclin E is up-regulated in pin1 / cells and these cells show increased genomic instability at early passage after immortalization, we hypothesized that deletion of Pin1 might lead to changes in the genome that are sufficient to promote more advanced cellular transformation of MEFs. In support of this hypothesis, we have previously reported that c-Myc is stabilized in the absence of Pin1 (5). Since Myc and Ras collaborate to transform rodent cells (47), it seemed plausible that the addition of oncogenic Ras alone to Pin1 /-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	The Absence of Pin1 >>> Ras alone to Pin1 /
154	primary MEFs might induce spontaneous transformation. However, expression of RasG12V alone in pin1 / cells caused the early passage primary MEFs to senesce, similar to what we found to occur in wild type MEFs (data not shown).-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	primary MEFs might i>>>Fs (data not shown).
155	Previous reports indicate that whereas cooperating oncogenes such as Myc and Ras can spontaneously transform rodent cells, several lines of evidence suggest that the expression of these oncogenes co-selects for the loss of p53 and INK4a (71, 72). Boehm et al. (73) recently demonstrated that p53DD collaborates with Myc and Ras to efficiently transform MEFs. These authors demonstrated that whereas p53DD and Ras alone can result in transformation of wild type MEFs, the transforma--->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Previous reports ind>>>EFs, the transforma-
156	FIGURE 4. Immortalization of Pin1 / MEFs with a dominant negative p53 results in a more rapid progression toward genomic instability. A, creation of stable cell line expressing p53DD. Primary MEFs were infected with retrovirus containing the dominant negative form of p53, p53DD. Cell lysates were analyzed by Western blotting (IB) and probed for the expression of p53DD using anti-p53 antibody. p53DD is expressed as a truncated form of p53 protein and runs at a lower Mr (middle) than native p53 protein (top). B, Pin1 null cells show increased formation of micronuclei. Cells were plated on coverslips prior to methanol fixation and staining with 4 ,6-diamidino-2-phenylindole. Cells were visualized by fluorescence microscopy and scored by counting total number of cells ( 300 cells counted per cell type in three individual experiments; see figure for p value) compared with the number of cells containing micronuclei. C, immortalization of Pin1 / MEFs with a dominant negative p53 results in a more rapid progression toward aneuploidy. Primary MEFs (passage 3) and p53DD MEFs (passage 3 after the addition of p53DD) were fixed and stained with propidium iodide prior to analysis by single parameter flow cytometry to determine DNA content. Primary pin1 / and pin1 / MEFs did not display aneuploidy, whereas immortalized cells of both genotypes resulted in a higher number of cells containing4NDNA content indicating aneuploidy. pin1 / cells contained the highest percentage of cells with 4 N DNA content (compare rows B and D, M5 and M6). KO, knock-out.-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	FIGURE 4. Immortaliz>>> M6). KO, knock-out.
157	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
158	JANUARY 6, 2006 · VOLUME 281 · NUMBER 1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	JANUARY 6, 2006 · VO>>>OLUME 281 · NUMBER 1
159	JOURNAL OF BIOLOGICAL CHEMISTRY 247-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->note	JOURNAL OF BIOLOGICA>>>OGICAL CHEMISTRY 247
160	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
161	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
162	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
163	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
164	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
165	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
166	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
167	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
168	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
169	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
170	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
171	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
172	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	from>>>from
173	tion of these cells can be accelerated by the addition of another oncoprotein, such as Myc. Thus, MEFs expressing p53DD and Ras were less efficiently transformed compared with MEFs expressing p53DD, Ras, and Myc, and the MEFs only containing p53DD and Ras formed significantly fewer colonies in soft agar than MEFs containing p53DD, Ras, and Myc (73). These results demonstrate that the serial introduction of oncogenic components (p53DD, Ras, Myc) can result in an additive effect on transformation. In Fig. 5A, we demonstrate that expression of oncogenic Ras results in the ability of p53DD, pin1 / MEFs to form colonies in soft agar to a higher extent than pin1 / MEFs expressing the same components (Fig. 5A). The fact that these latter cells would form colonies to a markedly lower extent is consistent with the conclusions of the study by Boehm et al. (73). These results show that deletion of Pin1 can sensitize cells to a more aggressive Ras-induced transformation relative to pin1 / cells. We postulate that the deregulation of cell cycle proteins, such as cyclin E and c-Myc, in pin1 / cells may be at least in part responsible for this difference.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	tion of these cells >>>for this difference.
174	In order to test the in vivo relevance of Pin1 depletion in tumor formation, we injected immunocompromised mice with four cell types: pin1 / MEFs expressing p53DD alone, pin1 / MEFs expressing p53DD and Ras, pin1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	In order to test the>>> p53DD and Ras, pin1
175	/ MEFs expressing p53DD alone, and pin1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	/ MEFs expressing p5>>>53DD alone, and pin1
176	/-->id=9, page=0, size=6, fam=Times, col=#231f20, type=parrnote, textLines=61--->[]--->parr	/>>>/
177	MEFs expressing p53DD and Ras. Consistent with our demonstration that loss of Pin1 promotes a more aggressive type of transformation in conjunction with loss of p53 function and oncogenic Ras when compared with WT cells, we found the same to be true in our tumorigenesis assay. Whereas both pin1 / MEFs expressing p53DD and Ras and pin1 / MEFs expressing p53DD and Ras supported tumor formation in immunocompromised mice (Fig. 5B), the tumors that developed from the pin1 / MEFs expressing p53DD and Ras were significantly larger in volume (Fig. 5C) and size (Fig. 5D) than their WT counterparts just 1 week postinjection. In fact, two of the five SCID mice injected with pin1 / MEFs expressing p53DD alone formed tumors (Fig. 5B). These data support the idea that cells can exhibit varying levels of oncogenic potency based on their ability to select for specific mutations, such as loss of p53 or Myc overexpression. Thus, Ras is more efficient in causing tumorigenesis when Pin1 null MEFs are immortalized by expression of p53DD but not when cells contain normal levels of Pin1. Our study demonstrates that the loss of Pin1, perhaps due to a deregulation of Pin1 protein substrates such as cyclin E and Myc, has a profound effect on proliferation, genomic instability, and tumorigenesis of MEFs.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	MEFs expressing p53D>>>morigenesis of MEFs.
178	DISCUSSION-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	DISCUSSION>>>DISCUSSION
179	Here we show that cyclin E is a Pin1-binding protein and that the steady-state level of cyclin E protein is increased in Pin1-null MEFs isolated from mice in which the pin1 gene deletion is maintained in an isogenic C57BL6 background. This increased protein level seems to be-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Here we show that cy>>>in level seems to be
180	FIGURE 5. Pin1 depletion sensitizes cells to transformation and can initiate tumorigenesis in vivo. A, Pin1-null cells are more readily transformed in the presence of Ras. pin1 /-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	FIGURE 5. Pin1 deple>>>sence of Ras. pin1 /
181	or pin1 / cells were stably infected with p53DD alone or p53DD and RasG12V retrovirus as indicated. 50,000 cells were plated in 0.3% agar, incubated for 21 days, and assessed for colony growth in soft agar. The mean and S.D. represent two independent experiments performed in triplicate (p 0.0001, Student's t test). The pin1 / p53DD Ras cells formed colonies in soft agar to a much greater extent than pin1 / p53DD Ras cells. B, summary of in vivo tumor formation. Mice were injected with pin1-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->capfig	or pin1 / cells were>>>e injected with pin1
182	/ and pin1 / MEF expressing-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->note	/ and pin1 / MEF exp>>>in1 / MEF expressing
183	p53DD alone and pin1-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->note	p53DD alone and pin1>>>p53DD alone and pin1
184	/ and pin1 / MEF expressing p53 DD and RasG12V (n 5 mice for each cell type). Injections were performed subcutaneously at the shoulder. C, mice injected with pin1 / MEF expressing p53DD and RasG12V formed more aggressive tumors than pin1 / MEF expressing p53DD and RasG12V. Tumor volume analysis represents the average tumor volume calculated from the five mice injected per cell type (p 0.0019; compare pin1 / and pin1 / MEF expressing p53DD and Ras G12V by Student's t test). The volumes (in mm3) of tumors derived from pin1 / MEFs are significantly larger than tumors derived from pin1 / MEFs. D, the tumors derived from pin1 / MEFs were markedly larger than the tumors derived from pin1 / MEFs. Images are representative of tumor size (indicated with average diameter; top) prior to and after excision (bottom).-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=347--->[]--->parr	/ and pin1 / MEF exp>>>r excision (bottom).
185	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
186	248 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	248 JOURNAL OF BIOLO>>>BIOLOGICAL CHEMISTRY
187	VOLUME 281 · NUMBER 1 · JANUARY 6, 2006-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	VOLUME 281 · NUMBER >>> 1 · JANUARY 6, 2006
188	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
189	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
190	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
191	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
192	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
193	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
194	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
195	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
196	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
197	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
198	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
199	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
200	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	from>>>from
201	predominantly due to a decreased rate of cyclin E turnover. Additionally, we show that Pin1 binds to the cyclin E-Cdk2 complex in a phosphorylation-dependent manner; the interaction requires an intact Ser384 and is decreased by inhibition of Cdk2 activity. Although antibodies that specifically recognize Ser(P)384 are not available, these data argue that Ser384 may need to be phosphorylated in order for Pin1 to efficiently bind the cyclin E-Cdk2 complex. The fact that Ser384 is phosphorylated by the Cdk2 component of the cyclin E-Cdk2 complex (12) and this phosphorylation is required for SCFCdc4-dependent degradation of cyclin E (12, 33, 35) but not for formation of the cyclin E-Cdk2 complex suggests the possibility that Pin1 binds to cyclin E and facilitates its ubiquitylation in preparation for its degradation via the 26 S proteosome. This hypothesis is consistent with our previous study, which demonstrated that Myc is a phosphorylation-dependent Pin1binding protein, the loss of Pin1 increased the steady state level of Myc in MEFs, and the deregulation of Myc protein was due to a decreased rate of its ubiquitylation and degradation (5). Since both cyclin E and Myc are degraded by SCFCdc4 (6, 7, 33, 36, 74) and the "phosphodegrons" recognized by Cdc4 and Pin1 are remarkably similar, our data support the idea that a mechanism with many common features controls the degradation of these two important cell regulatory proteins.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	predominantly due to>>>regulatory proteins.
202	On the other hand the precise way in which Pin1 participates in the Cdc4-dependent degradation of cyclin E and Myc may be different. In the case of Myc, the binding of Pin1 required phosphorylation of Thr58, which has been proposed to constitute the primary Cdc4 recognition site (6, 7, 74). Pin1 binding resulted in a conformational change in the phosphodegron that presented Ser(P)62 as a substrate for PP2A (5). Since ubiquitylated Myc contained only Thr(P)58, we concluded that Pin1/PP2A-mediated dephosphorylation of Ser62 was important for ubiquitylation and/or degradation (5). We show in the current study that Pin1 binding does not require phosphorylation of Thr380 in cyclin E, which is equivalent to Thr58 in Myc and also constitutes the primary binding site for Cdc4 based on crystallographic analysis of Cdc4 in complex with a peptide mimic of the cyclin E phosphodegron (3). Rather, effective Pin1 binding to cyclin E is dependent on phosphorylation of-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	On the other hand th>>>n phosphorylation of
203	Ser384, which is in a location in the phosphodegron equivalent to Ser62 in-->id=9, page=0, size=6, fam=Times, col=#231f20, type=parrnote, textLines=61--->[]--->parr	Ser384, which is in >>>uivalent to Ser62 in
204	Myc. Regardless of these differences, the outcome of Pin1 binding to a phosphorylated form of cyclin E and Myc is the same, since this interaction promotes Cdc4-dependent degradation in each case. Clarifying the precise mechanism by which Pin1 facilitates the turnover of cyclin E will be an important future endeavor.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Myc. Regardless of t>>>ant future endeavor.
205	There is considerable information demonstrating a role for Pin1 in the G0/G1 to S phase transition, and many of its proposed targets are deregulated in human cancer (4, 5, 75­91). Because cyclin E and Myc levels (5) are increased in MEFs null for Pin1 and deregulation of cyclin E and Myc have been linked to processes involved in tumorigenesis (16, 20, 22, 25, 27, 41­ 44, 46, 92), such as hyperproliferation and genomic instability, we evaluated whether Pin1 null MEFs would be more susceptible to Ras-dependent transformation after they were immortalized by conditional inactivation of p53. Indeed, we found that the absence of Pin1 led to increased genomic instability, and in the presence of p53DD these cells could be more aggressively transformed by oncogenic Ras than wild type cells. Thus, at least in a C57BL6 genetic background, loss of function of Pin1 sensitizes MEFs to more extensive and aggressive transformation and tumorigenesis.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	There is considerabl>>>n and tumorigenesis.
206	Contrary to our results, loss of Pin1 has been suggested to decrease sensitivity to cancer (81). It would follow that mice null for Pin1 might be "protected" from oncogene-mediated tumorigenesis. Wulf et al. (89) tested this hypothesis by breeding FVB mice containing a transgene expressing MMTV-Neu or MMTV-H-Ras with Pin1-null mice maintained in SvJae/129/C57BL6 mixed genetic background. MMTV-Neu and MMTV-Ras have previously been shown to form spontaneous breast tumors, resulting in decreased survival (93, 94). Wulf et al. (67) hypothesized that the resulting pin1 / /ras or pin1 / /neu transgenic populations would be less susceptible to tumor formation and have an increased chance of survival. These authors found that the absence of Pin1 prevented oncogenic Neu or Ras from inducing breast cancer and attributed these effects to cyclin D deregulation due to the loss of Pin1. Correlating with these observations, MEFs from the Pin1 null mice maintained in the SVJae/129/C57BL6 background demonstrated decreased protein levels of cyclin D1, c-Jun, and -catenin (4, 79, 88). Therefore, it is difficult to contend that depletion of Pin1 cannot also decrease susceptibility to mammary cancer, at least in a FBV/129/SvJae/ C57BL6 mixed genetic background.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	Contrary to our resu>>> genetic background.
207	How can one reconcile these apparently opposite outcomes of the role for Pin1 in cancer? The differences in protein expression in the SVJae/129/C57BL6 mixed background (e.g. c-Jun, cyclin D1, and -catenin) versus the isogenic C57BL6 (e.g. c-Myc and cyclin E) predict differential sensitivity of the two lines of mice to oncogenesis, and it is highly unlikely that these are the only differences that exist between cells derived from these two genetic backgrounds. It is certainly not surprising that identical mutations can have different biological effects when studied in distinct genetic backgrounds, which probably reflect differential expression of potential modifier genes, and this could explain the conundrum of the role of Pin1 in tumorigenesis. This idea was clearly demonstrated by Reilly et al. (95), who proposed that the susceptibility of mice to development of astrocytoma is intimately linked to the genetic strain of mice in which the experiments are conducted. These authors engineered different strains of mice (namely 129, 129/C57BL6, 129/SvJae, or C57BL6) to express mutant Nf1 or TP53 (95). The mutant mice maintained in the isogenic C57BL6 genetic background always formed astrocytomas. Conversely, the same mutations in mice maintained in 129, 129/C57BL6, or 129/SvJae backgrounds did not result in astrocytoma formation. These data provoked the authors to suggest that the study of specific gene mutations in mixed genetic backgrounds, especially those including 129, might conceal the functions of certain genes. Moreover, proteins other than Pin1 have also been reported to have conflicting roles in tumorigenesis. For example, transforming growth factor has been described as functioning either to suppress or exacerbate invasive and/or metastatic behavior of tumor cells (39, 96, 97). Similarly, studies on the protein DCC demonstrate that the loss of DCC occurs frequently in colorectal cancers, whereas forced expression of the DCC ligand, netrin-1, promotes intestinal tumor development (31). As a result of these observations, it was suggested that DCC and netrin-1 be classified as "conditional" tumor suppressors to distinguish them from "classical" tumor suppressors, such as p53 or Rb, which always inhibit tumor formation due to their role in the cell cycle. Based on this analysis, it is tempting to speculate that, at least in the context of a C57BL6 genetic background, Pin1 can also function as a conditional tumor suppressor due in part to its role in regulating the timely Cdc4dependent degradation of cyclin E and c-Myc.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->[]--->parr	How can one reconcil>>> cyclin E and c-Myc.
208	Acknowledgments--We thank Chris Counter for providing housing for the mice used to perform the in vivo tumor analysis. In addition, we thank Tom Ribar for performing the animal injections required to carry out these experiments.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->parr	Acknowledgments--We >>>t these experiments.
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214	at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[u'a']--->note	at>>>at
215	Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Centro>>>Centro
216	Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Nacional>>>Nacional
217	de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	de>>>de
218	Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Investigaciones>>>Investigaciones
219	Oncológicas-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Oncológicas>>>Oncológicas
220	on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	on>>>on
221	September-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	September>>>September
222	11,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	11,>>>11,
223	2006-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	2006>>>2006
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297	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
298	250 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	250 JOURNAL OF BIOLO>>>BIOLOGICAL CHEMISTRY
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311	Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	Downloaded>>>Downloaded
312	from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	from>>>from
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327	Loss of Pin1 Deregulates Cyclin E-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=10--->[]--->note	Loss of Pin1 Deregul>>>Deregulates Cyclin E
328	JANUARY 6, 2006 · VOLUME 281 · NUMBER 1-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=799--->['U']--->note	JANUARY 6, 2006 · VO>>>OLUME 281 · NUMBER 1
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340	www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=150--->[]--->note	www.jbc.org>>>www.jbc.org
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