0	Familial FTDP-17 Missense Mutations Inhibit Microtubule, otherFeat=[]-->, belongsTo=title
1	Assembly-promoting Activity of Tau by Increasing, otherFeat=[]-->, belongsTo=title
2	Phosphorylation at Ser202 in Vitro*S, otherFeat=[]-->, belongsTo=title
3	Received for publication, February 17, 2009, and in revised form, March 17, 2009 Published, JBC Papers in Press, March 19, 2009, DOI 10.1074/jbc.M901095200, otherFeat=[]-->, belongsTo=parrnote
4	Dong Han1, Hamid Y. Qureshi2, Yifan Lu, and Hemant K. Paudel?3, otherFeat=[]-->, belongsTo=title
5	From the Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General, otherFeat=[]-->, belongsTo=title
6	Hospital, and the ?Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3T 1E2, Canada, otherFeat=[]-->, belongsTo=title
7	In Alzheimer disease (AD), frontotemporal dementia and, otherFeat=[]-->, belongsTo=parrnote
8	parkinsonism linked to chromosome 17 (FTDP-17) and other, otherFeat=[]-->, belongsTo=parrnote
9	tauopathies, tau accumulates and forms paired helical filaments, otherFeat=[]-->, belongsTo=parrnote
10	(PHFs) in the brain. Tau isolated from PHFs is phosphorylated, otherFeat=[]-->, belongsTo=parrnote
11	at a number of sites, migrates as 60-, 64-, and 68-kDa bands on, otherFeat=[]-->, belongsTo=parrnote
12	SDS-gel, and does not promote microtubule assembly. Upon, otherFeat=[]-->, belongsTo=parrnote
13	dephosphorylation, the PHF-tau migrates as 50 ? 60-kDa, otherFeat=[]-->, belongsTo=parrnote
14	bands on SDS-gels in a manner similar to tau that is isolated, otherFeat=[]-->, belongsTo=parrnote
15	from normal brain and promotes microtubule assembly. The, otherFeat=[]-->, belongsTo=parrnote
16	site(s) that inhibits microtubule assembly-promoting activity, otherFeat=[]-->, belongsTo=parrnote
17	when phosphorylated in the diseased brain is not known. In this, otherFeat=[]-->, belongsTo=parrnote
18	study, when tau was phosphorylated by Cdk5 in vitro, its mobil-, otherFeat=[]-->, belongsTo=parrnote
19	ity shifted from 60-kDa bands to 64- and 68-kDa bands in a, otherFeat=[]-->, belongsTo=parrnote
20	time-dependent manner. This mobility shift correlated with, otherFeat=[]-->, belongsTo=parrnote
21	phosphorylation at Ser202, and Ser202 phosphorylation inhibited, otherFeat=[]-->, belongsTo=parrnote
22	tau microtubule-assembly promoting activity. When several tau, otherFeat=[]-->, belongsTo=parrnote
23	point mutants were analyzed, G272V, P301L, V337M, and, otherFeat=[]-->, belongsTo=parrnote
24	R406W mutations associated with FTDP-17, but not nonspe-, otherFeat=[]-->, belongsTo=parrnote
25	cific mutations S214A and S262A, promoted Ser202 phosphoryl-, otherFeat=[]-->, belongsTo=parrnote
26	ation and mobility shift to a 68-kDa band. Furthermore, Ser202, otherFeat=[]-->, belongsTo=parrnote
27	phosphorylation inhibited the microtubule assembly-promot-, otherFeat=[]-->, belongsTo=parrnote
28	ing activity of FTDP-17 mutants more than of WT. Our data, otherFeat=[]-->, belongsTo=parrnote
29	indicate that FTDP-17 missense mutations, by promoting phos-, otherFeat=[]-->, belongsTo=parrnote
30	phorylation at Ser202, inhibit the microtubule assembly-pro-, otherFeat=[]-->, belongsTo=parrnote
31	moting activity of tau in vitro, suggesting that Ser202 phospho-, otherFeat=[]-->, belongsTo=parrnote
32	rylation plays a major role in the development of NFT pathology, otherFeat=[]-->, belongsTo=parrnote
33	in AD and related tauopathies., otherFeat=[]-->, belongsTo=parrnote
34	Neurofibrillary tangles (NFTs)4 and senile plaques are the, otherFeat=[]-->, belongsTo=parr
35	two characteristic neuropathological lesions found in the, otherFeat=[]-->, belongsTo=parr
36	brains of patients suffering from Alzheimer disease (AD)., otherFeat=[]-->, belongsTo=parr
37	The major fibrous component of NFTs are paired helical fila-, otherFeat=[]-->, belongsTo=parr
38	ments (PHFs) (for reviews see Refs. 1?3). Initially, PHFs were, otherFeat=[]-->, belongsTo=parr
39	found to be composed of a protein component referred to as, otherFeat=[]-->, belongsTo=parr
40	"A68" (4). Biochemical analysis reveled that A68 is identical to, otherFeat=[]-->, belongsTo=parr
41	the microtubule-associated protein, tau (4, 5). Some character-, otherFeat=[]-->, belongsTo=parr
42	istic features of tau isolated from PHFs (PHF-tau) are that it is, otherFeat=[]-->, belongsTo=parr
43	abnormally hyperphosphorylated (phosphorylated on more, otherFeat=[]-->, belongsTo=parr
44	sites than the normal brain tau), does not bind to microtubules,, otherFeat=[]-->, belongsTo=parr
45	and does not promote microtubule assembly in vitro. Upon, otherFeat=[]-->, belongsTo=parr
46	dephosphorylation, PHF-tau regains its ability to bind to and, otherFeat=[]-->, belongsTo=parr
47	promote microtubule assembly (6, 7). Tau hyperphosphoryla-, otherFeat=[]-->, belongsTo=parr
48	tion is suggested to cause microtubule instability and PHF for-, otherFeat=[]-->, belongsTo=parr
49	mation, leading to NFT pathology in the brain (1?3)., otherFeat=[]-->, belongsTo=parr
50	PHF-tau is phosphorylated on at least 21 proline-directed, otherFeat=[]-->, belongsTo=parr
51	and non-proline-directed sites (8, 9). The individual contri-, otherFeat=[]-->, belongsTo=parr
52	bution of these sites in converting tau to PHFs is not entirely, otherFeat=[]-->, belongsTo=parr
53	clear. However, some sites are only partially phosphorylated, otherFeat=[]-->, belongsTo=parr
54	in PHFs (8), whereas phosphorylation on specific sites inhib-, otherFeat=[]-->, belongsTo=parr
55	its the microtubule assembly-promoting activity of tau (6,, otherFeat=[]-->, belongsTo=parr
56	10). These observations suggest that phosphorylation on a, otherFeat=[]-->, belongsTo=parr
57	few sites may be responsible and sufficient for causing tau, otherFeat=[]-->, belongsTo=parr
58	dysfunction in AD., otherFeat=[]-->, belongsTo=parr
59	Tau purified from the human brain migrates as 50 ? 60-, otherFeat=[]-->, belongsTo=parr
60	kDa bands on SDS-gel due to the presence of six isoforms that, otherFeat=[]-->, belongsTo=parr
61	are phosphorylated to different extents (2). PHF-tau isolated, otherFeat=[]-->, belongsTo=parr
62	from AD brain, on the other hand, displays 60-, 64-, and 68, otherFeat=[]-->, belongsTo=parr
63	kDa-bands on an SDS-gel (4, 5, 11). Studies have shown that, otherFeat=[]-->, belongsTo=parr
64	64- and 68-kDa tau bands (the authors have described the, otherFeat=[]-->, belongsTo=parr
65	68-kDa tau band as an 69-kDa band in these studies) are, otherFeat=[]-->, belongsTo=parr
66	present only in brain areas affected by NFT degeneration (12,, otherFeat=[]-->, belongsTo=parr
67	13). Their amount is correlated with the NFT densities at the, otherFeat=[]-->, belongsTo=parr
68	affected brain regions. Moreover, the increase in the amount of, otherFeat=[]-->, belongsTo=parr
69	64- and 68-kDa band tau in the brain correlated with a decline, otherFeat=[]-->, belongsTo=parr
70	in the intellectual status of the patient. The 64- and 68-kDa, otherFeat=[]-->, belongsTo=parr
71	tau bands were suggested to be the pathological marker of AD, otherFeat=[]-->, belongsTo=parr
72	(12, 13). Biochemical analyses determined that 64- and, otherFeat=[]-->, belongsTo=parr
73	68-kDa bands are hyperphosphorylated tau, which upon, otherFeat=[]-->, belongsTo=parr
74	dephosphorylation, migrated as normal tau on SDS-gel (4, 5,, otherFeat=[]-->, belongsTo=parr
75	11). Tau sites involved in the tau mobility shift to 64- and, otherFeat=[]-->, belongsTo=parr
76	68-kDa bands were suggested to have a role in AD pathology, otherFeat=[]-->, belongsTo=parr
77	(12, 13). It is not known whether phosphorylation at all 21 PHF-, otherFeat=[]-->, belongsTo=parr
78	sites is required for the tau mobility shift in AD. However, in, otherFeat=[]-->, belongsTo=parr
79	vitro the tau mobility shift on SDS-gel is sensitive to phospho-, otherFeat=[]-->, belongsTo=parr
80	rylation only on some sites (6, 14). It is therefore possible that in, otherFeat=[]-->, belongsTo=parr
81	the AD brain, phosphorylation on some sites also causes a tau, otherFeat=[]-->, belongsTo=parr
82	* This work was supported by grants from the Canadian Institute for Health, otherFeat=[]-->, belongsTo=parrnote
83	Research, the Alzheimer Society of Canada, and the National Science and, otherFeat=[]-->, belongsTo=parrnote
84	Engineering Research Council of Canada., otherFeat=[]-->, belongsTo=parrnote
85	S The on-line version of this article (available at http://www.jbc.org) contains, otherFeat=[]-->, belongsTo=parrnote
86	supplemental Tables S1?S3., otherFeat=[]-->, belongsTo=parrnote
87	1 Recipient of a Ph.D. studentship from the Alzheimer Society of Canada., otherFeat=[]-->, belongsTo=parrnote
88	2 Postdoctoral scholar of the Parkinson Society of Canada., otherFeat=[]-->, belongsTo=parrnote
89	3 To whom correspondence should be addressed: Lady Davis Inst. for Medical, otherFeat=[]-->, belongsTo=parrnote
90	Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste, otherFeat=[]-->, belongsTo=parrnote
91	Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8222, Ext., otherFeat=[]-->, belongsTo=parrnote
92	4866; Fax: 514-340-7502; E-mail: hemant.paudel@mcgill.ca., otherFeat=[]-->, belongsTo=parrnote
93	4 The abbreviations used are: NFT, neurofibrillary tangle; AD, Alzheimer dis-, otherFeat=[]-->, belongsTo=parrnote
94	ease; Cdk5, cyclin-dependent protein kinase 5; AU, absorption unit; FTDP-, otherFeat=[]-->, belongsTo=parrnote
95	17, frontotemporal dementia and Parkinsonism linked to chromosome 17;, otherFeat=[]-->, belongsTo=parrnote
96	PHF, paired helical filament; PKA, cAMP-dependent protein kinase; Pipes,, otherFeat=[]-->, belongsTo=parrnote
97	1,4-piperazinediethanesulfonic acid; WT, wild type., otherFeat=[]-->, belongsTo=parrnote
98	THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 20, pp. 13422?13433, May 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
99	? 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A., otherFeat=[]-->, belongsTo=nota_cab_pie
100	13422 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
101	VOLUME 284 ? NUMBER 20 ? MAY 15, 2009, otherFeat=['U']-->, belongsTo=nota_cab_pie
102	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
103	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
104	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
105	de, otherFeat=[]-->, belongsTo=nota_cab_pie
106	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
107	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
108	on, otherFeat=[]-->, belongsTo=nota_cab_pie
109	May, otherFeat=[]-->, belongsTo=nota_cab_pie
110	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
111	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
112	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
113	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
114	from, otherFeat=[]-->, belongsTo=nota_cab_pie
115	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
116	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
117	mobility shift. Identification of such sites will significantly, otherFeat=[]-->, belongsTo=parr
118	enhance our knowledge of how NFT pathology develops in the, otherFeat=[]-->, belongsTo=parr
119	brain., otherFeat=[]-->, belongsTo=parr
120	PHFs are also the major component of NFTs found in the, otherFeat=[]-->, belongsTo=parr
121	brains of patients suffering from a group of neurodegenerative, otherFeat=[]-->, belongsTo=parr
122	disorders collectively called tauopathies (2, 11). These disor-, otherFeat=[]-->, belongsTo=parr
123	ders include frontotemporal dementia and Parkinsonism, otherFeat=[]-->, belongsTo=parr
124	linked to chromosome 17 (FTDP-17), corticobasal degenera-, otherFeat=[]-->, belongsTo=parr
125	tion, progressive supranuclear palsy, and Pick disease. Each, otherFeat=[]-->, belongsTo=parr
126	PHF-tau isolated from autopsied brains of patients suffering, otherFeat=[]-->, belongsTo=parr
127	from various tauopathies is hyperphosphorylated, displays, otherFeat=[]-->, belongsTo=parr
128	60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of, otherFeat=[]-->, belongsTo=parr
129	binding to microtubules. Upon dephosphorylation, the above, otherFeat=[]-->, belongsTo=parr
130	referenced PHF-tau migrates as a normal tau on SDS-gel, binds, otherFeat=[]-->, belongsTo=parr
131	to microtubules, and promotes microtubule assembly (2, 11)., otherFeat=[]-->, belongsTo=parr
132	These observations suggest that the mechanisms of NFT, otherFeat=[]-->, belongsTo=parr
133	pathology in various tauopathies may be similar and the phos-, otherFeat=[]-->, belongsTo=parr
134	phorylation-dependent mobility shift of tau on SDS-gel may be, otherFeat=[]-->, belongsTo=parr
135	an indicator of the disease. The tau gene is mutated in familial, otherFeat=[]-->, belongsTo=parr
136	FTDP-17, and these mutations accelerate NFT pathology in the, otherFeat=[]-->, belongsTo=parr
137	brain (15?18). Understanding how FTDP-17 mutations pro-, otherFeat=[]-->, belongsTo=parr
138	mote tau phosphorylation can provide a better understanding, otherFeat=[]-->, belongsTo=parr
139	of how NFT pathology develops in AD and various tauopathies., otherFeat=[]-->, belongsTo=parr
140	However, when expressed in CHO cells, G272V, R406W,, otherFeat=[]-->, belongsTo=parr
141	V337M, and P301L tau mutations reduce tau phosphorylation, otherFeat=[]-->, belongsTo=parr
142	(19, 20). In COS cells, although G272V, P301L, and V337M, otherFeat=[]-->, belongsTo=parr
143	mutations do not show any significant affect, the R406W muta-, otherFeat=[]-->, belongsTo=parr
144	tion caused a reduction in tau phosphorylation (21, 22). When, otherFeat=[]-->, belongsTo=parr
145	expressed in SH-SY5Y cells subsequently differentiated into, otherFeat=[]-->, belongsTo=parr
146	neurons, the R406W, P301L, and V337M mutations reduce tau, otherFeat=[]-->, belongsTo=parr
147	phosphorylation (23). In contrast, in hippocampal neurons,, otherFeat=[]-->, belongsTo=parr
148	R406W increases tau phosphorylation (24). When phosphoryl-, otherFeat=[]-->, belongsTo=parr
149	ated by recombinant GSK3 in vitro, the P301L and V337M, otherFeat=[]-->, belongsTo=parr
150	mutations do not have any effect, and the R406W mutation, otherFeat=[]-->, belongsTo=parr
151	inhibits phosphorylation (25). However, when incubated with, otherFeat=[]-->, belongsTo=parr
152	rat brain extract, all of the G272V, P301L, V337M, and R406W, otherFeat=[]-->, belongsTo=parr
153	mutations stimulate tau phosphorylation (26). The mechanism, otherFeat=[]-->, belongsTo=parr
154	by which FTDP-17 mutations promote tau phosphorylation, otherFeat=[]-->, belongsTo=parr
155	leading to development of NFT pathology has remained, otherFeat=[]-->, belongsTo=parr
156	unclear., otherFeat=[]-->, belongsTo=parr
157	Cyclin-dependent protein kinase 5 (Cdk5) is one of the major, otherFeat=[]-->, belongsTo=parr
158	kinases that phosphorylates tau in the brain (27, 28). In this, otherFeat=[]-->, belongsTo=parr
159	study, to determine how FTDP-17 missense mutations affect, otherFeat=[]-->, belongsTo=parr
160	tau phosphorylation, we phosphorylated four FTDP-17 tau, otherFeat=[]-->, belongsTo=parr
161	mutants (G272V, P301L, V337M, and R406W) by Cdk5. We, otherFeat=[]-->, belongsTo=parr
162	have found that phosphorylation of tau by Cdk5 causes a tau, otherFeat=[]-->, belongsTo=parr
163	mobility shift to 64- and 68 kDa-bands. Although the mobility, otherFeat=[]-->, belongsTo=parr
164	shift to a 64-kDa band is achieved by phosphorylation at, otherFeat=[]-->, belongsTo=parr
165	Ser396/404 or Ser202, the mobility shift to a 68-kDa band occurs, otherFeat=[]-->, belongsTo=parr
166	only in response to phosphorylation at Ser202. We show that in, otherFeat=[]-->, belongsTo=parr
167	vitro, FTDP-17 missense mutations, by promoting phosphoryl-, otherFeat=[]-->, belongsTo=parr
168	ation at Ser202, enhance the mobility shift to 64- and 68-kDa, otherFeat=[]-->, belongsTo=parr
169	bands and inhibit the microtubule assembly-promoting activity, otherFeat=[]-->, belongsTo=parr
170	of tau. Our data suggest that Ser202 phosphorylation is the, otherFeat=[]-->, belongsTo=parr
171	major event leading to NFT pathology in AD and related, otherFeat=[]-->, belongsTo=parr
172	tauopathies., otherFeat=[]-->, belongsTo=parr
173	MATERIALS AND METHODS, otherFeat=['U']-->, belongsTo=title
174	cDNA Cloning--The longest isoform of human tau and, otherFeat=[]-->, belongsTo=parr
175	FTDP-17 tau mutants G272V, P301L, V337M, and R406W,, otherFeat=[]-->, belongsTo=parr
176	each in the pQE32 vector, were gifts from Dr. Peter Davies, otherFeat=[]-->, belongsTo=parr
177	(Albert Einstein College of Medicine, Bronx, NY). Cloning of, otherFeat=[]-->, belongsTo=parr
178	tau mutants S202A, T231A, and S396A in pcDNA3.1 vector is, otherFeat=[]-->, belongsTo=parr
179	described previously (29). Tau mutants S262A and S214A in, otherFeat=[]-->, belongsTo=parr
180	pcDNA3.1 vector were gifts from Dr. Nicole Leclerc (University, otherFeat=[]-->, belongsTo=parr
181	of Montreal). Each DNA fragment from the WT or mutant tau, otherFeat=[]-->, belongsTo=parr
182	was amplified by PCR using pfu DNA polymerase (Stratagene),, otherFeat=[]-->, belongsTo=parr
183	with a forward primer (5 -AAAAAACGCCATATGGCT-, otherFeat=[]-->, belongsTo=parr
184	GAGCCCCGC-3 ) that contained an NdeI site and a reverse, otherFeat=[]-->, belongsTo=parr
185	primer (5 -AAA AAA GGA TCC TCA CAA ACC CTG CTT, otherFeat=[]-->, belongsTo=parr
186	GG-3 ) that contained a BamHI site, and subcloned into bacte-, otherFeat=[]-->, belongsTo=parr
187	rial expression vector pET9a (Promega). Various double, otherFeat=[]-->, belongsTo=parr
188	mutants, each containing the indicated FTDP-17 and S202A, otherFeat=[]-->, belongsTo=parr
189	mutations, were cloned by PCR using their respective FTDP-17, otherFeat=[]-->, belongsTo=parr
190	mutant in pET9a vector as the template and the QuikChange II, otherFeat=[]-->, belongsTo=parr
191	site-specific mutagenesis kit (Stratagene) following the manu-, otherFeat=[]-->, belongsTo=parr
192	facturer's instruction manual. Primers used for PCR were, otherFeat=[]-->, belongsTo=parr
193	5 -CAG CGG CTA CAG CAG CCC CGG CGC CCC AGG, otherFeat=[]-->, belongsTo=parr
194	CAC TCC CGG CAG CCG C-3 and 5 -GCG GCT GCC GGG, otherFeat=[]-->, belongsTo=parr
195	AGT GCC TGG GGC GCC GGG GCT GCT GTA GCC GCT G-, otherFeat=[]-->, belongsTo=parr
196	3 . All cDNA clones and mutations were confirmed by DNA, otherFeat=[]-->, belongsTo=parr
197	sequencing., otherFeat=[]-->, belongsTo=parr
198	Proteins and Enzymes--Tau(WT) and various tau mutants, otherFeat=[]-->, belongsTo=parr
199	were purified from lysates of Escherichia coli overexpressing, otherFeat=[]-->, belongsTo=parr
200	their respective tau species essentially as described previously, otherFeat=[]-->, belongsTo=parr
201	(28). Briefly, tau expression was induced by adding isopropyl, otherFeat=[]-->, belongsTo=parr
202	1-thio- -D-galactopyranoside (0.2 mM) to the overnight bacte-, otherFeat=[]-->, belongsTo=parr
203	rial culture. The culture containing isopropyl 1-thio- -D-galac-, otherFeat=[]-->, belongsTo=note
204	topyranoside was allowed to grow for3hat37 ?C with shaking, otherFeat=[]-->, belongsTo=parr
205	and then was centrifuged. The pellet was suspended in Pipes, otherFeat=[]-->, belongsTo=parr
206	buffer (100 mM Pipes (pH 6.8), 1 mM EGTA, 1 mM MgSO4), otherFeat=[]-->, belongsTo=note
207	containing 5 mg/ml benzamidine, 1 g/ml leupeptine, 1 g/ml, otherFeat=[]-->, belongsTo=parr
208	pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 20 g/ml, otherFeat=[]-->, belongsTo=parr
209	lysozyme. The bacterial suspension was lysed by sonication and, otherFeat=[]-->, belongsTo=parr
210	then clarified by centrifugation (15,000 rpm, 15 min at 4 ?C)., otherFeat=[]-->, belongsTo=parr
211	The supernatant was placed in a boiling water bath for 20 min, otherFeat=[]-->, belongsTo=parr
212	and subsequently centrifuged. The heat-stable proteins in the, otherFeat=[]-->, belongsTo=parr
213	supernatant were loaded onto a Q-Sepharose Fast Flow column, otherFeat=[]-->, belongsTo=parr
214	( 1 ml; Amersham Biosciences) equilibrated previously in, otherFeat=[]-->, belongsTo=parr
215	Pipes buffer. The flow-through containing tau was loaded onto, otherFeat=[]-->, belongsTo=parr
216	an SP-Sepharose Fast Flow column ( 1 ml) equilibrated in, otherFeat=[]-->, belongsTo=parr
217	Pipes buffer. The column was washed with 20 column vol-, otherFeat=[]-->, belongsTo=parr
218	umes of the Pipes buffer and then eluted with Pipes buffer con-, otherFeat=[]-->, belongsTo=parr
219	taining 0.2 M NaCl. Fractions containing tau were pooled, con-, otherFeat=[]-->, belongsTo=parr
220	centrated with Aquacade III (Calbiochem) by dialysis, dialyzed, otherFeat=[]-->, belongsTo=parr
221	against Hepes buffer (25 mM Hepes (pH 7.2), 0.1 mM EDTA, 0.5, otherFeat=[]-->, belongsTo=parr
222	mM dithiothreitol, and 100 mM NaCl), and stored at 80 ?C, otherFeat=[]-->, belongsTo=parr
223	until use. Microtubules were purified from fresh bovine brain, otherFeat=[]-->, belongsTo=parr
224	extract by three cycles of temperature-induced microtubule, otherFeat=[]-->, belongsTo=parr
225	polymerization and depolymerization as described previously, otherFeat=[]-->, belongsTo=parr
226	(28, 30). Tubulin was purified from purified microtubules, otherFeat=[]-->, belongsTo=parr
227	through phosphocellulose chromatography (28, 30). Mono-, otherFeat=[]-->, belongsTo=parr
228	clonal tau 5 antibody against total tau and tau phosphorylation-, otherFeat=[]-->, belongsTo=parr
229	dependent monoclonal antibodies AT8, PHF-1, MC6, and TG3, otherFeat=[]-->, belongsTo=parr
230	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
231	MAY 15, 2009 ? VOLUME 284 ? NUMBER 20, otherFeat=['U']-->, belongsTo=nota_cab_pie
232	JOURNAL OF BIOLOGICAL CHEMISTRY 13423, otherFeat=[]-->, belongsTo=nota_cab_pie
233	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
234	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
235	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
236	de, otherFeat=[]-->, belongsTo=nota_cab_pie
237	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
238	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
239	on, otherFeat=[]-->, belongsTo=nota_cab_pie
240	May, otherFeat=[]-->, belongsTo=nota_cab_pie
241	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
242	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
243	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
244	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
245	from, otherFeat=[]-->, belongsTo=nota_cab_pie
246	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
247	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
248	have been described previously (29, 31). Polyclonal antibodies, otherFeat=[]-->, belongsTo=parr
249	pS202 and pT212 against tau phosphorylated at Ser202 and, otherFeat=[]-->, belongsTo=parr
250	Thr212, respectively, were purchased from BIOSOURCE. Cdk5, otherFeat=[]-->, belongsTo=parr
251	was purified from the extract of fresh bovine brain (28). The, otherFeat=[]-->, belongsTo=parr
252	active catalytic subunit of PKA was purchased from Sigma-Al-, otherFeat=[]-->, belongsTo=parr
253	drich. Purification of protein phosphatase 1 (PP1) from E. coli, otherFeat=[]-->, belongsTo=parr
254	extract overexpressing human PP1 has been described previ-, otherFeat=[]-->, belongsTo=parr
255	ously (32, 33)., otherFeat=[]-->, belongsTo=parr
256	Protein Concentrations--Tau(WT) concentration is based, otherFeat=[]-->, belongsTo=parr
257	on its absorption at A280 nm as described previously (28). The, otherFeat=[]-->, belongsTo=parr
258	concentrations of various FTDP-17 tau mutants were deter-, otherFeat=[]-->, belongsTo=parr
259	mined by Bio-Rad protein assay using tau(WT) as the standard., otherFeat=[]-->, belongsTo=parr
260	Concentrations of phosphorylated tau and tau mutants were, otherFeat=[]-->, belongsTo=parr
261	also determined by Bio-Rad protein assay using tau(WT) as the, otherFeat=[]-->, belongsTo=parr
262	standard. The concentration of Cdk5 is based on its activity, otherFeat=[]-->, belongsTo=parr
263	(28). PKA concentration was determined by its dry weight. The, otherFeat=[]-->, belongsTo=parr
264	concentrations of all other proteins were determined by Bio-, otherFeat=[]-->, belongsTo=parr
265	Rad proteins assay using bovine serum albumin as the standard., otherFeat=[]-->, belongsTo=parr
266	Tau Phosphorylation--Tau(WT) and various tau mutants, otherFeat=[]-->, belongsTo=parr
267	were phosphorylated by Cdk5 under identical conditions. Each, otherFeat=[]-->, belongsTo=parr
268	phosphorylation mixture contained 25 mM Hepes (pH 7.2), 0.1, otherFeat=[]-->, belongsTo=parr
269	mM EDTA, 0.2 mM dithiothreitol, 0.1 M NaCl, 10 mM MgCl2, 0.5, otherFeat=[]-->, belongsTo=note
270	mM [ 32P] ATP, 1.0 mg/ml tau, and 400 units/ml Cdk5. The, otherFeat=[]-->, belongsTo=parr
271	reaction was initiated by adding an aliquot of Cdk5 to a vial, otherFeat=[]-->, belongsTo=parr
272	containing the rest of the phosphorylation mixture at 30 ?C. At, otherFeat=[]-->, belongsTo=parr
273	the indicated time points, aliquots were withdrawn and ana-, otherFeat=[]-->, belongsTo=parr
274	lyzed for phosphorylation by filter paper assay (34) or subjected, otherFeat=[]-->, belongsTo=parr
275	to SDS-PAGE followed by Western blot analysis. Gel and blot, otherFeat=[]-->, belongsTo=parr
276	bands were scanned, and the band intensity values were used to, otherFeat=[]-->, belongsTo=parr
277	determine the relative amounts of various proteins. Phospho-, otherFeat=[]-->, belongsTo=parr
278	rylation of tau and tau mutants by PKA was also performed as, otherFeat=[]-->, belongsTo=parr
279	described above, except Cdk5 was replaced by PKA at a con-, otherFeat=[]-->, belongsTo=parr
280	centration of 10 g/ml each., otherFeat=[]-->, belongsTo=parr
281	Microtubule Assembly Assay--The microtubule assembly, otherFeat=[]-->, belongsTo=parr
282	was monitored by measuring the increase of A350 by a spectro-, otherFeat=[]-->, belongsTo=parr
283	photometer (35). The vial containing all of the components of, otherFeat=[]-->, belongsTo=parr
284	the assay except tau was incubated at 37 ?C for 1 min in a water, otherFeat=[]-->, belongsTo=parr
285	bath. To the incubated vial, the indicated prewarmed tau spe-, otherFeat=[]-->, belongsTo=parr
286	cies was added. After gentle mixing, the content of the vial was, otherFeat=[]-->, belongsTo=parr
287	transferred immediately to a quartz cuvette placed in a spectro-, otherFeat=[]-->, belongsTo=parr
288	photometer at 37 ?C. The increase in the A350 of the transferred, otherFeat=[]-->, belongsTo=parr
289	sample was recorded at 1-min intervals for 32 min. The final, otherFeat=[]-->, belongsTo=parr
290	concentrations of various components in the assay were 0.75, otherFeat=[]-->, belongsTo=parr
291	mg/ml tubulin, 100 mM Pipes (pH 6.8), 1 mM EGTA, 1 mM, otherFeat=[]-->, belongsTo=parr
292	dithiothreitol, 2 mM MgSO4,1mM GTP, 10 M taxol, and 0.2, otherFeat=[]-->, belongsTo=parr
293	mg/ml tau (indicated species). The lag phase of polymerization, otherFeat=[]-->, belongsTo=parr
294	is defined as the time at which the rise in the A350 is detected, otherFeat=[]-->, belongsTo=parr
295	since initiation of the assay. The rate of polymerization is the, otherFeat=[]-->, belongsTo=parr
296	A350 at the steady state divided by the minimum time required, otherFeat=[]-->, belongsTo=parr
297	to achieve the steady state after the lag phase, and it is expressed, otherFeat=[]-->, belongsTo=parr
298	as absorption units per min (AU/min). The amount of micro-, otherFeat=[]-->, belongsTo=parr
299	tubule formed corresponds to the maximum A350 reached dur-, otherFeat=[]-->, belongsTo=parr
300	ing the assay., otherFeat=[]-->, belongsTo=parr
301	RESULTS, otherFeat=[]-->, belongsTo=title
302	Effect of Phosphorylation on Microtubule Assembly-promot-, otherFeat=[]-->, belongsTo=parr
303	ing Activity of FTDP-17 Tau Mutants--In vitro, purified tubu-, otherFeat=[]-->, belongsTo=parr
304	lin polymerizes in the presence of GTP/Mg2 and assembles, otherFeat=[]-->, belongsTo=parr
305	into microtubules. This process consists of an initial lag phase, otherFeat=[]-->, belongsTo=parr
306	during which microtubules nucleate (35, 36). Following nucle-, otherFeat=[]-->, belongsTo=parr
307	ation, microtubules polymerize and reach the steady state. In, otherFeat=[]-->, belongsTo=parr
308	vitro microtubule assembly can be monitored spectrophoto-, otherFeat=[]-->, belongsTo=parr
309	metrically by the light scattering technique, which measures, otherFeat=[]-->, belongsTo=parr
310	the turbidity of the solution at 350 nm, which increases as the, otherFeat=[]-->, belongsTo=parr
311	microtubules assemble (35, 37). When present, tau promotes, otherFeat=[]-->, belongsTo=parr
312	microtubule assembly by influencing some or all of these, otherFeat=[]-->, belongsTo=parr
313	parameters (35?37). To determine how phosphorylation, otherFeat=[]-->, belongsTo=parr
314	affects the microtubule assembly promoting activity of various, otherFeat=[]-->, belongsTo=parr
315	tau species, we phosphorylated WT and FTDP-17 mutants by, otherFeat=[]-->, belongsTo=parr
316	Cdk5 under identical conditions. Each phosphorylated and, otherFeat=[]-->, belongsTo=parr
317	nonphosphorylated tau species was included in the microtu-, otherFeat=[]-->, belongsTo=parr
318	bule assembly mixture, and the assembly was monitored by, otherFeat=[]-->, belongsTo=parr
319	light scattering assay. The concentration of tubulin was kept, otherFeat=[]-->, belongsTo=parr
320	low so that no detectable turbidity was observed in the absence, otherFeat=[]-->, belongsTo=parr
321	of tau., otherFeat=[]-->, belongsTo=parr
322	Microtubules in the presence of tau(WT) assembled with a, otherFeat=[]-->, belongsTo=parr
323	lag time of 2 min at a rate of 0.0636 AU/min. When P301L was, otherFeat=[]-->, belongsTo=parr
324	used, the lag phase was extended to 6 min and the polymeriza-, otherFeat=[]-->, belongsTo=parr
325	tion rate was reduced to 0.0363 AU/min (Fig. 1 and  supplemen-, otherFeat=[]-->, belongsTo=parr
326	tal Table S1). These data determined that in the presence of, otherFeat=[]-->, belongsTo=parr
327	P301L, microtubule nucleation and polymerization occurred 3, otherFeat=[]-->, belongsTo=parr
328	and 1.75 times, respectively, slower than in the presence of, otherFeat=[]-->, belongsTo=parr
329	tau(WT). This, in turn, indicates that the microtubule nucle-, otherFeat=[]-->, belongsTo=parr
330	ation-promoting activity of P301L is 33.3% of that of the WT., otherFeat=[]-->, belongsTo=parr
331	Likewise, microtubule polymerization-promoting activity of, otherFeat=[]-->, belongsTo=parr
332	P301L is 57% of that of the WT. In the presence of the WT,, otherFeat=[]-->, belongsTo=parr
333	microtubule polymerization plateaued at A350 0.70, which, otherFeat=[]-->, belongsTo=parr
334	represents the amount of microtubules formed  (supplemental, otherFeat=[]-->, belongsTo=parr
335	Table S1). This value is reduced to 0.40 in the presence of, otherFeat=[]-->, belongsTo=parr
336	P301L. These data indicate that the microtubule formation-, otherFeat=[]-->, belongsTo=parr
337	promoting activity of P301L is 57.1% of that of the WT. The, otherFeat=[]-->, belongsTo=parr
338	FIGURE 1. Microtubule assembly in the presence of phosphorylated, otherFeat=[]-->, belongsTo=fig_caption
339	and nonphosphorylated tau(WT) or FTDP-17 tau mutants. Microtu-, otherFeat=[]-->, belongsTo=fig_caption
340	bule assembly was monitored by the light scattering technique described, otherFeat=[]-->, belongsTo=fig_caption
341	under "Materials and Methods" in the presence of the indicated tau spe-, otherFeat=[]-->, belongsTo=fig_caption
342	cies. Phosphorylated tau is indicated by -P. Changes in the A350 were, otherFeat=[]-->, belongsTo=fig_caption
343	recorded every min., otherFeat=[]-->, belongsTo=fig_caption
344	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
345	13424 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
346	VOLUME 284 ? NUMBER 20 ? MAY 15, 2009, otherFeat=['U']-->, belongsTo=nota_cab_pie
347	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
348	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
349	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
350	de, otherFeat=[]-->, belongsTo=nota_cab_pie
351	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
352	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
353	on, otherFeat=[]-->, belongsTo=nota_cab_pie
354	May, otherFeat=[]-->, belongsTo=nota_cab_pie
355	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
356	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
357	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
358	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
359	from, otherFeat=[]-->, belongsTo=nota_cab_pie
360	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
361	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
362	microtubule nucleation, polymerization, and formation-pro-, otherFeat=[]-->, belongsTo=parr
363	moting activities of V337M and R406W are also significantly, otherFeat=[]-->, belongsTo=parr
364	less than that of the WT (Fig. 1 and  supplemental Table S1)., otherFeat=[]-->, belongsTo=parr
365	This observation is consistent with previous reports (37, 38), otherFeat=[]-->, belongsTo=parr
366	and indicates that FTDP-17 mutations impair the microtubule, otherFeat=[]-->, belongsTo=parr
367	assembly-promoting activity of tau., otherFeat=[]-->, belongsTo=parr
368	Phosphorylation reduced the microtubule nucleation,, otherFeat=[]-->, belongsTo=parr
369	polymerization, and formation-promoting activities of, otherFeat=[]-->, belongsTo=parr
370	tau(WT) to 33.3, 29.5, and 28.6%, respectively (Fig. 1 and  sup-, otherFeat=[]-->, belongsTo=parr
371	plemental Table S1). Compared with its nonphosphorylated, otherFeat=[]-->, belongsTo=parr
372	counterpart, the microtubule nucleation, polymerization, and, otherFeat=[]-->, belongsTo=parr
373	formation-promoting activities of phosphorylated P301L were, otherFeat=[]-->, belongsTo=parr
374	54.6, 30.5, and 50.0%, respectively, those of V337M were 36.4,, otherFeat=[]-->, belongsTo=parr
375	31.7, and 50.0%, respectively, and those of R406W were 58.5,, otherFeat=[]-->, belongsTo=parr
376	36.0, and 45.9%, respectively. Thus, phosphorylation also, otherFeat=[]-->, belongsTo=parr
377	reduced the microtubule assembly-promoting activities of all, otherFeat=[]-->, belongsTo=parr
378	FTDP-17 tau mutants., otherFeat=[]-->, belongsTo=parr
379	Effect of FTDP-17 Mutations on Tau Phosphorylation--As, otherFeat=[]-->, belongsTo=parr
380	shown in  supplemental Table S1, the rate of microtubule nucle-, otherFeat=[]-->, belongsTo=parr
381	ation and polymerization and the amount of microtubules, otherFeat=[]-->, belongsTo=parr
382	formed are significantly less in the presence of all phosphoryl-, otherFeat=[]-->, belongsTo=parr
383	ated FTDP-17 mutants when compared with that of the phos-, otherFeat=[]-->, belongsTo=parr
384	phorylated WT. One likely explanation for this observation is, otherFeat=[]-->, belongsTo=parr
385	that the FTDP-17 mutation and phosphorylation act additively, otherFeat=[]-->, belongsTo=parr
386	to reduce the microtubule assembly-promoting activity of tau., otherFeat=[]-->, belongsTo=parr
387	However, some FTDP-17 mutations may promote tau phos-, otherFeat=[]-->, belongsTo=parr
388	phorylation and by doing so may further affect the microtubule, otherFeat=[]-->, belongsTo=parr
389	assembly-promoting activity of tau. To understand how phos-, otherFeat=[]-->, belongsTo=parr
390	phorylation affects the microtubule assembly-promoting activ-, otherFeat=[]-->, belongsTo=parr
391	ity of various FTDP-17 tau mutants, we set out to determine, otherFeat=[]-->, belongsTo=parr
392	how each FTDP-17 mutation affects tau phosphorylation. We, otherFeat=[]-->, belongsTo=parr
393	phosphorylated tau(WT) and various FTDP-17 tau mutants by, otherFeat=[]-->, belongsTo=parr
394	Cdk5 under identical conditions and analyzed them on an SDS-, otherFeat=[]-->, belongsTo=parr
395	gel (Fig. 2). Phosphorylated tau(WT) displayed two bands of, otherFeat=[]-->, belongsTo=parr
396	sizes 60 and 64 kDa (lane 2). Control tau(WT), incubated, otherFeat=[]-->, belongsTo=parr
397	with all of the components of the phosphorylation mixture, otherFeat=[]-->, belongsTo=parr
398	except Cdk5, migrated as one 60-kDa band (lane 6), which is, otherFeat=[]-->, belongsTo=parr
399	consistent with a previous report (39) and shows that Cdk5, otherFeat=[]-->, belongsTo=parr
400	phosphorylation causes a mobility shift of tau(WT) from a, otherFeat=[]-->, belongsTo=parr
401	60- to 64-kDa band on an SDS-gel. Interestingly, when phos-, otherFeat=[]-->, belongsTo=parr
402	phorylated P301L was analyzed, it displayed three bands of sizes, otherFeat=[]-->, belongsTo=parr
403	60, 64, and 68 kDa (lane 3). Phosphorylated V337M and, otherFeat=[]-->, belongsTo=parr
404	R406W also displayed 60-, 64-, and 68-kDa bands (lanes 4, 5)., otherFeat=[]-->, belongsTo=parr
405	When a portion of each sample used to generate Fig. 2 was, otherFeat=[]-->, belongsTo=parr
406	Western blotted using anti-tau antibody, in addition to the, otherFeat=[]-->, belongsTo=parr
407	60- and 64-kDa bands, phosphorylated tau(WT) also, otherFeat=[]-->, belongsTo=parr
408	showed a faint 68-kDa band (data not shown). All FTDP-17, otherFeat=[]-->, belongsTo=parr
409	mutants, on the other hand, displayed prominent 60-, 64-,, otherFeat=[]-->, belongsTo=parr
410	and 68-kDa bands. When monoclonal antibody AT8, which, otherFeat=[]-->, belongsTo=parr
411	recognizes phosphorylated tau, was used in Western blot anal-, otherFeat=[]-->, belongsTo=parr
412	ysis, all 60-, 64-, and 68-kDa bands of WT and FTDP-17, otherFeat=[]-->, belongsTo=parr
413	mutants displayed immunoreactivity (data not shown but see, otherFeat=[]-->, belongsTo=parr
414	below). When phosphorylated, various tau species were incu-, otherFeat=[]-->, belongsTo=parr
415	bated with protein phosphatase 1 and the products analyzed by, otherFeat=[]-->, belongsTo=parr
416	SDS-PAGE. All of the WT and FTDP-17 mutants almost com-, otherFeat=[]-->, belongsTo=parr
417	pletely lost the 64- and 68-kDa bands and displayed a major, otherFeat=[]-->, belongsTo=parr
418	60-kDa band (data not shown). Based on these results, we, otherFeat=[]-->, belongsTo=parr
419	concluded that phosphorylation by Cdk5 caused a mobility, otherFeat=[]-->, belongsTo=parr
420	shift of tau(WT) to 64- and 68-kDa bands and that the, otherFeat=[]-->, belongsTo=parr
421	FTDP-17 missense mutations P301L, V337M, and R406W pro-, otherFeat=[]-->, belongsTo=parr
422	moted a phosphorylation-induced mobility shift., otherFeat=[]-->, belongsTo=parr
423	We reasoned that there were two possibilities to explain the, otherFeat=[]-->, belongsTo=parr
424	increased mobility shift of FTDP-17 tau mutants upon phos-, otherFeat=[]-->, belongsTo=parr
425	phorylation. Because all FTDP-17 mutants tested displayed, otherFeat=[]-->, belongsTo=parr
426	more mobility shift than the WT, the first possibility is that, otherFeat=[]-->, belongsTo=parr
427	FTDP-17 mutations promote phosphorylation-induced mobil-, otherFeat=[]-->, belongsTo=parr
428	ity shift. As we did not include a nonspecific point mutant in our, otherFeat=[]-->, belongsTo=parr
429	assay, the second possibility is that structural change caused by, otherFeat=[]-->, belongsTo=parr
430	any point mutation causes mobility shift of tau upon phospho-, otherFeat=[]-->, belongsTo=parr
431	rylation by Cdk5. To discriminate between the above two pos-, otherFeat=[]-->, belongsTo=parr
432	sibilities, we decided to examine another FTDP-17 tau mutant,, otherFeat=[]-->, belongsTo=parr
433	G272V, and two nonspecific mutants, S214A and S262A., otherFeat=[]-->, belongsTo=parr
434	Because FTDP-17 mutant P301L, V337M, and R406W are, otherFeat=[]-->, belongsTo=parr
435	located at the C terminus, we selected G272V, situated at the, otherFeat=[]-->, belongsTo=parr
436	central region of the tau molecule. We chose S214A and S262A, otherFeat=[]-->, belongsTo=parr
437	because these sites are neither mutated in any tauopathies (2), otherFeat=[]-->, belongsTo=parr
438	nor are they phosphorylated by Cdk5 (27). Moreover, Ser262 is, otherFeat=[]-->, belongsTo=parr
439	located only 10 residues away from Gly272, and S262A is a good, otherFeat=[]-->, belongsTo=parr
440	control against G272V. We phosphorylated WT, S214A,, otherFeat=[]-->, belongsTo=parr
441	S262A, G272V, P301L, V337M, and R406W by Cdk5 under, otherFeat=[]-->, belongsTo=parr
442	identical conditions and analyzed the products by SDS-PAGE., otherFeat=[]-->, belongsTo=parr
443	Phosphorylated WT showed 60- and 64-kDa bands, whereas, otherFeat=[]-->, belongsTo=parr
444	phosphorylated P301L, V337M, and R406W all showed 60-,, otherFeat=[]-->, belongsTo=parr
445	64-, and 68-kDa bands. Phosphorylated S214A and S262A, on, otherFeat=[]-->, belongsTo=parr
446	the other hand, migrated as 60- and 64-kDa bands in a man-, otherFeat=[]-->, belongsTo=parr
447	ner similar to the phosphorylated WT (data not shown, but see, otherFeat=[]-->, belongsTo=parr
448	Fig. 3A). More importantly, phosphorylated G272V displayed, otherFeat=[]-->, belongsTo=parr
449	FIGURE 2. SDS-PAGE of phosphorylated tau(WT) and FTDP-17 tau, otherFeat=[]-->, belongsTo=fig_caption
450	mutants. Indicated tau species (5 g each) phosphorylated by Cdk5 under, otherFeat=[]-->, belongsTo=fig_caption
451	identical conditions for 60 min (lanes 2?5) or control samples incubated with, otherFeat=[]-->, belongsTo=fig_caption
452	all the components of the phosphorylation mixture except Cdk5 (lanes 6 ?9), otherFeat=[]-->, belongsTo=fig_caption
453	were subjected to 10% SDS-PAGE. The resulting gel was stained with Coo-, otherFeat=[]-->, belongsTo=fig_caption
454	massie Brilliant Blue. M (lane 1) represents standard molecular weight marker., otherFeat=[]-->, belongsTo=fig_caption
455	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
456	MAY 15, 2009 ? VOLUME 284 ? NUMBER 20, otherFeat=['U']-->, belongsTo=nota_cab_pie
457	JOURNAL OF BIOLOGICAL CHEMISTRY 13425, otherFeat=[]-->, belongsTo=nota_cab_pie
458	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
459	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
460	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
461	de, otherFeat=[]-->, belongsTo=nota_cab_pie
462	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
463	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
464	on, otherFeat=[]-->, belongsTo=nota_cab_pie
465	May, otherFeat=[]-->, belongsTo=nota_cab_pie
466	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
467	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
468	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
469	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
470	from, otherFeat=[]-->, belongsTo=nota_cab_pie
471	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
472	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
473	60-, 64-, and 68-kDa bands similar to those shown by phos-, otherFeat=[]-->, belongsTo=parr
474	phorylated P301L, V337M, and R406W (data not shown). Thus,, otherFeat=[]-->, belongsTo=parr
475	among the three new mutants tested (G272V, S214A, and, otherFeat=[]-->, belongsTo=parr
476	S262A), only G272V associated with FTDP-17 promoted tau a, otherFeat=[]-->, belongsTo=parr
477	mobility shift to a 68-kDa band upon phosphorylation by, otherFeat=[]-->, belongsTo=parr
478	Cdk5. These data are consistent with the idea that FTDP-17, otherFeat=[]-->, belongsTo=parr
479	mutations promote tau mobility shift upon phosphorylation by, otherFeat=[]-->, belongsTo=parr
480	Cdk5., otherFeat=[]-->, belongsTo=parr
481	To determine the basis of the mobility shift, we wanted to, otherFeat=[]-->, belongsTo=parr
482	know first whether FTDP-17 mutations stimulate tau phospho-, otherFeat=[]-->, belongsTo=parr
483	rylation. We phosphorylated tau(WT) and various FTDP-17, otherFeat=[]-->, belongsTo=parr
484	tau mutants by Cdk5 under identical conditions for different, otherFeat=[]-->, belongsTo=parr
485	time periods and determined the number of phosphate incor-, otherFeat=[]-->, belongsTo=parr
486	porated into each tau species. Tau(WT) incorporated 1.1 mol, otherFeat=[]-->, belongsTo=parr
487	of phosphate/mol of protein in 15 min. This value increased to, otherFeat=[]-->, belongsTo=parr
488	2.4 in 30 min and became 4.1 in 60 min. On SDS-gel,, otherFeat=[]-->, belongsTo=parr
489	tau(WT) phosphorylated for 15 min migrated as a 60-kDa, otherFeat=[]-->, belongsTo=parr
490	band but became 60- and 64-kDa bands after 60 min of phos-, otherFeat=[]-->, belongsTo=parr
491	phorylation (data not shown). G272V incorporated 1.5, 3.4,, otherFeat=[]-->, belongsTo=parr
492	and 5.2 mol of phosphate/mol of protein in 15, 30, and 60 min,, otherFeat=[]-->, belongsTo=parr
493	respectively. In 15, 30, and 60 min, P301L contained 1.3, 2.5,, otherFeat=[]-->, belongsTo=parr
494	and 4 mol of phosphate/mol of protein, respectively, whereas, otherFeat=[]-->, belongsTo=parr
495	V337M incorporated 1.2, 3.2, and 3.9 mol of phosphate/mol, otherFeat=[]-->, belongsTo=parr
496	of protein, respectively. R406W, on the other hand, incorpo-, otherFeat=[]-->, belongsTo=parr
497	rated 1.2, 2.5, and 3.2 mol of phosphate/mol of protein in 15,, otherFeat=[]-->, belongsTo=parr
498	30, and 60 min, respectively. Thus after 60 min, G272V incor-, otherFeat=[]-->, belongsTo=parr
499	porated more phosphate, whereas P301L and V337M incorpo-, otherFeat=[]-->, belongsTo=parr
500	rated amounts similar to the WT. R406W, on the other hand,, otherFeat=[]-->, belongsTo=parr
501	incorporated less phosphate than the WT. On SDS-gel, how-, otherFeat=[]-->, belongsTo=parr
502	ever, all FTDP-17 mutants phosphorylated for 60 min migrated, otherFeat=[]-->, belongsTo=parr
503	as 60-, 64-, and 68-kDa bands (data not shown, but see Fig. 2)., otherFeat=[]-->, belongsTo=parr
504	These data indicated that the increased mobility shift of phos-, otherFeat=[]-->, belongsTo=parr
505	phorylated FTDP-17 mutants was not due to the higher extent, otherFeat=[]-->, belongsTo=parr
506	of total phosphorylation of mutants compared with the WT., otherFeat=[]-->, belongsTo=parr
507	Effect of FTDP-17 Mutations on Site-specific Phosphorylation, otherFeat=[]-->, belongsTo=parr
508	of Tau--To evaluate whether FTDP-17 mutations, by promot-, otherFeat=[]-->, belongsTo=parr
509	ing tau phosphorylation at any specific site(s), increase the tau, otherFeat=[]-->, belongsTo=parr
510	mobility shift, we phosphorylated tau(WT) and various, otherFeat=[]-->, belongsTo=parr
511	FTDP-17 tau mutants by Cdk5 under identical conditions., otherFeat=[]-->, belongsTo=parr
512	Controls were S214A and S262A. Phosphorylated products, otherFeat=[]-->, belongsTo=parr
513	were analyzed by Western blot using antibodies that recognize, otherFeat=[]-->, belongsTo=parr
514	phosphorylated tau on proline-directed sites Ser396, Ser404,, otherFeat=[]-->, belongsTo=parr
515	Ser235, Thr231, Ser202/205, and Thr212, which are potential tar-, otherFeat=[]-->, belongsTo=parr
516	gets of Cdk5 (27)., otherFeat=[]-->, belongsTo=parr
517	As shown in Fig. 3, compared with the WT, G272V and, otherFeat=[]-->, belongsTo=parr
518	P301L are more phosphorylated and R406W is less phospho-, otherFeat=[]-->, belongsTo=parr
519	rylated at Ser396/404.AtSer235, G272V and V337M are more, otherFeat=[]-->, belongsTo=parr
520	phosphorylated and R406W is less phosphorylated than the, otherFeat=[]-->, belongsTo=parr
521	WT. At Thr231, WT and all mutants are phosphorylated to, otherFeat=[]-->, belongsTo=parr
522	similar extents. However, at Ser202, G272V, P301L, V337M, and, otherFeat=[]-->, belongsTo=parr
523	R406W are 1.8-, 2.2-. 2.1-, and 2.5-fold more phosphorylated, otherFeat=[]-->, belongsTo=parr
524	than the WT. At Thr212, on the other hand, neither WT nor any, otherFeat=[]-->, belongsTo=parr
525	of the FTDP-17 mutants was phosphorylated. Thus, at Ser235, otherFeat=[]-->, belongsTo=parr
526	and Ser396/404, some mutations promoted and some inhibited, otherFeat=[]-->, belongsTo=parr
527	phosphorylation. At Ser202, neither the S214A nor the S262A, otherFeat=[]-->, belongsTo=parr
528	control showed any effect, whereas all FTDP-17 mutations pro-, otherFeat=[]-->, belongsTo=parr
529	FIGURE 3. Site-specific phosphorylation of tau and FTDP-17 tau mutants by Cdk5. Tau(WT) and the indicated tau mutants were phosphorylated by Cdk5, otherFeat=[]-->, belongsTo=fig_caption
530	for 60 min. Each phosphorylated sample (1 g each) was subjected to Western blotting using antibody specific for total tau or tau phosphorylated at the, otherFeat=[]-->, belongsTo=fig_caption
531	indicated site. Blots were scanned, and based on the intensities of various bands relative tau phosphorylation was calculated. A, Western blots. B, relative, otherFeat=[]-->, belongsTo=fig_caption
532	phosphorylation. To calculate relative phosphorylation, the sum of the band intensity values of all bands in a lane of the blot of the indicated tau species, otherFeat=[]-->, belongsTo=fig_caption
533	representing the tau phosphorylated at indicated site was normalized against the sum of the band intensity values of all bands of that tau species in that lane, otherFeat=[]-->, belongsTo=fig_caption
534	of the blot representing the total tau. The resulting value of each species was further normalized against the resulting value of the WT to be expressed as, otherFeat=[]-->, belongsTo=fig_caption
535	percent of the WT. All values are the average of three determinations. Tau phosphorylated by GST-GSK3 was used as a positive control on lane 1 of pT212 blot., otherFeat=[]-->, belongsTo=fig_caption
536	Recombinant GST-GSK3 was purified as described (30, 53)., otherFeat=[]-->, belongsTo=fig_caption
537	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
538	13426 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
539	VOLUME 284 ? NUMBER 20 ? MAY 15, 2009, otherFeat=['U']-->, belongsTo=nota_cab_pie
540	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
541	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
542	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
543	de, otherFeat=[]-->, belongsTo=nota_cab_pie
544	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
545	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
546	on, otherFeat=[]-->, belongsTo=nota_cab_pie
547	May, otherFeat=[]-->, belongsTo=nota_cab_pie
548	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
549	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
550	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
551	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
552	from, otherFeat=[]-->, belongsTo=nota_cab_pie
553	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
554	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
555	moted phosphorylation. To substantiate these data, we ana-, otherFeat=[]-->, belongsTo=parr
556	lyzed all of the above phosphorylated samples by using a mono-, otherFeat=[]-->, belongsTo=parr
557	clonal AT8 antibody specific for Ser202-phosphorylated tau., otherFeat=[]-->, belongsTo=parr
558	Like pS202, AT8 antibody indicated that all FTDP-17 muta-, otherFeat=[]-->, belongsTo=parr
559	tions promote tau phosphorylation at Ser202 (data not shown)., otherFeat=[]-->, belongsTo=parr
560	When Western blotted using tau 5 antibody for total tau,, otherFeat=[]-->, belongsTo=parr
561	phosphorylated tau(WT) showed two major bands of sizes 60, otherFeat=[]-->, belongsTo=parr
562	and 64 kDa and a faint 68-kDa band, whereas V337M,, otherFeat=[]-->, belongsTo=parr
563	R406W, P301L, and G272V displayed prominent 60-, 64-,, otherFeat=[]-->, belongsTo=parr
564	and 68-kDa bands (Fig. 3A). PHF-1 antibody, specific for, otherFeat=[]-->, belongsTo=parr
565	Ser396/404-phosphorylated tau, stained 60- and 64-kDa bands, otherFeat=[]-->, belongsTo=parr
566	and failed to recognize the 68-kDa band of all tau species., otherFeat=[]-->, belongsTo=parr
567	Similarly, MC6 and TG3 antibodies, which recognize tau phos-, otherFeat=[]-->, belongsTo=parr
568	phorylated at Ser235 and Thr231, respectively, stained only a, otherFeat=[]-->, belongsTo=parr
569	60-kDa band for WT and all FTDP-17 mutants. However,, otherFeat=[]-->, belongsTo=parr
570	pS202 antibody, specific for tau phosphorylated at Ser202, rec-, otherFeat=[]-->, belongsTo=parr
571	ognized all three, the 60-, 64-, and 68-kDa bands. These data, otherFeat=[]-->, belongsTo=parr
572	indicate that the 60-kDa band of all tau species is phospho-, otherFeat=[]-->, belongsTo=parr
573	rylated at Ser396/404, Ser235, Thr231, and Ser202. Similarly, the, otherFeat=[]-->, belongsTo=parr
574	64-kDa band of all tau species is phosphorylated at Ser396/404, otherFeat=[]-->, belongsTo=parr
575	and Ser202, and the 68-kDa band is phosphorylated at Ser202, otherFeat=[]-->, belongsTo=parr
576	but not at Ser396/404, Ser235,orThr231., otherFeat=[]-->, belongsTo=parrnote
577	Next, we phosphorylated tau and various FTDP-17, otherFeat=[]-->, belongsTo=parr
578	mutants at different time points and analyzed them by West-, otherFeat=[]-->, belongsTo=parr
579	ern blot using AT8 antibody. Tau(WT) phosphorylated by, otherFeat=[]-->, belongsTo=parr
580	Cdk5 for 5 min became immunoreactive (Fig. 4A). At 30 min,, otherFeat=[]-->, belongsTo=parr
581	tau(WT) displayed mobility shift, and at 60 min 60-, 64-,, otherFeat=[]-->, belongsTo=parr
582	and 68-kDa bands became visible. G272V, P301L, V337M,, otherFeat=[]-->, belongsTo=parr
583	and R406W also displayed Ser202-phosphorylated 60-,, otherFeat=[]-->, belongsTo=parr
584	64-, and 68-kDa bands. Blot band quantification determined, otherFeat=[]-->, belongsTo=parr
585	that at the 60-min time point, out of all Ser202-phosphoryl-, otherFeat=[]-->, belongsTo=parr
586	ated tau(WT), 40% remained as a 60-kDa band and the, otherFeat=[]-->, belongsTo=parr
587	rest shifted to 64 ( 50%)- and 68 ( 10%)-kDa bands (Fig., otherFeat=[]-->, belongsTo=parr
588	4B). G272V and P301L, on the other hand, displayed a 68-, otherFeat=[]-->, belongsTo=parr
589	kDa band with a relative amount (48 and 49%, respectively), otherFeat=[]-->, belongsTo=parr
590	of the total. The relative amount of 68-kDa bands was 45, otherFeat=[]-->, belongsTo=parr
591	and 44% of the total in V337M and R406W, respectively., otherFeat=[]-->, belongsTo=parr
592	When compared with the WT, G272V, P301L, V337M, and, otherFeat=[]-->, belongsTo=parr
593	R406W displayed, respectively 4.8-, 4.9-, 4.5-, and 4.4-, otherFeat=[]-->, belongsTo=parr
594	fold more Ser202-phosphorylated 68-kDa band upon phos-, otherFeat=[]-->, belongsTo=parr
595	phorylation by Cdk5 (Fig. 4B). Thus, all FTDP-17 mutations, otherFeat=[]-->, belongsTo=parr
596	promoted phosphorylation at Ser202 as well as mobility shift, otherFeat=[]-->, belongsTo=parr
597	to a 68-kDa band., otherFeat=[]-->, belongsTo=parr
598	Effect of Ser202 Phosphorylation on Tau Structure--Tau(WT), otherFeat=[]-->, belongsTo=parr
599	displayed a mobility shift that correlated with phosphorylation, otherFeat=[]-->, belongsTo=parr
600	at Ser202, and FTDP-17 mutations accelerated this process, otherFeat=[]-->, belongsTo=parr
601	(Figs. 3 and 4). To determine the significance of this phenome-, otherFeat=[]-->, belongsTo=parr
602	non, we wanted to know the role of Ser202 phosphorylation on, otherFeat=[]-->, belongsTo=parr
603	the tau mobility shift on SDS-gel/Western blot. We phospho-, otherFeat=[]-->, belongsTo=parr
604	rylated tau(WT) and the site-specific tau mutant S202A under, otherFeat=[]-->, belongsTo=parr
605	identical conditions and analyzed the products by Western blot, otherFeat=[]-->, belongsTo=parr
606	analysis. Tau(WT) became increasingly phosphorylated with, otherFeat=[]-->, belongsTo=parr
607	time (Fig. 5A, lanes 2?5). At 15 min, tau(WT) displayed mobil-, otherFeat=[]-->, belongsTo=parr
608	ity shift to 64 kDa, which became prominent at 60 min (Fig., otherFeat=[]-->, belongsTo=parr
609	5B, lanes 2? 4). At 120 min, the 60-kDa band of tau(WT), otherFeat=[]-->, belongsTo=parr
610	faded significantly with the appearance of a 68-kDa band (Fig., otherFeat=[]-->, belongsTo=parr
611	5B, lane 5). S202A showed mobility shift, and a 64-kDa band, otherFeat=[]-->, belongsTo=parr
612	became visible at 15 min. With increasing time, the relative, otherFeat=[]-->, belongsTo=parr
613	amount of 64-kDa band increased progressively, and that of, otherFeat=[]-->, belongsTo=parr
614	the 60-kDa band decreased. However, even with phosphoryl-, otherFeat=[]-->, belongsTo=parr
615	ation at 120 min the 68-kDa band was not formed signifi-, otherFeat=[]-->, belongsTo=parr
616	cantly (Fig. 5B, lane 11). Thus, unlike tau(WT), S202A failed to, otherFeat=[]-->, belongsTo=parr
617	display the 68-kDa mobility shift in response to Cdk5 phos-, otherFeat=[]-->, belongsTo=parr
618	phorylation. These data indicate that tau does not display, otherFeat=[]-->, belongsTo=parr
619	mobility shift to the 68-kDa band upon phosphorylation by, otherFeat=[]-->, belongsTo=parr
620	Cdk5 if phosphorylation at Ser202 is blocked., otherFeat=[]-->, belongsTo=parr
621	Tau is phosphorylated at multiple sites (8). Studies have, otherFeat=[]-->, belongsTo=parr
622	shown that tau phosphorylation on some sites affect subse-, otherFeat=[]-->, belongsTo=parr
623	quent phosphorylation on other sites (29). To determine, otherFeat=[]-->, belongsTo=parr
624	whether blocking Ser202 phosphorylation may inhibit phospho-, otherFeat=[]-->, belongsTo=parr
625	rylation at other sites, which may prevent mobility shift, phos-, otherFeat=[]-->, belongsTo=parr
626	phorylated tau(WT) and S202A were Western blotted using, otherFeat=[]-->, belongsTo=parr
627	antibodies that recognize phosphorylated tau (Fig. 6). Tau(WT), otherFeat=[]-->, belongsTo=parr
628	was phosphorylated at Ser202, Ser396/404, Ser235, and Thr231, otherFeat=[]-->, belongsTo=parr
629	(Fig. 6, lane 3). S202A, on the other hand, was phosphorylated, otherFeat=[]-->, belongsTo=parr
630	on all of the above sites except Ser202 (Fig. 6, lane 4). Thus,, otherFeat=[]-->, belongsTo=parr
631	blocking Ser202 phosphorylation did not prevent phosphoryla-, otherFeat=[]-->, belongsTo=parr
632	tion at any of the major sites known to be phosphorylated by, otherFeat=[]-->, belongsTo=parr
633	Cdk5., otherFeat=[]-->, belongsTo=parr
634	Cdk5 phosphorylates tau on a number of sites (27). Among, otherFeat=[]-->, belongsTo=parr
635	these sites, Ser396 and Thr231 were reported to cause tau con-, otherFeat=[]-->, belongsTo=parr
636	formational change (6, 29, 40). To evaluate whether mobility, otherFeat=[]-->, belongsTo=parr
637	shift caused by Cdk5 phosphorylation is specific for Ser202,we, otherFeat=[]-->, belongsTo=parr
638	phosphorylated tau(WT), S202A, S396A, and T231A by Cdk5, otherFeat=[]-->, belongsTo=parr
639	and analyzed the products by Western blot analysis (Fig. 7)., otherFeat=[]-->, belongsTo=parr
640	Phosphorylated tau(WT) migrated as 60-, 64-, and 68-kDa, otherFeat=[]-->, belongsTo=parr
641	bands (Fig. 7, lanes 12?15). Phosphorylated S202A was not, otherFeat=[]-->, belongsTo=parr
642	phosphorylated at Ser202, and it displayed 60- and 64-kDa but, otherFeat=[]-->, belongsTo=parr
643	not 68-kDa bands (Fig. 7, lanes 7?10). S396A was phospho-, otherFeat=[]-->, belongsTo=parr
644	FIGURE 4. Phosphorylation of tau and FTDP-17 tau mutants by Cdk5 on, otherFeat=[]-->, belongsTo=fig_caption
645	Ser202. The indicated tau species were phosphorylated by Cdk5. At the indi-, otherFeat=[]-->, belongsTo=fig_caption
646	cated time points, aliquots were withdrawn, and 1 g of each sample was, otherFeat=[]-->, belongsTo=fig_caption
647	subjected to Western blot analysis using AT8 antibody that recognizes tau, otherFeat=[]-->, belongsTo=fig_caption
648	phosphorylated at Ser202. Based on the intensities of various bands at the, otherFeat=[]-->, belongsTo=fig_caption
649	60-min time point, the relative amount of a 68-kDa band of each sample, otherFeat=[]-->, belongsTo=fig_caption
650	was determined. A, Western blots. B, relative amount. To calculate the relative, otherFeat=[]-->, belongsTo=fig_caption
651	amount, the intensity value of the 68-kDa band of each sample in each blot, otherFeat=[]-->, belongsTo=fig_caption
652	was normalized against the sum of the band intensity values of 60-, 64-, and, otherFeat=[]-->, belongsTo=fig_caption
653	68-kDa bands of that sample in that blot. The values are the average of three, otherFeat=[]-->, belongsTo=fig_caption
654	determinations., otherFeat=[]-->, belongsTo=fig_caption
655	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
656	MAY 15, 2009 ? VOLUME 284 ? NUMBER 20, otherFeat=['U']-->, belongsTo=nota_cab_pie
657	JOURNAL OF BIOLOGICAL CHEMISTRY 13427, otherFeat=[]-->, belongsTo=nota_cab_pie
658	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
659	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
660	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
661	de, otherFeat=[]-->, belongsTo=nota_cab_pie
662	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
663	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
664	on, otherFeat=[]-->, belongsTo=nota_cab_pie
665	May, otherFeat=[]-->, belongsTo=nota_cab_pie
666	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
667	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
668	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
669	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
670	from, otherFeat=[]-->, belongsTo=nota_cab_pie
671	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
672	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
673	rylated at Ser202 and showed 60-, 64-, and 68-kDa bands (Fig., otherFeat=[]-->, belongsTo=parr
674	7, lanes 1? 4). Similarly, phosphorylated T231A was phospho-, otherFeat=[]-->, belongsTo=parr
675	rylated at Ser202 and displayed 60-, 64-, and 68-kDa bands, otherFeat=[]-->, belongsTo=parr
676	(Fig. 7, lanes 17?20). Thus, blocking phosphorylation at Ser202,, otherFeat=[]-->, belongsTo=parr
677	but not at Ser396 or Thr231, prevented mobility shift to a 68-, otherFeat=[]-->, belongsTo=parr
678	kDa band upon Cdk5 phosphorylation., otherFeat=[]-->, belongsTo=parr
679	PKA is a non-proline-directed kinase and phosphorylates tau, otherFeat=[]-->, belongsTo=parr
680	at several sites including Ser214 but not at Ser202 (34, 41, 42). To, otherFeat=[]-->, belongsTo=parr
681	gain more evidence in favor of the idea that Ser202 phosphoryl-, otherFeat=[]-->, belongsTo=parr
682	ation is the major factor in the tau mobility shift on SDS-PAGE,, otherFeat=[]-->, belongsTo=parr
683	we phosphorylated tau WT and, otherFeat=[]-->, belongsTo=parr
684	S202A by Cdk5 or PKA for 120 min., otherFeat=[]-->, belongsTo=parr
685	Products were analyzed by Western, otherFeat=[]-->, belongsTo=parr
686	blot using tau 5 antibody (data not, otherFeat=[]-->, belongsTo=parr
687	included). Tau(WT) phosphoryla-, otherFeat=[]-->, belongsTo=parr
688	ted by Cdk5 for 120 min showed, otherFeat=[]-->, belongsTo=parr
689	60-, 64-, and 68-kDa bands., otherFeat=[]-->, belongsTo=parr
690	S202A phosphorylated by Cdk5, otherFeat=[]-->, belongsTo=parr
691	under identical conditions showed, otherFeat=[]-->, belongsTo=parr
692	only 60- and 64-kDa bands. Both, otherFeat=[]-->, belongsTo=parr
693	tau(WT) and S202A phosphoryla-, otherFeat=[]-->, belongsTo=parr
694	ted by PKA, on the other hand,, otherFeat=[]-->, belongsTo=parr
695	showed two bands of sizes 60 and, otherFeat=[]-->, belongsTo=parr
696	64 kDa (data not shown). Thus,, otherFeat=[]-->, belongsTo=parr
697	Cdk5 phosphorylates tau at Ser202, otherFeat=[]-->, belongsTo=parr
698	and causes mobility shift to a 68-, otherFeat=[]-->, belongsTo=parr
699	kDa band, and PKA, which does not, otherFeat=[]-->, belongsTo=parr
700	phosphorylate Ser202, does not, otherFeat=[]-->, belongsTo=parr
701	cause this shift. Based on these, otherFeat=[]-->, belongsTo=parr
702	results, we concluded that phospho-, otherFeat=[]-->, belongsTo=parr
703	rylation at Ser202 is a major determi-, otherFeat=[]-->, belongsTo=parr
704	nant for the tau mobility shift to the, otherFeat=[]-->, belongsTo=parr
705	68-kDa band upon Cdk5, otherFeat=[]-->, belongsTo=parr
706	phosphorylation., otherFeat=[]-->, belongsTo=parr
707	Effect of Ser202 Phosphorylation, otherFeat=[]-->, belongsTo=parrnote
708	on Microtubule Assembly-promot-, otherFeat=[]-->, belongsTo=parr
709	ing Activity of Tau--To determine why FTDP-17 mutations, otherFeat=[]-->, belongsTo=parr
710	promote phosphorylation at Ser202, we examined the effect of, otherFeat=[]-->, belongsTo=parr
711	Ser202 phosphorylation on the microtubule assembly-promot-, otherFeat=[]-->, belongsTo=parr
712	ing activity of tau. We phosphorylated WT and S202A by Cdk5., otherFeat=[]-->, belongsTo=parr
713	As controls, we phosphorylated T231A, S396S, and S214A., otherFeat=[]-->, belongsTo=parr
714	Thr231 and Ser396 are phosphorylated in vivo (8) and in vitro by, otherFeat=[]-->, belongsTo=parr
715	Cdk5 (27). Ser214 is not phosphorylated by Cdk5, and hence it, otherFeat=[]-->, belongsTo=parr
716	was used to monitor the effect of the Ser to Ala mutation on tau, otherFeat=[]-->, belongsTo=parr
717	activity. Microtubule assembly-promoting activities of non-, otherFeat=[]-->, belongsTo=parr
718	phosphorylated and phosphorylated tau were monitored., otherFeat=[]-->, belongsTo=parr
719	As shown in Fig. 8 and  supplemental Table S2, nonphospho-, otherFeat=[]-->, belongsTo=parr
720	rylated S202A, T231A, S396A, S214A, and WT promoted, otherFeat=[]-->, belongsTo=parr
721	microtubule assembly with similar nucleation time and polym-, otherFeat=[]-->, belongsTo=parr
722	erization rate, leading to the formation of similar amounts of, otherFeat=[]-->, belongsTo=parr
723	microtubules. Likewise, the microtubule assembly-promoting, otherFeat=[]-->, belongsTo=parr
724	activities of phosphorylated WT and phosphorylated S214A are, otherFeat=[]-->, belongsTo=parr
725	similar. These data demonstrate that mutation of any nonspe-, otherFeat=[]-->, belongsTo=parr
726	cific Ser to Ala does not significantly affect the microtubule, otherFeat=[]-->, belongsTo=parr
727	assembly-promoting activity of tau., otherFeat=[]-->, belongsTo=parr
728	The microtubule assembly-promoting activity of all phos-, otherFeat=[]-->, belongsTo=parr
729	phorylated WT, S214A, T231A, and S396A was significantly, otherFeat=[]-->, belongsTo=parr
730	less than their respective nonphosphorylated counterparts (Fig., otherFeat=[]-->, belongsTo=parr
731	8A). This observation indicated that phosphorylation inhibits, otherFeat=[]-->, belongsTo=parr
732	the microtubule assembly-promoting activities of all the above, otherFeat=[]-->, belongsTo=parr
733	tau species. Moreover, although the microtubule assembly-, otherFeat=[]-->, belongsTo=parr
734	promoting activity of phosphorylated T231A is similar to that, otherFeat=[]-->, belongsTo=parr
735	of phosphorylated WT, those of phosphorylated S396A and, otherFeat=[]-->, belongsTo=parr
736	S202A are higher (Fig. 8A). Thus, the microtubule assembly-, otherFeat=[]-->, belongsTo=parr
737	promoting activity of phosphorylated tau is not affected signif-, otherFeat=[]-->, belongsTo=parr
738	icantly by blocking Thr231 phosphorylation. Blocking phospho-, otherFeat=[]-->, belongsTo=parr
739	rylation at Ser396 or Ser202, however, increases the microtubule, otherFeat=[]-->, belongsTo=parr
740	assembly-promoting activity of phosphorylated tau. These, otherFeat=[]-->, belongsTo=parr
741	FIGURE 5. Effect of Ser202 phosphorylation on SDS-gel mobility of tau. Tau(WT) and tau(S202A) phospho-, otherFeat=[]-->, belongsTo=fig_caption
742	rylated by Cdk5 using [ 32P]ATP for the indicated time points were analyzed by Western blot (IB). The blot was, otherFeat=[]-->, belongsTo=fig_caption
743	subsequently autoradiographed to monitor radioactivity in each band., otherFeat=[]-->, belongsTo=fig_caption
744	FIGURE 6. Site-specific phosphorylation of tau(WT) and tau(S202A) by, otherFeat=[]-->, belongsTo=fig_caption
745	Cdk5. Tau(WT) and tau(S202A), phosphorylated for 120 min, were analyzed, otherFeat=[]-->, belongsTo=fig_caption
746	by Western blot (IB) using the indicated antibodies., otherFeat=[]-->, belongsTo=fig_caption
747	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
748	13428 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
749	VOLUME 284 ? NUMBER 20 ? MAY 15, 2009, otherFeat=['U']-->, belongsTo=nota_cab_pie
750	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
751	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
752	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
753	de, otherFeat=[]-->, belongsTo=nota_cab_pie
754	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
755	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
756	on, otherFeat=[]-->, belongsTo=nota_cab_pie
757	May, otherFeat=[]-->, belongsTo=nota_cab_pie
758	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
759	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
760	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
761	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
762	from, otherFeat=[]-->, belongsTo=nota_cab_pie
763	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
764	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
765	data, in turn, indicate that phosphorylation at Ser396 or Ser202, otherFeat=[]-->, belongsTo=parr
766	decreases the microtubule assembly-promoting activity of tau., otherFeat=[]-->, belongsTo=parr
767	Microtubules, in the presence of phosphorylated WT, nucle-, otherFeat=[]-->, belongsTo=parr
768	ated at 6 min  (supplemental Table S2). In the presence of phos-, otherFeat=[]-->, belongsTo=parr
769	phorylated S202A, nucleation time, otherFeat=[]-->, belongsTo=parr
770	was reduced to 5 min, an increase of, otherFeat=[]-->, belongsTo=parr
771	16.5% nucleation promoting activ-, otherFeat=[]-->, belongsTo=parr
772	ity. This means that Ser202 phos-, otherFeat=[]-->, belongsTo=parr
773	phorylation inhibits 16.6% of tau, otherFeat=[]-->, belongsTo=parr
774	microtubule nucleation-promoting, otherFeat=[]-->, belongsTo=parr
775	activity (Fig. 8B). In the presence of, otherFeat=[]-->, belongsTo=parr
776	phosphorylated WT, microtubules, otherFeat=[]-->, belongsTo=parr
777	polymerized with a rate of 0.016, otherFeat=[]-->, belongsTo=parr
778	AU/min. In the presence of phos-, otherFeat=[]-->, belongsTo=parr
779	phorylated S202A, the microtubule, otherFeat=[]-->, belongsTo=parr
780	polymerization rate was increased, otherFeat=[]-->, belongsTo=parr
781	1.5-fold to 0.024 AU/min. These, otherFeat=[]-->, belongsTo=parr
782	data indicate that Ser202 phospho-, otherFeat=[]-->, belongsTo=parr
783	rylation inhibits 50% of tau micro-, otherFeat=[]-->, belongsTo=parr
784	tubule polymerization-promoting, otherFeat=[]-->, belongsTo=parr
785	activity (Fig. 8B). Finally, the, otherFeat=[]-->, belongsTo=parr
786	amount of microtubules formed in, otherFeat=[]-->, belongsTo=parr
787	the presence of phosphorylated, otherFeat=[]-->, belongsTo=parr
788	S202A was 1.4 times more than that, otherFeat=[]-->, belongsTo=parr
789	formed in the presence of phospho-, otherFeat=[]-->, belongsTo=parr
790	rylated WT, indicating that Ser202, otherFeat=[]-->, belongsTo=parr
791	phosphorylation inhibits 40% of tau, otherFeat=[]-->, belongsTo=parr
792	microtubule formation-promoting, otherFeat=[]-->, belongsTo=parr
793	activity. Furthermore, the microtu-, otherFeat=[]-->, belongsTo=parr
794	bule nucleation polymerization and, otherFeat=[]-->, belongsTo=parr
795	formation-promoting activities of, otherFeat=[]-->, belongsTo=parr
796	phosphorylated S202A are higher, otherFeat=[]-->, belongsTo=parr
797	than that of phosphorylated Ser396, otherFeat=[]-->, belongsTo=parr
798	(supplemental Table S2 and Fig., otherFeat=[]-->, belongsTo=parr
799	8B). These data show that the, otherFeat=[]-->, belongsTo=parr
800	microtubule assembly-promoting, otherFeat=[]-->, belongsTo=parr
801	activity of tau is inhibited more by, otherFeat=[]-->, belongsTo=parr
802	phosphorylation at Ser202 than by, otherFeat=[]-->, belongsTo=parr
803	phosphorylation at Ser396. Taken, otherFeat=[]-->, belongsTo=parr
804	together, these data indicate that, otherFeat=[]-->, belongsTo=parr
805	phosphorylation at Ser202 has the, otherFeat=[]-->, belongsTo=parr
806	major inhibitory impact on the, otherFeat=[]-->, belongsTo=parr
807	microtubule assembly-promoting, otherFeat=[]-->, belongsTo=parr
808	activity of tau in vitro., otherFeat=[]-->, belongsTo=parr
809	Effect of Ser202 Phosphorylation, otherFeat=[]-->, belongsTo=parrnote
810	on SDS-gel Mobility Shift of, otherFeat=[]-->, belongsTo=parr
811	FTDP-17 Tau Mutants--To deter-, otherFeat=[]-->, belongsTo=parr
812	mine whether missense FTDP-17, otherFeat=[]-->, belongsTo=parr
813	mutations promote mobility shift by, otherFeat=[]-->, belongsTo=parr
814	promoting phosphorylation at, otherFeat=[]-->, belongsTo=parr
815	Ser202, we mutated Ser202 of each of, otherFeat=[]-->, belongsTo=parr
816	the FTDP-17 mutants to Ala. All, otherFeat=[]-->, belongsTo=parr
817	double mutants were phosphoryla-, otherFeat=[]-->, belongsTo=parr
818	ted along with their corresponding, otherFeat=[]-->, belongsTo=parr
819	FTDP-17 single mutants, WT, and, otherFeat=[]-->, belongsTo=parr
820	S202A by Cdk5. All phosphorylated, otherFeat=[]-->, belongsTo=parr
821	proteins were then analyzed by, otherFeat=[]-->, belongsTo=parr
822	Western blot., otherFeat=[]-->, belongsTo=parr
823	All nonphosphorylated single and double mutants migrated, otherFeat=[]-->, belongsTo=parr
824	as single 60-kDa band on SDS-gel (Fig. 9A). Phosphorylated, otherFeat=[]-->, belongsTo=parr
825	WT migrated as 60- and 64-kDa bands and a relatively weak, otherFeat=[]-->, belongsTo=parr
826	68-kDa band (Fig. 9B, lane 3), whereas phosphorylated S202A, otherFeat=[]-->, belongsTo=parr
827	FIGURE 7. Effect of Ser396, Thr231, and Ser202 phosphorylation on SDS-gel mobility of tau. The indicated, otherFeat=[]-->, belongsTo=fig_caption
828	tau species phosphorylated by Cdk5 were analyzed by Western blot using the indicated antibodies., otherFeat=[]-->, belongsTo=fig_caption
829	FIGURE 8. Effect of Ser202, Ser396, and Thr231 phosphorylation on microtubule assembly-promoting, otherFeat=[]-->, belongsTo=fig_caption
830	activity of tau. Microtubule assembly was monitored in the presence of the indicated tau species. From the, otherFeat=[]-->, belongsTo=fig_caption
831	light scattering data, microtubule nucleation lag time, polymerization rate, and the amount of microtubules, otherFeat=[]-->, belongsTo=fig_caption
832	formed were calculated as described under "Materials and Methods"  (supplemental Table S2) and were used to, otherFeat=[]-->, belongsTo=fig_caption
833	determine the inhibition of various parameters of the microtubule assembly. To determine the percent inhi-, otherFeat=[]-->, belongsTo=fig_caption
834	bition of microtubule nucleation by Ser202 phosphorylation, the lag time value of phosphorylated S202A was, otherFeat=[]-->, belongsTo=fig_caption
835	subtracted from the lag time value of phosphorylated WT. The resulting value was then normalized against the, otherFeat=[]-->, belongsTo=fig_caption
836	lag time value of phosphorylated WT. Note that this value is the gain in the microtubule nucleation-promoting, otherFeat=[]-->, belongsTo=fig_caption
837	activity of phosphorylated WT upon blocking Ser202 phosphorylation. This, in turn, is the contribution of Ser202, otherFeat=[]-->, belongsTo=fig_caption
838	phosphorylation in inhibiting the nucleation-promoting activity of tau. To determine the percent inhibition of, otherFeat=[]-->, belongsTo=fig_caption
839	microtubule polymerization by Ser202 phosphorylation, the polymerization rate of phosphorylated WT was, otherFeat=[]-->, belongsTo=fig_caption
840	subtracted from that of phosphorylated S202A. The resulting value was normalized against the polymerization, otherFeat=[]-->, belongsTo=fig_caption
841	rate of phosphorylated WT. Likewise, the percent inhibition of the microtubule amount formed by Ser202, otherFeat=[]-->, belongsTo=fig_caption
842	phosphorylation was calculated as described above for microtubule polymerization, except microtubule, otherFeat=[]-->, belongsTo=fig_caption
843	amounts for phosphorylated WT and phosphorylated S202A were used. The percent inhibition of microtubule, otherFeat=[]-->, belongsTo=fig_caption
844	nucleation, polymerization, and microtubule formation by Ser396 and Thr231 phosphorylation were calculated, otherFeat=[]-->, belongsTo=fig_caption
845	in the same manner using the values of phosphorylated S396A and phosphorylated T231A, respectively., otherFeat=[]-->, belongsTo=fig_caption
846	A, microtubule assembly. B, inhibition of microtubule assembly by phosphorylation at Ser202, Thr231, and, otherFeat=[]-->, belongsTo=fig_caption
847	Ser396. Values in B are an average of three determinations., otherFeat=[]-->, belongsTo=fig_caption
848	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
849	MAY 15, 2009 ? VOLUME 284 ? NUMBER 20, otherFeat=['U']-->, belongsTo=nota_cab_pie
850	JOURNAL OF BIOLOGICAL CHEMISTRY 13429, otherFeat=[]-->, belongsTo=nota_cab_pie
851	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
852	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
853	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
854	de, otherFeat=[]-->, belongsTo=nota_cab_pie
855	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
856	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
857	on, otherFeat=[]-->, belongsTo=nota_cab_pie
858	May, otherFeat=[]-->, belongsTo=nota_cab_pie
859	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
860	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
861	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
862	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
863	from, otherFeat=[]-->, belongsTo=nota_cab_pie
864	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
865	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
866	migrated as 60- and 64-kDa bands (Fig. 9B, lane 2). Likewise,, otherFeat=[]-->, belongsTo=parr
867	each phosphorylated G272V, P301L, V337M, and R406W dis-, otherFeat=[]-->, belongsTo=parr
868	played prominent 60-, 64-, and 68-kDa bands (Fig. 9B, lanes, otherFeat=[]-->, belongsTo=parr
869	4?7). These data are consistent with the data of Fig. 2 and show, otherFeat=[]-->, belongsTo=parr
870	that FTDP-17 mutations promote mobility shift of tau on SDS-, otherFeat=[]-->, belongsTo=parr
871	gel. However, the phosphorylated double mutant G272V/, otherFeat=[]-->, belongsTo=parr
872	S202A migrated as 60- and 64-kDa bands and failed to display, otherFeat=[]-->, belongsTo=parr
873	any significant 68-kDa mobility shift (Fig. 9B, lane 8). Simi-, otherFeat=[]-->, belongsTo=parr
874	larly, phosphorylated P301L/S202A, V337M/S202A, and, otherFeat=[]-->, belongsTo=parr
875	R406W/S202A also showed only 60- and 64-kDa bands (Fig., otherFeat=[]-->, belongsTo=parr
876	9B, lanes 9 ?11). Thus, blocking Ser202 phosphorylation, otherFeat=[]-->, belongsTo=parr
877	blocked the phosphorylation-induced 68-kDa mobility shift, otherFeat=[]-->, belongsTo=parr
878	of all FTDP-17 tau mutants. These, otherFeat=[]-->, belongsTo=parr
879	data indicate that FTDP-17 muta-, otherFeat=[]-->, belongsTo=parr
880	tions lose their abilities to promote, otherFeat=[]-->, belongsTo=parr
881	mobility shift to a 68-kDa band if, otherFeat=[]-->, belongsTo=parr
882	phosphorylation at Ser202 is, otherFeat=[]-->, belongsTo=parr
883	blocked. This in turn indicates that, otherFeat=[]-->, belongsTo=parr
884	FTDP-17 mutations promote the, otherFeat=[]-->, belongsTo=parr
885	68-kDa band mobility shift by, otherFeat=[]-->, belongsTo=parr
886	enhancing phosphorylation at, otherFeat=[]-->, belongsTo=parr
887	Ser202., otherFeat=[]-->, belongsTo=parr
888	Effect of Ser202 Phosphorylation, otherFeat=[]-->, belongsTo=parrnote
889	on Microtubule Assembly-promot-, otherFeat=[]-->, belongsTo=parr
890	ing Activity of FTDP-17 Tau, otherFeat=[]-->, belongsTo=parr
891	Mutants--Finally, we monitored, otherFeat=[]-->, belongsTo=parr
892	the microtubule assembly-promot-, otherFeat=[]-->, belongsTo=parr
893	ing activities of phosphorylated, otherFeat=[]-->, belongsTo=parr
894	double mutants and their respective, otherFeat=[]-->, belongsTo=parr
895	FTDP-17 single mutants. Nonphos-, otherFeat=[]-->, belongsTo=parr
896	phorylated WT and WT(S202A), otherFeat=[]-->, belongsTo=parr
897	promoted microtubule assembly, otherFeat=[]-->, belongsTo=parr
898	with similar nucleation time and, otherFeat=[]-->, belongsTo=parr
899	polymerization rate and caused the, otherFeat=[]-->, belongsTo=parr
900	formation of similar amounts of, otherFeat=[]-->, belongsTo=parr
901	microtubules. Likewise, nonphos-, otherFeat=[]-->, belongsTo=parr
902	phorylated double mutants G272V/, otherFeat=[]-->, belongsTo=parr
903	S202A, P301L/S202A, V337M/, otherFeat=[]-->, belongsTo=parr
904	S202A, and R406W/S202A sup-, otherFeat=[]-->, belongsTo=parr
905	ported microtubule assembly in a, otherFeat=[]-->, belongsTo=parr
906	manner similar to their respective, otherFeat=[]-->, belongsTo=parr
907	FTDP-17 single mutants (data not, otherFeat=[]-->, belongsTo=parr
908	included). Thus, mutation of Ser202, otherFeat=[]-->, belongsTo=parr
909	to Ala did not affect the microtubule, otherFeat=[]-->, belongsTo=parr
910	assembly-promoting activity of WT, otherFeat=[]-->, belongsTo=parr
911	or any of the FTDP-17 mutants., otherFeat=[]-->, belongsTo=parr
912	Phosphorylated WT(S202A) dis-, otherFeat=[]-->, belongsTo=parr
913	played microtubule nucleation,, otherFeat=[]-->, belongsTo=parr
914	polymerization, and formation-pro-, otherFeat=[]-->, belongsTo=parr
915	moting activities higher than that of, otherFeat=[]-->, belongsTo=parr
916	phosphorylated WT (Fig. 10A and, otherFeat=[]-->, belongsTo=parr
917	supplemental Table S3). These data, otherFeat=[]-->, belongsTo=parr
918	are consistent with the observation, otherFeat=[]-->, belongsTo=parr
919	made in Fig. 8, demonstrating that, otherFeat=[]-->, belongsTo=parr
920	blocking Ser202 phosphorylation, otherFeat=[]-->, belongsTo=parr
921	enhances the microtubule assem-, otherFeat=[]-->, belongsTo=parr
922	bly-promoting activity of phospho-, otherFeat=[]-->, belongsTo=parr
923	rylated tau. As shown in Fig. 10A, otherFeat=[]-->, belongsTo=parr
924	and  supplemental Table S3, microtubule nucleation-promot-, otherFeat=[]-->, belongsTo=parr
925	ing activity of phosphorylated G272V/S202A is more than that, otherFeat=[]-->, belongsTo=parr
926	of phosphorylated G272V. Likewise, microtubule polymeriza-, otherFeat=[]-->, belongsTo=parr
927	tion and formation-promoting activities of phosphorylated, otherFeat=[]-->, belongsTo=parr
928	G272V/S202A are higher than that of phosphorylated G272V., otherFeat=[]-->, belongsTo=parr
929	Moreover, microtubule nucleation, polymerization, and forma-, otherFeat=[]-->, belongsTo=parr
930	tion-promoting activities of phosphorylated P301L/S202A,, otherFeat=[]-->, belongsTo=parr
931	V337M/S202A, and R406W/S202A are higher than those of, otherFeat=[]-->, belongsTo=parr
932	their respective phosphorylated FTDP-17 single mutants., otherFeat=[]-->, belongsTo=parr
933	Thus, as with phosphorylated WT, blocking Ser202 phosphoryl-, otherFeat=[]-->, belongsTo=parr
934	ation increased the microtubule assembly-promoting activity, otherFeat=[]-->, belongsTo=parr
935	of all phosphorylated FTDP-17 mutants. This indicates that, otherFeat=[]-->, belongsTo=parr
936	FIGURE 9. Effect of Ser202 phosphorylation on the SDS-gel mobility of phosphorylated FTDP-17 tau, otherFeat=[]-->, belongsTo=fig_caption
937	mutants. Indicated tau mutants phosphorylated by Cdk5 were Western blotted using tau 5 antibody. A, non-, otherFeat=[]-->, belongsTo=fig_caption
938	phosphorylated tau species. B, phosphorylated tau species., otherFeat=[]-->, belongsTo=fig_caption
939	FIGURE 10. Effect of Ser202 phosphorylation on microtubule assembly-promoting activities of FTDP-17, otherFeat=[]-->, belongsTo=fig_caption
940	tau mutants. Microtubule assembly was monitored in the presence of the indicated tau species by light, otherFeat=[]-->, belongsTo=fig_caption
941	scattering as described in the legend for Fig. 1A. Based on the light scattering data, the nucleation lag, micro-, otherFeat=[]-->, belongsTo=fig_caption
942	tubule polymerization rate, and microtubule amount formed were calculated  (supplemental Table S3), and, otherFeat=[]-->, belongsTo=fig_caption
943	these values were used to determine the inhibition of microtubule assembly-promoting activity of WT and its, otherFeat=[]-->, belongsTo=fig_caption
944	FTDP-17 mutants by Ser202 phosphorylation. A, microtubule assembly. B and C, inhibition of microtubule, otherFeat=[]-->, belongsTo=fig_caption
945	assembly. By using values shown in  supplemental Table S3, panel B was generated as described for Ser202, otherFeat=[]-->, belongsTo=fig_caption
946	phosphorylation in the legend for Fig. 8. To generate panel C, the value of each tau species in each section of B, otherFeat=[]-->, belongsTo=fig_caption
947	was normalized against the value of the WT in that section of B to be expressed as the -fold of WT. The values, otherFeat=[]-->, belongsTo=fig_caption
948	are an average of three determinations., otherFeat=[]-->, belongsTo=fig_caption
949	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
950	13430 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
951	VOLUME 284 ? NUMBER 20 ? MAY 15, 2009, otherFeat=['U']-->, belongsTo=nota_cab_pie
952	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
953	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
954	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
955	de, otherFeat=[]-->, belongsTo=nota_cab_pie
956	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
957	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
958	on, otherFeat=[]-->, belongsTo=nota_cab_pie
959	May, otherFeat=[]-->, belongsTo=nota_cab_pie
960	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
961	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
962	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
963	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
964	from, otherFeat=[]-->, belongsTo=nota_cab_pie
965	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
966	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
967	Ser202 phosphorylation inhibits the microtubule assembly-pro-, otherFeat=[]-->, belongsTo=parr
968	moting activity of tau(WT) and all its FTDP-17 mutants., otherFeat=[]-->, belongsTo=parr
969	Microtubule nucleation-promoting activity of phosphoryla-, otherFeat=[]-->, belongsTo=parr
970	ted WT is 6 min, whereas that of phosphorylated WT(S202A) is, otherFeat=[]-->, belongsTo=parr
971	5 min  (supplemental Table S3). This means that blocking Ser202, otherFeat=[]-->, belongsTo=parr
972	phosphorylation increases the nucleation activity of phospho-, otherFeat=[]-->, belongsTo=parr
973	rylated WT by 16.6%. This, in turn, indicates that 16.6% of the, otherFeat=[]-->, belongsTo=parr
974	microtubule nucleation-promoting activity of tau(WT) is, otherFeat=[]-->, belongsTo=parr
975	inhibited by Ser202 phosphorylation. As shown in Fig. 10B,, otherFeat=[]-->, belongsTo=parr
976	Ser202 phosphorylation inhibits the microtubule nucleation-, otherFeat=[]-->, belongsTo=parr
977	promoting activity of G272V, P301L, V337M, and R406W by, otherFeat=[]-->, belongsTo=parr
978	22.2, 18.2, 27.3, and 25.0%, respectively. Ser202 phosphorylation, otherFeat=[]-->, belongsTo=parr
979	inhibits the microtubule polymerization-promoting activity of, otherFeat=[]-->, belongsTo=parr
980	WT by 37.5% and that of G272V, P301L, V337M, and R406W, otherFeat=[]-->, belongsTo=parr
981	by 45.0, 63.6, 44.2, and 70.0%, respectively. Similarly, Ser202, otherFeat=[]-->, belongsTo=parr
982	phosphorylation inhibits the microtubule formation-promot-, otherFeat=[]-->, belongsTo=parr
983	ing activity of WT by 40.6%, which is increased in G272V,, otherFeat=[]-->, belongsTo=parr
984	P301L, V337M, and R406W mutants to 53.8, 71.4, 65.2, and, otherFeat=[]-->, belongsTo=parr
985	88.9%, respectively. Thus, compared with the WT, Ser202 phos-, otherFeat=[]-->, belongsTo=parr
986	phorylation inhibits the microtubule nucleation-promoting, otherFeat=[]-->, belongsTo=parr
987	activity of G272V, P301L, V337M, and R406W 1.34-, 1.10-,, otherFeat=[]-->, belongsTo=parr
988	1.64-, and 1.50-fold more, respectively, microtubule polymeriza-, otherFeat=[]-->, belongsTo=parr
989	tion-promoting activity 1.32-, 1.75-, 1.60-, and 2.20-fold more,, otherFeat=[]-->, belongsTo=parr
990	respectively, and microtubule formation-promoting activity 1.32-,, otherFeat=[]-->, belongsTo=parr
991	1.75-, 1.6-, and 2.20-fold more, respectively (Fig. 10C). These data, otherFeat=[]-->, belongsTo=parr
992	indicate that phosphorylation at Ser202 has a more profound inhib-, otherFeat=[]-->, belongsTo=parr
993	itory effect on the microtubule assembly-promoting activity of, otherFeat=[]-->, belongsTo=parr
994	FTDP-17 mutants than on that of the WT., otherFeat=[]-->, belongsTo=parr
995	DISCUSSION, otherFeat=[]-->, belongsTo=title
996	The presence of 64- and 68-kDa tau bands is a character-, otherFeat=[]-->, belongsTo=parr
997	istic feature of the AD brain, and studies suggest that the, otherFeat=[]-->, belongsTo=parr
998	appearance of these species correlates with the disease progres-, otherFeat=[]-->, belongsTo=parr
999	sion (12, 13). Because these tau species are formed due to, otherFeat=[]-->, belongsTo=parr
1000	abnormal tau phosphorylation, tau sites that are responsible for, otherFeat=[]-->, belongsTo=parr
1001	causing their formation are suggested to be involved in the, otherFeat=[]-->, belongsTo=parr
1002	development of AD pathology in brain (12, 13)., otherFeat=[]-->, belongsTo=parr
1003	Cdk5 is one of the kinases suggested to phosphorylate tau, otherFeat=[]-->, belongsTo=parr
1004	in the AD brain (27, 39). In vitro, Cdk5 phosphorylates tau on, otherFeat=[]-->, belongsTo=parr
1005	several sites that are phosphorylated in PHFs including, otherFeat=[]-->, belongsTo=parr
1006	Ser202, Thr231, and Ser396 (27). In this study, nonphosphoryl-, otherFeat=[]-->, belongsTo=parr
1007	ated tau migrated as a 60-kDa band on SDS-gel. Upon, otherFeat=[]-->, belongsTo=parr
1008	phosphorylation by Cdk5, tau migrated as 60-, 64-, and, otherFeat=[]-->, belongsTo=parr
1009	68-kDa bands (Fig. 4). These data indicate that phosphorylation, otherFeat=[]-->, belongsTo=parr
1010	by Cdk5 cause mobility shift of tau to 64- and 68-kDa bands., otherFeat=[]-->, belongsTo=parr
1011	Although phosphorylation at Ser396/404 also promoted tau, otherFeat=[]-->, belongsTo=parr
1012	mobility shift to a 64-kDa band, only Ser202-phosphorylated, otherFeat=[]-->, belongsTo=parr
1013	tau displayed both 64- and 68-kDa bands, and phosphoryla-, otherFeat=[]-->, belongsTo=parr
1014	tion at Ser202 correlated with their formation (Figs. 3 and 4)., otherFeat=[]-->, belongsTo=parr
1015	Furthermore, blocking Thr231 or Ser396 did not affect tau, otherFeat=[]-->, belongsTo=parr
1016	mobility shift to 64- or 68-kDa bands. Blocking Ser202 phos-, otherFeat=[]-->, belongsTo=parr
1017	phorylation blocked mobility shift to a 68-kDa band (Fig. 7)., otherFeat=[]-->, belongsTo=parr
1018	Our data indicate that tau mobility shift to a 64-kDa band can, otherFeat=[]-->, belongsTo=parr
1019	occur without Ser202 phosphorylation. However, mobility shift, otherFeat=[]-->, belongsTo=parr
1020	to a 68-kDa band in response to Cdk5 phosphorylation, otherFeat=[]-->, belongsTo=parr
1021	requires Ser202 phosphorylation., otherFeat=[]-->, belongsTo=parrnote
1022	As shown in Fig. 3, the 68-kDa band formed by Cdk5 phos-, otherFeat=[]-->, belongsTo=parr
1023	phorylation is phosphorylated on Ser202 but not at Ser396/404,, otherFeat=[]-->, belongsTo=parr
1024	Thr231, Ser235,orThr212. This indicates that the 68-kDa band, otherFeat=[]-->, belongsTo=parr
1025	is formed as a result of phosphorylation at Ser202 but not at, otherFeat=[]-->, belongsTo=parr
1026	Ser396/404, Thr231, Ser235,orThr212. Similarly, the 64-kDa, otherFeat=[]-->, belongsTo=parr
1027	band is phosphorylated at Ser202 and Ser396/404 but not at, otherFeat=[]-->, belongsTo=parr
1028	Thr231, Ser235, and Thr212, indicating that the 64-kDa band is, otherFeat=[]-->, belongsTo=parr
1029	formed due to phosphorylation at Ser202 and Ser396/404 and not, otherFeat=[]-->, belongsTo=parr
1030	at Thr231, Ser235,orThr212. Furthermore, the Ser202-phospho-, otherFeat=[]-->, belongsTo=parr
1031	rylated tau first migrates as a 60-kDa band (Fig. 4A, lane 2)., otherFeat=[]-->, belongsTo=parr
1032	With the increase in phosphorylation time, 64-kDa band fol-, otherFeat=[]-->, belongsTo=parr
1033	lowed by 68-kDa band appear. These data suggest that Ser202-, otherFeat=[]-->, belongsTo=parr
1034	phosphorylated tau migrates as a 60-kDa band and that phos-, otherFeat=[]-->, belongsTo=parr
1035	phorylation on this site alone does not cause a tau band shift., otherFeat=[]-->, belongsTo=parr
1036	The mobility shift of tau may, therefore, occur by a sequential, otherFeat=[]-->, belongsTo=parr
1037	mechanism. First-step phosphorylation at Ser202 may allow, otherFeat=[]-->, belongsTo=parr
1038	Cdk5 to perform second-step phosphorylation on new sites, otherFeat=[]-->, belongsTo=parr
1039	that are not accessible in non-Ser202-phosphorylated tau. First-, otherFeat=[]-->, belongsTo=parr
1040	plus second-step-phosphorylated tau may then migrate as a, otherFeat=[]-->, belongsTo=parr
1041	64-kDa band. The 64-kDa phosphorylated tau may then, otherFeat=[]-->, belongsTo=parr
1042	undergo third-step phosphorylation on additional sites and, otherFeat=[]-->, belongsTo=parr
1043	become hyperphosphorylated. The hyperphosphorylated tau, otherFeat=[]-->, belongsTo=parr
1044	may then migrate as a 68-kDa band. Note that in this mech-, otherFeat=[]-->, belongsTo=parr
1045	anism, the 68-kDa band will be expected to contain phos-, otherFeat=[]-->, belongsTo=parr
1046	phate on all the sites, including Ser202, that are involved in the, otherFeat=[]-->, belongsTo=parr
1047	mobility shift from 60- to 64-kDa and then to 68-kDa bands., otherFeat=[]-->, belongsTo=parr
1048	As shown in Fig. 3, the 68-kDa band is phosphorylated at, otherFeat=[]-->, belongsTo=parr
1049	Ser202 but not at Ser396/404, Ser235, Thr231,orThr212. This, otherFeat=[]-->, belongsTo=parr
1050	observation suggests that phosphorylation on the new, addi-, otherFeat=[]-->, belongsTo=parr
1051	tional sites that occurs after phosphorylation at Ser202, and, otherFeat=[]-->, belongsTo=parr
1052	results in a 60 ? 68-kDa band shift, has to occur at sites, otherFeat=[]-->, belongsTo=parr
1053	other than Ser396/404, Ser235, Thr231, and Thr212. In fact, in, otherFeat=[]-->, belongsTo=parr
1054	addition to the sites mentioned above, PHF-tau is phospho-, otherFeat=[]-->, belongsTo=parr
1055	rylated on a number of proline-directed sites, including, otherFeat=[]-->, belongsTo=parr
1056	Ser199, Thr181, Thr217, and Ser222, which are potential targets, otherFeat=[]-->, belongsTo=parr
1057	of Cdk5 (8)., otherFeat=[]-->, belongsTo=parr
1058	Our data have demonstrated that Thr231 phosphorylation, otherFeat=[]-->, belongsTo=parr
1059	did not have a significant effect on microtubule nucleation or, otherFeat=[]-->, belongsTo=parr
1060	formation-promoting activities and only slightly affected the, otherFeat=[]-->, belongsTo=parr
1061	microtubule polymerization-promoting activity of tau (Fig., otherFeat=[]-->, belongsTo=parr
1062	8A). Likewise, Ser396 phosphorylation inhibited microtubule, otherFeat=[]-->, belongsTo=parr
1063	polymerization and formation-promoting activities but did not, otherFeat=[]-->, belongsTo=parr
1064	affect microtubule nucleation-promoting activity (Fig. 8A and, otherFeat=[]-->, belongsTo=parr
1065	supplemental Table S2). Ser202 phosphorylation, on the other, otherFeat=[]-->, belongsTo=parr
1066	hand, not only inhibited microtubule nucleation-promoting, otherFeat=[]-->, belongsTo=parr
1067	activity, but it inhibited microtubule polymerization and for-, otherFeat=[]-->, belongsTo=parr
1068	mation-promoting activities 2- and 1.5-fold more than Ser396, otherFeat=[]-->, belongsTo=parr
1069	phosphorylation (Fig. 8B). Our results indicate that Ser202, otherFeat=[]-->, belongsTo=parr
1070	phosphorylation significantly inhibits the microtubule assem-, otherFeat=[]-->, belongsTo=parr
1071	bly-promoting activity of tau in vitro. It should be noted that, otherFeat=[]-->, belongsTo=parr
1072	under our experimental conditions, Cdk5 may have phospho-, otherFeat=[]-->, belongsTo=parr
1073	rylated tau at Ser202 to a higher extent than at Ser396 and Thr231., otherFeat=[]-->, belongsTo=parrnote
1074	The observed difference in the ability of each of the above sites, otherFeat=[]-->, belongsTo=parr
1075	to inhibit tau microtubule assembly-promoting activity may be, otherFeat=[]-->, belongsTo=parr
1076	due, in part, to the differences in extent of phosphorylation., otherFeat=[]-->, belongsTo=parr
1077	The pathological significance of Ser202 phosphorylation in, otherFeat=[]-->, belongsTo=parr
1078	the brain is not very clear. Studies suggest that mild memory, otherFeat=[]-->, belongsTo=parr
1079	impairment is the earliest clinical feature of AD and is associ-, otherFeat=[]-->, belongsTo=parr
1080	ated with subtle cytoskeletal alterations in pre-tangle neurons., otherFeat=[]-->, belongsTo=parr
1081	This alteration can be detected by immunohistochemical anal-, otherFeat=[]-->, belongsTo=parr
1082	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
1083	MAY 15, 2009 ? VOLUME 284 ? NUMBER 20, otherFeat=['U']-->, belongsTo=nota_cab_pie
1084	JOURNAL OF BIOLOGICAL CHEMISTRY 13431, otherFeat=[]-->, belongsTo=nota_cab_pie
1085	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
1086	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
1087	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1088	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1089	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1090	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1091	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1092	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1093	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1094	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
1095	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
1096	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
1097	from, otherFeat=[]-->, belongsTo=nota_cab_pie
1098	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
1099	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
1100	ysis of the brain using AT8 monoclonal antibody (43, 44). The, otherFeat=[]-->, belongsTo=parr
1101	AT8 epitope first appears in the pre-AD brain areas that do not, otherFeat=[]-->, belongsTo=parr
1102	show any brain degeneration and are devoid of NFTs. But, otherFeat=[]-->, belongsTo=parr
1103	slowly as the disease progresses and the brain begins to degen-, otherFeat=[]-->, belongsTo=parr
1104	erate, the intensity of AT8 reactivity increases (43? 45). Among, otherFeat=[]-->, belongsTo=parr
1105	several tau phosphorylation-sensitive antibodies tested, AD, otherFeat=[]-->, belongsTo=parr
1106	brains stain strongest with AT8 (46). When purified PHF-tau is, otherFeat=[]-->, belongsTo=parr
1107	Western blotted, 60-, 64-, and 68-kDa tau bands cross-react, otherFeat=[]-->, belongsTo=parr
1108	with AT8 antibody (47). AT8 immunoreactivity in the brain is, otherFeat=[]-->, belongsTo=parr
1109	regarded as the abnormal cytoskeletal change that occurs dur-, otherFeat=[]-->, belongsTo=parr
1110	ing the AD development (43, 44). In vitro AT8 specifically rec-, otherFeat=[]-->, belongsTo=parr
1111	ognizes tau phosphorylated at Ser202 and/or Ser205 (48). How-, otherFeat=[]-->, belongsTo=parr
1112	ever, mass spectrometric studies have determined that PHF-tau, otherFeat=[]-->, belongsTo=parr
1113	is phosphorylated at Ser202 but not at Ser205 (8, 9). Thus, AT8, otherFeat=[]-->, belongsTo=parr
1114	immunoreactivity in the brain represents tau phosphorylated at, otherFeat=[]-->, belongsTo=parr
1115	Ser202. Moreover, among several sites tested, only phosphoryl-, otherFeat=[]-->, belongsTo=parr
1116	ation at Ser202 correlates with mobility shift of tau to 64- and, otherFeat=[]-->, belongsTo=parr
1117	68-kDa bands upon Cdk5 phosphorylation in vitro (Figs. 3 and, otherFeat=[]-->, belongsTo=parr
1118	4). Also, all pathogenic FTDP-17 tau missense mutations that, otherFeat=[]-->, belongsTo=parr
1119	accelerate NFT pathology in the brain promote phosphoryla-, otherFeat=[]-->, belongsTo=parr
1120	tion at Ser202 (Figs. 3 and 4). Taken together, these observations, otherFeat=[]-->, belongsTo=parr
1121	suggest that Ser202 phosphorylation is the major pathological, otherFeat=[]-->, belongsTo=parr
1122	event in the brain leading to brain degeneration in AD., otherFeat=[]-->, belongsTo=parr
1123	Exonic and intronic mutations have been discovered in the, otherFeat=[]-->, belongsTo=parr
1124	familial type of FTDP-17. Although intronic mutations inter-, otherFeat=[]-->, belongsTo=parr
1125	fere with pre-mRNA splicing leading to an increase in four-, otherFeat=[]-->, belongsTo=parr
1126	repeat tau in the brain, the mechanism by which exonic muta-, otherFeat=[]-->, belongsTo=parr
1127	tions promote NFT pathology is an area of current research in, otherFeat=[]-->, belongsTo=parr
1128	neurobiology. Studies using antibodies directed against tau, otherFeat=[]-->, belongsTo=parr
1129	phosphorylated at various sites have determined that V337M, otherFeat=[]-->, belongsTo=parr
1130	FTDP-17 brains stain most intensely with AT8 (Ser202), less, otherFeat=[]-->, belongsTo=parr
1131	intensely with PHF-1 (Ser396/404), AT100 (Thr212/Ser214),, otherFeat=[]-->, belongsTo=parrnote
1132	AT180 (Thr231/Ser235), AT270 (Ser181), and 12E8 (Ser262), indi-, otherFeat=[]-->, belongsTo=parr
1133	cating that V337M in the brain is most strongly phosphorylated, otherFeat=[]-->, belongsTo=parr
1134	at Ser202 and less strongly at Ser396/404, Thr212/214, Thr231/, otherFeat=[]-->, belongsTo=parrnote
1135	Ser235, Ser181, and Ser262 (46). Likewise, P301L in the brain is, otherFeat=[]-->, belongsTo=parr
1136	most extensively phosphorylated at Ser202, Ser396/404, and, otherFeat=[]-->, belongsTo=parr
1137	Thr212/Ser214 and less extensively at Thr231/Ser235 and Ser262, otherFeat=[]-->, belongsTo=parr
1138	(49, 50). G272V in the brain is most extensively phosphorylated, otherFeat=[]-->, belongsTo=parr
1139	at Ser202, less extensively at Ser396/404 and Thr231/Ser235, and, otherFeat=[]-->, belongsTo=parr
1140	not at all at Ser262 (17). Finally, R406W in the brain is most, otherFeat=[]-->, belongsTo=parr
1141	strongly phosphorylated at Ser202, Ser396/404, and Thr212/Ser214, otherFeat=[]-->, belongsTo=parr
1142	(50). Thus, among all of the different sites examined, all, otherFeat=[]-->, belongsTo=parr
1143	FTDP-17 tau mutants in the brain are most extensively phos-, otherFeat=[]-->, belongsTo=parr
1144	phorylated at Ser202 recognized by AT8 antibody. In addition,, otherFeat=[]-->, belongsTo=parr
1145	PHF-tau isolated from all FTDP-17 mutant brains migrates as, otherFeat=[]-->, belongsTo=parr
1146	60-, 64-, and 68-kDa bands on SDS-gel (11, 17, 46, 49, 50), and, otherFeat=[]-->, belongsTo=parr
1147	in vitro Ser202 phosphorylation promotes tau mobility shift to, otherFeat=[]-->, belongsTo=parr
1148	64- and 68-kDa bands (Fig. 4). These studies indicate that all, otherFeat=[]-->, belongsTo=parr
1149	missense FTDP-17 mutations promote tau phosphorylation at, otherFeat=[]-->, belongsTo=parr
1150	Ser202 in the brain., otherFeat=[]-->, belongsTo=parr
1151	In this study, we examined four FTDP-17 tau mutations,, otherFeat=[]-->, belongsTo=parr
1152	G272V, P301L, V337M, and R406W, and found that these, otherFeat=[]-->, belongsTo=parr
1153	mutations, by promoting phosphorylation at some sites and, otherFeat=[]-->, belongsTo=parr
1154	inhibiting at some sites, do not significantly affect the total, otherFeat=[]-->, belongsTo=parr
1155	amount of phosphate incorporated into the tau molecule., otherFeat=[]-->, belongsTo=parr
1156	Although phosphorylation at Thr231 is not influenced by any, otherFeat=[]-->, belongsTo=parr
1157	mutation, phosphorylation at Ser396/404 is promoted by G272V, otherFeat=[]-->, belongsTo=parr
1158	and P301L but inhibited is by R406W (Fig. 3). Ser235 phospho-, otherFeat=[]-->, belongsTo=parr
1159	rylation is promoted by V337M, G272V, and P301L but is, otherFeat=[]-->, belongsTo=parr
1160	inhibited by R406W. Phosphorylation at Ser202, on the other, otherFeat=[]-->, belongsTo=parr
1161	hand, is promoted by all of the FTDP-17 missense mutations,, otherFeat=[]-->, belongsTo=parr
1162	and by enhancing phosphorylation at Ser202 each mutation pro-, otherFeat=[]-->, belongsTo=parr
1163	moted a tau mobility shift to 64- and 68-kDa bands (Figs. 3, 4,, otherFeat=[]-->, belongsTo=parr
1164	and 9). Furthermore, compared with the phosphorylated WT,, otherFeat=[]-->, belongsTo=parr
1165	each phosphorylated FTDP-17 mutant displayed reduced, otherFeat=[]-->, belongsTo=parr
1166	microtubule assembly-promoting activity (Fig. 10). When, otherFeat=[]-->, belongsTo=parr
1167	Ser202 phosphorylation was blocked, each phosphorylated, otherFeat=[]-->, belongsTo=parr
1168	mutant recovered more relative amounts of microtubule, otherFeat=[]-->, belongsTo=parr
1169	assembly-promoting activity than the phosphorylated WT (Fig., otherFeat=[]-->, belongsTo=parr
1170	10A and  supplemental Table S3). Moreover, compared with the, otherFeat=[]-->, belongsTo=parr
1171	WT, G272V is 175% more phosphorylated at Ser202 (Fig. 3) and, otherFeat=[]-->, belongsTo=parr
1172	causes 1.4 times less microtubule formation than the phospho-, otherFeat=[]-->, belongsTo=parr
1173	rylated WT  (supplemental Table S3). Likewise, P301L, V337M,, otherFeat=[]-->, belongsTo=parr
1174	and R406W are phosphorylated 225, 219, and 259% more than, otherFeat=[]-->, belongsTo=parr
1175	the WT at Ser202. The amount of microtubules formed in the, otherFeat=[]-->, belongsTo=parr
1176	presence of phosphorylated P301L, V337M, and R406W is 1.7-,, otherFeat=[]-->, belongsTo=parr
1177	1.5-, and 2.2-fold less than formed in the presence of phospho-, otherFeat=[]-->, belongsTo=parr
1178	rylated WT. Thus there is a correlation between the amount of, otherFeat=[]-->, belongsTo=parr
1179	Ser202 phosphorylation and the amount of loss in the microtu-, otherFeat=[]-->, belongsTo=parr
1180	bule formation-promoting activity of various FTDP-17 tau, otherFeat=[]-->, belongsTo=parr
1181	mutants. Our data indicate that FTDP-17 missense mutations, otherFeat=[]-->, belongsTo=parr
1182	inhibit tau microtubule assembly-promoting activity by pro-, otherFeat=[]-->, belongsTo=parr
1183	moting tau phosphorylation at Ser202, suggesting that FTDP-17, otherFeat=[]-->, belongsTo=parr
1184	mutations may accelerate NFT pathology by increasing Ser202, otherFeat=[]-->, belongsTo=parr
1185	phosphorylation in the brain., otherFeat=[]-->, belongsTo=parr
1186	Previous studies have shown that 60-, 64-, and 68-kDa tau, otherFeat=[]-->, belongsTo=parr
1187	bands of AD brain are also immunoreactive to PHF-1 antibody, otherFeat=[]-->, belongsTo=parr
1188	specific for Ser396/404-phosphorylated tau (51). This observa-, otherFeat=[]-->, belongsTo=parr
1189	tion suggests that Ser396/404 phosphorylation may also promote, otherFeat=[]-->, belongsTo=parr
1190	the formation of 60-, 64-, and 68-kDa tau bands in the brain., otherFeat=[]-->, belongsTo=parr
1191	However, phosphorylation by Cdk5 at Ser396/404 does not cause, otherFeat=[]-->, belongsTo=parr
1192	tau mobility shift to the 68-kDa band (Fig. 3). Our data indi-, otherFeat=[]-->, belongsTo=parr
1193	cate that Cdk5 phosphorylation alone is not sufficient for the, otherFeat=[]-->, belongsTo=parr
1194	Ser396/404-phosphorylated 68-kDa mobility shift. It is possible, otherFeat=[]-->, belongsTo=parr
1195	that for the formation of the Ser396/404-phosphorylated 68-, otherFeat=[]-->, belongsTo=parr
1196	kDa tau band, in addition to Cdk5, phosphorylation by another, otherFeat=[]-->, belongsTo=parr
1197	or other brain kinases may be required. Alternatively, the, otherFeat=[]-->, belongsTo=parr
1198	Ser396/404-phosphorylated 68-kDa band in the brain may, otherFeat=[]-->, belongsTo=parr
1199	result from phosphorylation by kinases other than Cdk5., otherFeat=[]-->, belongsTo=parr
1200	In this study we used the longest human tau isoform, which, otherFeat=[]-->, belongsTo=parr
1201	migrates as a 60-kDa band on SDS-gels. We showed that, otherFeat=[]-->, belongsTo=parr
1202	upon Cdk5 phosphorylation, this isoform migrates as 60-,, otherFeat=[]-->, belongsTo=parr
1203	64-, and 68-kDa bands on an SDS-gel. We also demonstrated, otherFeat=[]-->, belongsTo=parr
1204	that various FTDP-17 mutations enhance the mobility shift of, otherFeat=[]-->, belongsTo=parr
1205	this isoform from 60- to 64-kDa and 68-kDa bands upon, otherFeat=[]-->, belongsTo=parr
1206	Cdk5 phosphorylation. Because PHF-tau isolated from AD, otherFeat=[]-->, belongsTo=parr
1207	brain migrates as 60-, 64-, and 68-kDa bands on an SDS-gel, otherFeat=[]-->, belongsTo=parr
1208	(4, 5, 11), we argued that Cdk5 phosphorylation converts tau to, otherFeat=[]-->, belongsTo=parr
1209	a PHF-like state. However, in adult human brain there are six, otherFeat=[]-->, belongsTo=parr
1210	tau isoforms, and all are present in PHFs (52). Migration of, otherFeat=[]-->, belongsTo=parr
1211	PHF-tau as 60-, 64-, and 68-kDa bands may also be due in part, otherFeat=[]-->, belongsTo=parr
1212	to the difference in the sizes of various tau isoforms present in, otherFeat=[]-->, belongsTo=parr
1213	PHFs. More studies will be required to determine how Cdk5, otherFeat=[]-->, belongsTo=parr
1214	phosphorylation affects the SDS-gel mobility of tau isoforms, otherFeat=[]-->, belongsTo=parr
1215	not analyzed in this study., otherFeat=[]-->, belongsTo=parr
1216	Phosphorylation of FTDP-17 Tau Mutants, otherFeat=[]-->, belongsTo=nota_cab_pie
1217	13432 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie
1218	VOLUME 284 ? NUMBER 20 ? MAY 15, 2009, otherFeat=['U']-->, belongsTo=nota_cab_pie
1219	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
1220	Centro, otherFeat=[]-->, belongsTo=nota_cab_pie
1221	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1222	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1223	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1224	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1225	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1226	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1227	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1228	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
1229	www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie
1230	Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie
1231	from, otherFeat=[]-->, belongsTo=nota_cab_pie
1232	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie
1233	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
1234	Acknowledgment--We thank Dr. Peter Davies of Albert Einstein Col-, otherFeat=[]-->, belongsTo=parrnote
1235	lege of Medicine (Bronx, NY) for providing plasmids containing vari-, otherFeat=[]-->, belongsTo=parrnote
1236	ous FTDP-17 tau mutants and PHF1, TG3, and MC6 antibodies., otherFeat=[]-->, belongsTo=parrnote
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1360	JOURNAL OF BIOLOGICAL CHEMISTRY 13433, otherFeat=[]-->, belongsTo=nota_cab_pie
1361	at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie
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1363	Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie
1364	de, otherFeat=[]-->, belongsTo=nota_cab_pie
1365	Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie
1366	Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie
1367	on, otherFeat=[]-->, belongsTo=nota_cab_pie
1368	May, otherFeat=[]-->, belongsTo=nota_cab_pie
1369	28,, otherFeat=[]-->, belongsTo=nota_cab_pie
1370	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
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1375	Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie
==============================
0	Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro*S-->id=0, page=0, size=27, fam=Times, col=#231f20, type=title, textLines=3--->[]--->title	Familial FTDP-17 Mis>>>at Ser202 in Vitro*S
1	Received for publication, February 17, 2009, and in revised form, March 17, 2009 Published, JBC Papers in Press, March 19, 2009, DOI 10.1074/jbc.M901095200-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Received for publica>>>.1074/jbc.M901095200
2	Dong Han1, Hamid Y. Qureshi2, Yifan Lu, and Hemant K. Paudel§3 From the Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and the §Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3T 1E2, Canada-->id=6, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->[]--->note	Dong Han1, Hamid Y. >>>ebec H3T 1E2, Canada
3	In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as 60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as 50 ­ 60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from 60-kDa bands to 64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser202, and Ser202 phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser202 phosphorylation and mobility shift to a 68-kDa band. Furthermore, Ser202 phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser202, inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser202 phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->parr	In Alzheimer disease>>>related tauopathies.
4	Neurofibrillary tangles (NFTs)4 and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD).-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Neurofibrillary tang>>>heimer disease (AD).
5	The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1­3). Initially, PHFs were found to be composed of a protein component referred to as "A68" (4). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4, 5). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6, 7). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1­3).-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	The major fibrous co>>> in the brain (1­3).
6	PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8, 9). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6, 10). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	PHF-tau is phosphory>>>u dysfunction in AD.
7	Tau purified from the human brain migrates as 50 ­ 60kDa bands on SDS-gel due to the presence of six isoforms that are phosphorylated to different extents (2). PHF-tau isolated from AD brain, on the other hand, displays 60-, 64-, and 68 kDa-bands on an SDS-gel (4, 5, 11). Studies have shown that 64- and 68-kDa tau bands (the authors have described the 68-kDa tau band as an 69-kDa band in these studies) are present only in brain areas affected by NFT degeneration (12, 13). Their amount is correlated with the NFT densities at the affected brain regions. Moreover, the increase in the amount of 64- and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The 64- and 68-kDa tau bands were suggested to be the pathological marker of AD (12, 13). Biochemical analyses determined that 64- and 68-kDa bands are hyperphosphorylated tau, which upon dephosphorylation, migrated as normal tau on SDS-gel (4, 5, 11). Tau sites involved in the tau mobility shift to 64- and 68-kDa bands were suggested to have a role in AD pathology (12, 13). It is not known whether phosphorylation at all 21 PHFsites is required for the tau mobility shift in AD. However, in vitro the tau mobility shift on SDS-gel is sensitive to phosphorylation only on some sites (6, 14). It is therefore possible that in the AD brain, phosphorylation on some sites also causes a tau mobility shift. Identification of such sites will significantly enhance our knowledge of how NFT pathology develops in the brain.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Tau purified from th>>>velops in the brain.
8	* This work was supported by grants from the Canadian Institute for Health Research, the Alzheimer Society of Canada, and the National Science and Engineering Research Council of Canada.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->parr	* This work was supp>>>h Council of Canada.
9	S The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1­S3.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->parr	S The on-line versio>>>mental Tables S1­S3.
10	1 Recipient of a Ph.D. studentship from the Alzheimer Society of Canada. 2 Postdoctoral scholar of the Parkinson Society of Canada.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->parr	1 Recipient of a Ph.>>>n Society of Canada.
11	3 To whom correspondence should be addressed: Lady Davis Inst. for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8222, Ext. 4866; Fax: 514-340-7502; E-mail: hemant.paudel@mcgill.ca.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->parr	3 To whom correspond>>>nt.paudel@mcgill.ca.
12	4 The abbreviations used are: NFT, neurofibrillary tangle; AD, Alzheimer disease; Cdk5, cyclin-dependent protein kinase 5; AU, absorption unit; FTDP17, frontotemporal dementia and Parkinsonism linked to chromosome 17; PHF, paired helical filament; PKA, cAMP-dependent protein kinase; Pipes, 1,4-piperazinediethanesulfonic acid; WT, wild type.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->parr	4 The abbreviations >>>acid; WT, wild type.
13	THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 20, pp. 13422­13433, May 15, 2009-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	THE JOURNAL OF BIOLO>>>­13433, May 15, 2009
14	© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	© 2009 by The Americ>>>rinted in the U.S.A.
15	13422 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	13422 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
16	VOLUME 284 · NUMBER 20 · MAY 15, 2009-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	VOLUME 284 · NUMBER >>>ER 20 · MAY 15, 2009
17	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
18	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
19	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
20	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
21	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
22	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
23	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
24	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
25	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
26	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
27	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
28	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
29	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
30	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
31	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
32	PHFs are also the major component of NFTs found in the brains of patients suffering from a group of neurodegenerative disorders collectively called tauopathies (2, 11). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays 60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2, 11). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15­18). Understanding how FTDP-17 mutations promote tau phosphorylation can provide a better understanding of how NFT pathology develops in AD and various tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and P301L tau mutations reduce tau phosphorylation (19, 20). In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21, 22). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24). When phosphorylated by recombinant GSK3 in vitro, the P301L and V337M mutations do not have any effect, and the R406W mutation inhibits phosphorylation (25). However, when incubated with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations stimulate tau phosphorylation (26). The mechanism by which FTDP-17 mutations promote tau phosphorylation leading to development of NFT pathology has remained unclear.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	PHFs are also the ma>>>as remained unclear.
33	Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27, 28). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility shift to 64- and 68 kDa-bands. Although the mobility shift to a 64-kDa band is achieved by phosphorylation at Ser396/404 or Ser202, the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser202. We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser202, enhance the mobility shift to 64- and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser202 phosphorylation is the major event leading to NFT pathology in AD and related tauopathies.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Cyclin-dependent pro>>>related tauopathies.
34	MATERIALS AND METHODS-->id=6, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->['U']--->title	MATERIALS AND METHOD>>>ATERIALS AND METHODS
35	cDNA Cloning--The longest isoform of human tau and FTDP-17 tau mutants G272V, P301L, V337M, and R406W, each in the pQE32 vector, were gifts from Dr. Peter Davies (Albert Einstein College of Medicine, Bronx, NY). Cloning of tau mutants S202A, T231A, and S396A in pcDNA3.1 vector is described previously (29). Tau mutants S262A and S214A in pcDNA3.1 vector were gifts from Dr. Nicole Leclerc (University of Montreal). Each DNA fragment from the WT or mutant tau was amplified by PCR using pfu DNA polymerase (Stratagene), with a forward primer (5 -AAAAAACGCCATATGGCTGAGCCCCGC-3 ) that contained an NdeI site and a reverse primer (5 -AAA AAA GGA TCC TCA CAA ACC CTG CTT GG-3 ) that contained a BamHI site, and subcloned into bacterial expression vector pET9a (Promega). Various double mutants, each containing the indicated FTDP-17 and S202A mutations, were cloned by PCR using their respective FTDP-17 mutant in pET9a vector as the template and the QuikChange II site-specific mutagenesis kit (Stratagene) following the manufacturer's instruction manual. Primers used for PCR were 5 -CAG CGG CTA CAG CAG CCC CGG CGC CCC AGG CAC TCC CGG CAG CCG C-3 and 5 -GCG GCT GCC GGG AGT GCC TGG GGC GCC GGG GCT GCT GTA GCC GCT G3 . All cDNA clones and mutations were confirmed by DNA sequencing.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	cDNA Cloning--The lo>>>d by DNA sequencing.
36	Proteins and Enzymes--Tau(WT) and various tau mutants were purified from lysates of Escherichia coli overexpressing their respective tau species essentially as described previously (28). Briefly, tau expression was induced by adding isopropyl 1-thio- -D-galactopyranoside (0.2 mM) to the overnight bactehave been described previously (29, 31). Polyclonal antibodies pS202 and pT212 against tau phosphorylated at Ser202 and Thr212, respectively, were purchased from BIOSOURCE. Cdk5 was purified from the extract of fresh bovine brain (28). The active catalytic subunit of PKA was purchased from Sigma-Aldrich. Purification of protein phosphatase 1 (PP1) from E. coli extract overexpressing human PP1 has been described previously (32, 33).-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Proteins and Enzymes>>>previously (32, 33).
37	rial culture. The culture containing isopropyl 1-thio- -D-galac--->id=4, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=6--->[]--->note	rial culture. The cu>>>yl 1-thio- -D-galac-
38	topyranoside was allowed to grow for3hat37 °C with shaking and then was centrifuged. The pellet was suspended in Pipes-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	topyranoside was all>>>s suspended in Pipes
39	buffer (100 mM Pipes (pH 6.8), 1 mM EGTA, 1 mM MgSO4)-->id=4, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=6--->[]--->note	buffer (100 mM Pipes>>>mM EGTA, 1 mM MgSO4)
40	containing 5 mg/ml benzamidine, 1 g/ml leupeptine, 1 g/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 20 g/ml lysozyme. The bacterial suspension was lysed by sonication and then clarified by centrifugation (15,000 rpm, 15 min at 4 °C). The supernatant was placed in a boiling water bath for 20 min and subsequently centrifuged. The heat-stable proteins in the supernatant were loaded onto a Q-Sepharose Fast Flow column ( 1 ml; Amersham Biosciences) equilibrated previously in Pipes buffer. The flow-through containing tau was loaded onto an SP-Sepharose Fast Flow column ( 1 ml) equilibrated in Pipes buffer. The column was washed with 20 column volumes of the Pipes buffer and then eluted with Pipes buffer containing 0.2 M NaCl. Fractions containing tau were pooled, concentrated with Aquacade III (Calbiochem) by dialysis, dialyzed against Hepes buffer (25 mM Hepes (pH 7.2), 0.1 mM EDTA, 0.5 mM dithiothreitol, and 100 mM NaCl), and stored at 80 °C until use. Microtubules were purified from fresh bovine brain extract by three cycles of temperature-induced microtubule polymerization and depolymerization as described previously (28, 30). Tubulin was purified from purified microtubules through phosphocellulose chromatography (28, 30). Monoclonal tau 5 antibody against total tau and tau phosphorylationdependent monoclonal antibodies AT8, PHF-1, MC6, and TG3-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	containing 5 mg/ml b>>> PHF-1, MC6, and TG3
41	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
42	MAY 15, 2009 · VOLUME 284 · NUMBER 20-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	MAY 15, 2009 · VOLUM>>>LUME 284 · NUMBER 20
43	JOURNAL OF BIOLOGICAL CHEMISTRY 13423-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 13423
44	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
45	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
46	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
47	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
48	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
49	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
50	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
51	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
52	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
53	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
54	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
55	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
56	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
57	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
58	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
59	Protein Concentrations--Tau(WT) concentration is based on its absorption at A280 nm as described previously (28). The concentrations of various FTDP-17 tau mutants were determined by Bio-Rad protein assay using tau(WT) as the standard. Concentrations of phosphorylated tau and tau mutants were also determined by Bio-Rad protein assay using tau(WT) as the standard. The concentration of Cdk5 is based on its activity (28). PKA concentration was determined by its dry weight. The concentrations of all other proteins were determined by BioRad proteins assay using bovine serum albumin as the standard. Tau Phosphorylation--Tau(WT) and various tau mutants were phosphorylated by Cdk5 under identical conditions. Each phosphorylation mixture contained 25 mM Hepes (pH 7.2), 0.1 microtubule nucleation, polymerization, and formation-promoting activities of V337M and R406W are also significantly less than that of the WT (Fig. 1 and  supplemental Table S1). This observation is consistent with previous reports (37, 38) and indicates that FTDP-17 mutations impair the microtubule assembly-promoting activity of tau.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Protein Concentratio>>>ing activity of tau.
60	mM EDTA, 0.2 mM dithiothreitol, 0.1 M NaCl, 10 mM MgCl2, 0.5-->id=4, page=0, size=8, fam=Times, col=#231f20, type=note, textLines=6--->[]--->note	mM EDTA, 0.2 mM dith>>>Cl, 10 mM MgCl2, 0.5
61	mM [ 32P] ATP, 1.0 mg/ml tau, and 400 units/ml Cdk5. The reaction was initiated by adding an aliquot of Cdk5 to a vial containing the rest of the phosphorylation mixture at 30 °C. At the indicated time points, aliquots were withdrawn and analyzed for phosphorylation by filter paper assay (34) or subjected to SDS-PAGE followed by Western blot analysis. Gel and blot bands were scanned, and the band intensity values were used to determine the relative amounts of various proteins. Phosphorylation of tau and tau mutants by PKA was also performed as described above, except Cdk5 was replaced by PKA at a concentration of 10 g/ml each.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	mM [ 32P] ATP, 1.0 m>>>ion of 10 g/ml each.
62	Microtubule Assembly Assay--The microtubule assembly was monitored by measuring the increase of A350 by a spectrophotometer (35). The vial containing all of the components of the assay except tau was incubated at 37 °C for 1 min in a water bath. To the incubated vial, the indicated prewarmed tau species was added. After gentle mixing, the content of the vial was transferred immediately to a quartz cuvette placed in a spectrophotometer at 37 °C. The increase in the A350 of the transferred sample was recorded at 1-min intervals for 32 min. The final concentrations of various components in the assay were 0.75 mg/ml tubulin, 100 mM Pipes (pH 6.8), 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgSO4,1mM GTP, 10 M taxol, and 0.2 mg/ml tau (indicated species). The lag phase of polymerization is defined as the time at which the rise in the A350 is detected since initiation of the assay. The rate of polymerization is the A350 at the steady state divided by the minimum time required to achieve the steady state after the lag phase, and it is expressed as absorption units per min (AU/min). The amount of microtubule formed corresponds to the maximum A350 reached during the assay.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Microtubule Assembly>>>ed during the assay.
63	RESULTS-->id=6, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->[]--->title	RESULTS>>>RESULTS
64	Effect of Phosphorylation on Microtubule Assembly-promoting Activity of FTDP-17 Tau Mutants--In vitro, purified tubulin polymerizes in the presence of GTP/Mg2 and assembles into microtubules. This process consists of an initial lag phase during which microtubules nucleate (35, 36). Following nucleation, microtubules polymerize and reach the steady state. In vitro microtubule assembly can be monitored spectrophotometrically by the light scattering technique, which measures the turbidity of the solution at 350 nm, which increases as the microtubules assemble (35, 37). When present, tau promotes microtubule assembly by influencing some or all of these parameters (35­37). To determine how phosphorylation affects the microtubule assembly promoting activity of various tau species, we phosphorylated WT and FTDP-17 mutants by Cdk5 under identical conditions. Each phosphorylated and nonphosphorylated tau species was included in the microtubule assembly mixture, and the assembly was monitored by light scattering assay. The concentration of tubulin was kept low so that no detectable turbidity was observed in the absence of tau.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Effect of Phosphoryl>>> the absence of tau.
65	Microtubules in the presence of tau(WT) assembled with a lag time of 2 min at a rate of 0.0636 AU/min. When P301L was used, the lag phase was extended to 6 min and the polymerization rate was reduced to 0.0363 AU/min (Fig. 1 and  supplemental Table S1). These data determined that in the presence of P301L, microtubule nucleation and polymerization occurred 3 and 1.75 times, respectively, slower than in the presence of tau(WT). This, in turn, indicates that the microtubule nucleation-promoting activity of P301L is 33.3% of that of the WT. Likewise, microtubule polymerization-promoting activity of P301L is 57% of that of the WT. In the presence of the WT, microtubule polymerization plateaued at A350 0.70, which represents the amount of microtubules formed  (supplemental Table S1). This value is reduced to 0.40 in the presence of P301L. These data indicate that the microtubule formationpromoting activity of P301L is 57.1% of that of the WT. The-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Microtubules in the >>> that of the WT. The
66	FIGURE 1. Microtubule assembly in the presence of phosphorylated and nonphosphorylated tau(WT) or FTDP-17 tau mutants. Microtubule assembly was monitored by the light scattering technique described under "Materials and Methods" in the presence of the indicated tau species. Phosphorylated tau is indicated by -P. Changes in the A350 were recorded every min.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 1. Microtubul>>> recorded every min.
67	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
68	13424 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	13424 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
69	VOLUME 284 · NUMBER 20 · MAY 15, 2009-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	VOLUME 284 · NUMBER >>>ER 20 · MAY 15, 2009
70	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
71	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
72	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
73	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
74	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
75	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
76	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
77	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
78	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
79	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
80	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
81	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
82	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
83	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
84	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
85	Phosphorylation reduced the microtubule nucleation, polymerization, and formation-promoting activities of tau(WT) to 33.3, 29.5, and 28.6%, respectively (Fig. 1 and  supplemental Table S1). Compared with its nonphosphorylated counterpart, the microtubule nucleation, polymerization, and formation-promoting activities of phosphorylated P301L were 54.6, 30.5, and 50.0%, respectively, those of V337M were 36.4, 31.7, and 50.0%, respectively, and those of R406W were 58.5, 36.0, and 45.9%, respectively. Thus, phosphorylation also reduced the microtubule assembly-promoting activities of all FTDP-17 tau mutants.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Phosphorylation redu>>>FTDP-17 tau mutants.
86	Effect of FTDP-17 Mutations on Tau Phosphorylation--As shown in  supplemental Table S1, the rate of microtubule nucleation and polymerization and the amount of microtubules formed are significantly less in the presence of all phosphorylated FTDP-17 mutants when compared with that of the phosphorylated WT. One likely explanation for this observation is that the FTDP-17 mutation and phosphorylation act additively to reduce the microtubule assembly-promoting activity of tau. However, some FTDP-17 mutations may promote tau phosphorylation and by doing so may further affect the microtubule assembly-promoting activity of tau. To understand how phosphorylation affects the microtubule assembly-promoting activity of various FTDP-17 tau mutants, we set out to determine how each FTDP-17 mutation affects tau phosphorylation. We phosphorylated tau(WT) and various FTDP-17 tau mutants by Cdk5 under identical conditions and analyzed them on an SDSgel (Fig. 2). Phosphorylated tau(WT) displayed two bands of sizes 60 and 64 kDa (lane 2). Control tau(WT), incubated with all of the components of the phosphorylation mixture except Cdk5, migrated as one 60-kDa band (lane 6), which is consistent with a previous report (39) and shows that Cdk5 phosphorylation causes a mobility shift of tau(WT) from a 60- to 64-kDa band on an SDS-gel. Interestingly, when phosphorylated P301L was analyzed, it displayed three bands of sizes 60, 64, and 68 kDa (lane 3). Phosphorylated V337M and R406W also displayed 60-, 64-, and 68-kDa bands (lanes 4, 5). When a portion of each sample used to generate Fig. 2 was Western blotted using anti-tau antibody, in addition to the 60- and 64-kDa bands, phosphorylated tau(WT) also showed a faint 68-kDa band (data not shown). All FTDP-17 mutants, on the other hand, displayed prominent 60-, 64-, and 68-kDa bands. When monoclonal antibody AT8, which recognizes phosphorylated tau, was used in Western blot analysis, all 60-, 64-, and 68-kDa bands of WT and FTDP-17 mutants displayed immunoreactivity (data not shown but see below). When phosphorylated, various tau species were incubated with protein phosphatase 1 and the products analyzed by SDS-PAGE. All of the WT and FTDP-17 mutants almost completely lost the 64- and 68-kDa bands and displayed a major 60-kDa band (data not shown). Based on these results, we concluded that phosphorylation by Cdk5 caused a mobility shift of tau(WT) to 64- and 68-kDa bands and that the FTDP-17 missense mutations P301L, V337M, and R406W promoted a phosphorylation-induced mobility shift.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Effect of FTDP-17 Mu>>>uced mobility shift.
87	We reasoned that there were two possibilities to explain the increased mobility shift of FTDP-17 tau mutants upon phosphorylation. Because all FTDP-17 mutants tested displayed more mobility shift than the WT, the first possibility is that FTDP-17 mutations promote phosphorylation-induced mobility shift. As we did not include a nonspecific point mutant in our assay, the second possibility is that structural change caused by any point mutation causes mobility shift of tau upon phosphorylation by Cdk5. To discriminate between the above two possibilities, we decided to examine another FTDP-17 tau mutant, G272V, and two nonspecific mutants, S214A and S262A. Because FTDP-17 mutant P301L, V337M, and R406W are located at the C terminus, we selected G272V, situated at the central region of the tau molecule. We chose S214A and S262A because these sites are neither mutated in any tauopathies (2) nor are they phosphorylated by Cdk5 (27). Moreover, Ser262 is located only 10 residues away from Gly272, and S262A is a good control against G272V. We phosphorylated WT, S214A, S262A, G272V, P301L, V337M, and R406W by Cdk5 under identical conditions and analyzed the products by SDS-PAGE. Phosphorylated WT showed 60- and 64-kDa bands, whereas phosphorylated P301L, V337M, and R406W all showed 60-, 64-, and 68-kDa bands. Phosphorylated S214A and S262A, on the other hand, migrated as 60- and 64-kDa bands in a manner similar to the phosphorylated WT (data not shown, but see Fig. 3A). More importantly, phosphorylated G272V displayed 60-, 64-, and 68-kDa bands similar to those shown by phosphorylated P301L, V337M, and R406W (data not shown). Thus, among the three new mutants tested (G272V, S214A, and S262A), only G272V associated with FTDP-17 promoted tau a mobility shift to a 68-kDa band upon phosphorylation by Cdk5. These data are consistent with the idea that FTDP-17 mutations promote tau mobility shift upon phosphorylation by Cdk5.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	We reasoned that the>>>phorylation by Cdk5.
88	FIGURE 2. SDS-PAGE of phosphorylated tau(WT) and FTDP-17 tau mutants. Indicated tau species (5 g each) phosphorylated by Cdk5 under identical conditions for 60 min (lanes 2­5) or control samples incubated with all the components of the phosphorylation mixture except Cdk5 (lanes 6 ­9) were subjected to 10% SDS-PAGE. The resulting gel was stained with Coomassie Brilliant Blue. M (lane 1) represents standard molecular weight marker.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 2. SDS-PAGE o>>>cular weight marker.
89	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
90	MAY 15, 2009 · VOLUME 284 · NUMBER 20-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	MAY 15, 2009 · VOLUM>>>LUME 284 · NUMBER 20
91	JOURNAL OF BIOLOGICAL CHEMISTRY 13425-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 13425
92	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
93	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
94	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
95	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
96	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
97	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
98	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
99	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
100	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
101	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
102	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
103	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
104	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
105	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
106	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
107	To determine the basis of the mobility shift, we wanted to know first whether FTDP-17 mutations stimulate tau phosphorylation. We phosphorylated tau(WT) and various FTDP-17 tau mutants by Cdk5 under identical conditions for different time periods and determined the number of phosphate incorporated into each tau species. Tau(WT) incorporated 1.1 mol of phosphate/mol of protein in 15 min. This value increased to 2.4 in 30 min and became 4.1 in 60 min. On SDS-gel, tau(WT) phosphorylated for 15 min migrated as a 60-kDa band but became 60- and 64-kDa bands after 60 min of phosphorylation (data not shown). G272V incorporated 1.5, 3.4, and 5.2 mol of phosphate/mol of protein in 15, 30, and 60 min, respectively. In 15, 30, and 60 min, P301L contained 1.3, 2.5, and 4 mol of phosphate/mol of protein, respectively, whereas V337M incorporated 1.2, 3.2, and 3.9 mol of phosphate/mol of protein, respectively. R406W, on the other hand, incorporated 1.2, 2.5, and 3.2 mol of phosphate/mol of protein in 15, 30, and 60 min, respectively. Thus after 60 min, G272V incorporated more phosphate, whereas P301L and V337M incorporated amounts similar to the WT. R406W, on the other hand, incorporated less phosphate than the WT. On SDS-gel, however, all FTDP-17 mutants phosphorylated for 60 min migrated as 60-, 64-, and 68-kDa bands (data not shown, but see Fig. 2). These data indicated that the increased mobility shift of phosphorylated FTDP-17 mutants was not due to the higher extent of total phosphorylation of mutants compared with the WT.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	To determine the bas>>>ompared with the WT.
108	Effect of FTDP-17 Mutations on Site-specific Phosphorylation of Tau--To evaluate whether FTDP-17 mutations, by promoting tau phosphorylation at any specific site(s), increase the tau mobility shift, we phosphorylated tau(WT) and various FTDP-17 tau mutants by Cdk5 under identical conditions. Controls were S214A and S262A. Phosphorylated products were analyzed by Western blot using antibodies that recognize phosphorylated tau on proline-directed sites Ser396, Ser404, Ser235, Thr231, Ser202/205, and Thr212, which are potential targets of Cdk5 (27).-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Effect of FTDP-17 Mu>>>argets of Cdk5 (27).
109	As shown in Fig. 3, compared with the WT, G272V and P301L are more phosphorylated and R406W is less phosphorylated at Ser396/404.AtSer235, G272V and V337M are more phosphorylated and R406W is less phosphorylated than the WT. At Thr231, WT and all mutants are phosphorylated to similar extents. However, at Ser202, G272V, P301L, V337M, and R406W are 1.8-, 2.2-. 2.1-, and 2.5-fold more phosphorylated than the WT. At Thr212, on the other hand, neither WT nor any of the FTDP-17 mutants was phosphorylated. Thus, at Ser235 and Ser396/404, some mutations promoted and some inhibited phosphorylation. At Ser202, neither the S214A nor the S262A control showed any effect, whereas all FTDP-17 mutations probut not at Ser396/404, Ser235,orThr231.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	As shown in Fig. 3, >>>04, Ser235,orThr231.
110	FIGURE 3. Site-specific phosphorylation of tau and FTDP-17 tau mutants by Cdk5. Tau(WT) and the indicated tau mutants were phosphorylated by Cdk5 for 60 min. Each phosphorylated sample (1 g each) was subjected to Western blotting using antibody specific for total tau or tau phosphorylated at the indicated site. Blots were scanned, and based on the intensities of various bands relative tau phosphorylation was calculated. A, Western blots. B, relative phosphorylation. To calculate relative phosphorylation, the sum of the band intensity values of all bands in a lane of the blot of the indicated tau species representing the tau phosphorylated at indicated site was normalized against the sum of the band intensity values of all bands of that tau species in that lane of the blot representing the total tau. The resulting value of each species was further normalized against the resulting value of the WT to be expressed as percent of the WT. All values are the average of three determinations. Tau phosphorylated by GST-GSK3 was used as a positive control on lane 1 of pT212 blot. Recombinant GST-GSK3 was purified as described (30, 53).-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 3. Site-speci>>> described (30, 53).
111	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
112	13426 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	13426 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
113	VOLUME 284 · NUMBER 20 · MAY 15, 2009-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	VOLUME 284 · NUMBER >>>ER 20 · MAY 15, 2009
114	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
115	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
116	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
117	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
118	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
119	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
120	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
121	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
122	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
123	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
124	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
125	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
126	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
127	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
128	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
129	moted phosphorylation. To substantiate these data, we analyzed all of the above phosphorylated samples by using a monoclonal AT8 antibody specific for Ser202-phosphorylated tau. Like pS202, AT8 antibody indicated that all FTDP-17 mutations promote tau phosphorylation at Ser202 (data not shown). When Western blotted using tau 5 antibody for total tau, phosphorylated tau(WT) showed two major bands of sizes 60 and 64 kDa and a faint 68-kDa band, whereas V337M, R406W, P301L, and G272V displayed prominent 60-, 64-, and 68-kDa bands (Fig. 3A). PHF-1 antibody, specific for Ser396/404-phosphorylated tau, stained 60- and 64-kDa bands and failed to recognize the 68-kDa band of all tau species. Similarly, MC6 and TG3 antibodies, which recognize tau phosphorylated at Ser235 and Thr231, respectively, stained only a 60-kDa band for WT and all FTDP-17 mutants. However, pS202 antibody, specific for tau phosphorylated at Ser202, recognized all three, the 60-, 64-, and 68-kDa bands. These data indicate that the 60-kDa band of all tau species is phosphorylated at Ser396/404, Ser235, Thr231, and Ser202. Similarly, the 64-kDa band of all tau species is phosphorylated at Ser396/404 and Ser202, and the 68-kDa band is phosphorylated at Ser202-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	moted phosphorylatio>>>phorylated at Ser202
130	Next, we phosphorylated tau and various FTDP-17 mutants at different time points and analyzed them by Western blot using AT8 antibody. Tau(WT) phosphorylated by Cdk5 for 5 min became immunoreactive (Fig. 4A). At 30 min, tau(WT) displayed mobility shift, and at 60 min 60-, 64-, and 68-kDa bands became visible. G272V, P301L, V337M, and R406W also displayed Ser202-phosphorylated 60-, 64-, and 68-kDa bands. Blot band quantification determined that at the 60-min time point, out of all Ser202-phosphorylated tau(WT), 40% remained as a 60-kDa band and the rest shifted to 64 ( 50%)- and 68 ( 10%)-kDa bands (Fig. 4B). G272V and P301L, on the other hand, displayed a 68kDa band with a relative amount (48 and 49%, respectively) of the total. The relative amount of 68-kDa bands was 45 and 44% of the total in V337M and R406W, respectively. When compared with the WT, G272V, P301L, V337M, and R406W displayed, respectively 4.8-, 4.9-, 4.5-, and 4.4fold more Ser202-phosphorylated 68-kDa band upon phosphorylation by Cdk5 (Fig. 4B). Thus, all FTDP-17 mutations promoted phosphorylation at Ser202 as well as mobility shift to a 68-kDa band.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Next, we phosphoryla>>>ft to a 68-kDa band.
131	Effect of Ser202 Phosphorylation on Tau Structure--Tau(WT) displayed a mobility shift that correlated with phosphorylation at Ser202, and FTDP-17 mutations accelerated this process (Figs. 3 and 4). To determine the significance of this phenomenon, we wanted to know the role of Ser202 phosphorylation on the tau mobility shift on SDS-gel/Western blot. We phosphorylated tau(WT) and the site-specific tau mutant S202A under identical conditions and analyzed the products by Western blot analysis. Tau(WT) became increasingly phosphorylated with time (Fig. 5A, lanes 2­5). At 15 min, tau(WT) displayed mobility shift to 64 kDa, which became prominent at 60 min (Fig. 5B, lanes 2­ 4). At 120 min, the 60-kDa band of tau(WT) faded significantly with the appearance of a 68-kDa band (Fig. 5B, lane 5). S202A showed mobility shift, and a 64-kDa band became visible at 15 min. With increasing time, the relative amount of 64-kDa band increased progressively, and that of the 60-kDa band decreased. However, even with phosphorylation at 120 min the 68-kDa band was not formed significantly (Fig. 5B, lane 11). Thus, unlike tau(WT), S202A failed to display the 68-kDa mobility shift in response to Cdk5 phosphorylation. These data indicate that tau does not display mobility shift to the 68-kDa band upon phosphorylation by Cdk5 if phosphorylation at Ser202 is blocked.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Effect of Ser202 Pho>>>t Ser202 is blocked.
132	Tau is phosphorylated at multiple sites (8). Studies have shown that tau phosphorylation on some sites affect subsequent phosphorylation on other sites (29). To determine whether blocking Ser202 phosphorylation may inhibit phosphorylation at other sites, which may prevent mobility shift, phosphorylated tau(WT) and S202A were Western blotted using antibodies that recognize phosphorylated tau (Fig. 6). Tau(WT) was phosphorylated at Ser202, Ser396/404, Ser235, and Thr231 (Fig. 6, lane 3). S202A, on the other hand, was phosphorylated on all of the above sites except Ser202 (Fig. 6, lane 4). Thus, blocking Ser202 phosphorylation did not prevent phosphorylation at any of the major sites known to be phosphorylated by Cdk5.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Tau is phosphorylate>>>sphorylated by Cdk5.
133	Cdk5 phosphorylates tau on a number of sites (27). Among these sites, Ser396 and Thr231 were reported to cause tau conformational change (6, 29, 40). To evaluate whether mobility shift caused by Cdk5 phosphorylation is specific for Ser202,we phosphorylated tau(WT), S202A, S396A, and T231A by Cdk5 and analyzed the products by Western blot analysis (Fig. 7). Phosphorylated tau(WT) migrated as 60-, 64-, and 68-kDa bands (Fig. 7, lanes 12­15). Phosphorylated S202A was not phosphorylated at Ser202, and it displayed 60- and 64-kDa but not 68-kDa bands (Fig. 7, lanes 7­10). S396A was phosphorylated at Ser202 and showed 60-, 64-, and 68-kDa bands (Fig. 7, lanes 1­ 4). Similarly, phosphorylated T231A was phosphorylated at Ser202 and displayed 60-, 64-, and 68-kDa bands (Fig. 7, lanes 17­20). Thus, blocking phosphorylation at Ser202, but not at Ser396 or Thr231, prevented mobility shift to a 68kDa band upon Cdk5 phosphorylation.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Cdk5 phosphorylates >>>dk5 phosphorylation.
134	FIGURE 4. Phosphorylation of tau and FTDP-17 tau mutants by Cdk5 on Ser202. The indicated tau species were phosphorylated by Cdk5. At the indicated time points, aliquots were withdrawn, and 1 g of each sample was subjected to Western blot analysis using AT8 antibody that recognizes tau phosphorylated at Ser202. Based on the intensities of various bands at the 60-min time point, the relative amount of a 68-kDa band of each sample was determined. A, Western blots. B, relative amount. To calculate the relative amount, the intensity value of the 68-kDa band of each sample in each blot was normalized against the sum of the band intensity values of 60-, 64-, and 68-kDa bands of that sample in that blot. The values are the average of three determinations.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 4. Phosphoryl>>>hree determinations.
135	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
136	MAY 15, 2009 · VOLUME 284 · NUMBER 20-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	MAY 15, 2009 · VOLUM>>>LUME 284 · NUMBER 20
137	JOURNAL OF BIOLOGICAL CHEMISTRY 13427-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 13427
138	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
139	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
140	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
141	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
142	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
143	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
144	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
145	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
146	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
147	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
148	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
149	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
150	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
151	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
152	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
153	PKA is a non-proline-directed kinase and phosphorylates tau at several sites including Ser214 but not at Ser202 (34, 41, 42). To gain more evidence in favor of the idea that Ser202 phosphorylation is the major factor in the tau mobility shift on SDS-PAGE, we phosphorylated tau WT and S202A by Cdk5 or PKA for 120 min. Products were analyzed by Western blot using tau 5 antibody (data not included). Tau(WT) phosphorylated by Cdk5 for 120 min showed 60-, 64-, and 68-kDa bands. S202A phosphorylated by Cdk5 under identical conditions showed only 60- and 64-kDa bands. Both tau(WT) and S202A phosphorylated by PKA, on the other hand, showed two bands of sizes 60 and 64 kDa (data not shown). Thus, Cdk5 phosphorylates tau at Ser202 and causes mobility shift to a 68kDa band, and PKA, which does not phosphorylate Ser202, does not cause this shift. Based on these results, we concluded that phosphorylation at Ser202 is a major determinant for the tau mobility shift to the 68-kDa band upon Cdk5 phosphorylation.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	PKA is a non-proline>>>dk5 phosphorylation.
154	Effect of Ser202 Phosphorylation-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	Effect of Ser202 Pho>>>r202 Phosphorylation
155	on Microtubule Assembly-promoting Activity of Tau--To determine why FTDP-17 mutations promote phosphorylation at Ser202, we examined the effect of Ser202 phosphorylation on the microtubule assembly-promoting activity of tau. We phosphorylated WT and S202A by Cdk5. As controls, we phosphorylated T231A, S396S, and S214A. Thr231 and Ser396 are phosphorylated in vivo (8) and in vitro by Cdk5 (27). Ser214 is not phosphorylated by Cdk5, and hence it was used to monitor the effect of the Ser to Ala mutation on tau activity. Microtubule assembly-promoting activities of nonphosphorylated and phosphorylated tau were monitored.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	on Microtubule Assem>>> tau were monitored.
156	As shown in Fig. 8 and  supplemental Table S2, nonphosphorylated S202A, T231A, S396A, S214A, and WT promoted microtubule assembly with similar nucleation time and polymerization rate, leading to the formation of similar amounts of microtubules. Likewise, the microtubule assembly-promoting activities of phosphorylated WT and phosphorylated S214A are similar. These data demonstrate that mutation of any nonspecific Ser to Ala does not significantly affect the microtubule assembly-promoting activity of tau.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	As shown in Fig. 8 a>>>ing activity of tau.
157	The microtubule assembly-promoting activity of all phosphorylated WT, S214A, T231A, and S396A was significantly less than their respective nonphosphorylated counterparts (Fig. 8A). This observation indicated that phosphorylation inhibits the microtubule assembly-promoting activities of all the above tau species. Moreover, although the microtubule assemblypromoting activity of phosphorylated T231A is similar to that of phosphorylated WT, those of phosphorylated S396A and S202A are higher (Fig. 8A). Thus, the microtubule assemblypromoting activity of phosphorylated tau is not affected significantly by blocking Thr231 phosphorylation. Blocking phosphorylation at Ser396 or Ser202, however, increases the microtubule assembly-promoting activity of phosphorylated tau. These data, in turn, indicate that phosphorylation at Ser396 or Ser202 decreases the microtubule assembly-promoting activity of tau. Microtubules, in the presence of phosphorylated WT, nucleated at 6 min  (supplemental Table S2). In the presence of phosphorylated S202A, nucleation time was reduced to 5 min, an increase of 16.5% nucleation promoting activity. This means that Ser202 phosphorylation inhibits 16.6% of tau microtubule nucleation-promoting activity (Fig. 8B). In the presence of phosphorylated WT, microtubules polymerized with a rate of 0.016 AU/min. In the presence of phosphorylated S202A, the microtubule polymerization rate was increased 1.5-fold to 0.024 AU/min. These data indicate that Ser202 phosphorylation inhibits 50% of tau microtubule polymerization-promoting activity (Fig. 8B). Finally, the amount of microtubules formed in the presence of phosphorylated S202A was 1.4 times more than that formed in the presence of phosphorylated WT, indicating that Ser202 phosphorylation inhibits 40% of tau microtubule formation-promoting activity. Furthermore, the microtubule nucleation polymerization and formation-promoting activities of phosphorylated S202A are higher than that of phosphorylated Ser396 (supplemental Table S2 and Fig. 8B). These data show that the microtubule assembly-promoting activity of tau is inhibited more by phosphorylation at Ser202 than by phosphorylation at Ser396. Taken together, these data indicate that phosphorylation at Ser202 has the major inhibitory impact on the microtubule assembly-promoting activity of tau in vitro.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	The microtubule asse>>>ity of tau in vitro.
158	FIGURE 5. Effect of Ser202 phosphorylation on SDS-gel mobility of tau. Tau(WT) and tau(S202A) phosphorylated by Cdk5 using [ 32P]ATP for the indicated time points were analyzed by Western blot (IB). The blot was subsequently autoradiographed to monitor radioactivity in each band.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 5. Effect of >>>tivity in each band.
159	FIGURE 6. Site-specific phosphorylation of tau(WT) and tau(S202A) by Cdk5. Tau(WT) and tau(S202A), phosphorylated for 120 min, were analyzed by Western blot (IB) using the indicated antibodies.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 6. Site-speci>>>ndicated antibodies.
160	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
161	13428 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	13428 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
162	VOLUME 284 · NUMBER 20 · MAY 15, 2009-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	VOLUME 284 · NUMBER >>>ER 20 · MAY 15, 2009
163	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
164	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
165	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
166	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
167	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
168	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
169	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
170	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
171	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
172	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
173	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
174	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
175	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
176	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
177	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
178	Effect of Ser202 Phosphorylation-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	Effect of Ser202 Pho>>>r202 Phosphorylation
179	on SDS-gel Mobility Shift of FTDP-17 Tau Mutants--To determine whether missense FTDP-17 mutations promote mobility shift by promoting phosphorylation at Ser202, we mutated Ser202 of each of the FTDP-17 mutants to Ala. All double mutants were phosphorylated along with their corresponding FTDP-17 single mutants, WT, and S202A by Cdk5. All phosphorylated proteins were then analyzed by Western blot.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	on SDS-gel Mobility >>>zed by Western blot.
180	All nonphosphorylated single and double mutants migrated as single 60-kDa band on SDS-gel (Fig. 9A). Phosphorylated WT migrated as 60- and 64-kDa bands and a relatively weak 68-kDa band (Fig. 9B, lane 3), whereas phosphorylated S202A-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	All nonphosphorylate>>>phosphorylated S202A
181	FIGURE 7. Effect of Ser396, Thr231, and Ser202 phosphorylation on SDS-gel mobility of tau. The indicated tau species phosphorylated by Cdk5 were analyzed by Western blot using the indicated antibodies.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 7. Effect of >>>ndicated antibodies.
182	FIGURE 8. Effect of Ser202, Ser396, and Thr231 phosphorylation on microtubule assembly-promoting activity of tau. Microtubule assembly was monitored in the presence of the indicated tau species. From the light scattering data, microtubule nucleation lag time, polymerization rate, and the amount of microtubules formed were calculated as described under "Materials and Methods"  (supplemental Table S2) and were used to determine the inhibition of various parameters of the microtubule assembly. To determine the percent inhibition of microtubule nucleation by Ser202 phosphorylation, the lag time value of phosphorylated S202A was subtracted from the lag time value of phosphorylated WT. The resulting value was then normalized against the lag time value of phosphorylated WT. Note that this value is the gain in the microtubule nucleation-promoting activity of phosphorylated WT upon blocking Ser202 phosphorylation. This, in turn, is the contribution of Ser202 phosphorylation in inhibiting the nucleation-promoting activity of tau. To determine the percent inhibition of microtubule polymerization by Ser202 phosphorylation, the polymerization rate of phosphorylated WT was subtracted from that of phosphorylated S202A. The resulting value was normalized against the polymerization rate of phosphorylated WT. Likewise, the percent inhibition of the microtubule amount formed by Ser202 phosphorylation was calculated as described above for microtubule polymerization, except microtubule amounts for phosphorylated WT and phosphorylated S202A were used. The percent inhibition of microtubule nucleation, polymerization, and microtubule formation by Ser396 and Thr231 phosphorylation were calculated in the same manner using the values of phosphorylated S396A and phosphorylated T231A, respectively. A, microtubule assembly. B, inhibition of microtubule assembly by phosphorylation at Ser202, Thr231, and Ser396. Values in B are an average of three determinations.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 8. Effect of >>>hree determinations.
183	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
184	MAY 15, 2009 · VOLUME 284 · NUMBER 20-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	MAY 15, 2009 · VOLUM>>>LUME 284 · NUMBER 20
185	JOURNAL OF BIOLOGICAL CHEMISTRY 13429-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 13429
186	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
187	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
188	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
189	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
190	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
191	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
192	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
193	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
194	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
195	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
196	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
197	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
198	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
199	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
200	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
201	migrated as 60- and 64-kDa bands (Fig. 9B, lane 2). Likewise, each phosphorylated G272V, P301L, V337M, and R406W displayed prominent 60-, 64-, and 68-kDa bands (Fig. 9B, lanes 4­7). These data are consistent with the data of Fig. 2 and show that FTDP-17 mutations promote mobility shift of tau on SDSgel. However, the phosphorylated double mutant G272V/ S202A migrated as 60- and 64-kDa bands and failed to display any significant 68-kDa mobility shift (Fig. 9B, lane 8). Similarly, phosphorylated P301L/S202A, V337M/S202A, and R406W/S202A also showed only 60- and 64-kDa bands (Fig. 9B, lanes 9 ­11). Thus, blocking Ser202 phosphorylation blocked the phosphorylation-induced 68-kDa mobility shift of all FTDP-17 tau mutants. These data indicate that FTDP-17 mutations lose their abilities to promote mobility shift to a 68-kDa band if phosphorylation at Ser202 is blocked. This in turn indicates that FTDP-17 mutations promote the 68-kDa band mobility shift by enhancing phosphorylation at Ser202.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	migrated as 60- and >>>orylation at Ser202.
202	Effect of Ser202 Phosphorylation-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	Effect of Ser202 Pho>>>r202 Phosphorylation
203	on Microtubule Assembly-promoting Activity of FTDP-17 Tau Mutants--Finally, we monitored the microtubule assembly-promoting activities of phosphorylated double mutants and their respective FTDP-17 single mutants. Nonphosphorylated WT and WT(S202A) promoted microtubule assembly with similar nucleation time and polymerization rate and caused the formation of similar amounts of microtubules. Likewise, nonphosphorylated double mutants G272V/ S202A, P301L/S202A, V337M/ S202A, and R406W/S202A supported microtubule assembly in a manner similar to their respective FTDP-17 single mutants (data not included). Thus, mutation of Ser202 to Ala did not affect the microtubule assembly-promoting activity of WT or any of the FTDP-17 mutants.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	on Microtubule Assem>>>the FTDP-17 mutants.
204	Phosphorylated WT(S202A) displayed microtubule nucleation, polymerization, and formation-promoting activities higher than that of phosphorylated WT (Fig. 10A and supplemental Table S3). These data are consistent with the observation made in Fig. 8, demonstrating that blocking Ser202 phosphorylation enhances the microtubule assembly-promoting activity of phosphorylated tau. As shown in Fig. 10A and  supplemental Table S3, microtubule nucleation-promoting activity of phosphorylated G272V/S202A is more than that of phosphorylated G272V. Likewise, microtubule polymerization and formation-promoting activities of phosphorylated G272V/S202A are higher than that of phosphorylated G272V. Moreover, microtubule nucleation, polymerization, and formation-promoting activities of phosphorylated P301L/S202A, V337M/S202A, and R406W/S202A are higher than those of their respective phosphorylated FTDP-17 single mutants. Thus, as with phosphorylated WT, blocking Ser202 phosphorylation increased the microtubule assembly-promoting activity of all phosphorylated FTDP-17 mutants. This indicates that Ser202 phosphorylation inhibits the microtubule assembly-promoting activity of tau(WT) and all its FTDP-17 mutants.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Phosphorylated WT(S2>>>its FTDP-17 mutants.
205	FIGURE 9. Effect of Ser202 phosphorylation on the SDS-gel mobility of phosphorylated FTDP-17 tau mutants. Indicated tau mutants phosphorylated by Cdk5 were Western blotted using tau 5 antibody. A, nonphosphorylated tau species. B, phosphorylated tau species.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 9. Effect of >>>rylated tau species.
206	FIGURE 10. Effect of Ser202 phosphorylation on microtubule assembly-promoting activities of FTDP-17 tau mutants. Microtubule assembly was monitored in the presence of the indicated tau species by light scattering as described in the legend for Fig. 1A. Based on the light scattering data, the nucleation lag, microtubule polymerization rate, and microtubule amount formed were calculated  (supplemental Table S3), and these values were used to determine the inhibition of microtubule assembly-promoting activity of WT and its FTDP-17 mutants by Ser202 phosphorylation. A, microtubule assembly. B and C, inhibition of microtubule assembly. By using values shown in  supplemental Table S3, panel B was generated as described for Ser202 phosphorylation in the legend for Fig. 8. To generate panel C, the value of each tau species in each section of B was normalized against the value of the WT in that section of B to be expressed as the -fold of WT. The values are an average of three determinations.-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->capfig	FIGURE 10. Effect of>>>hree determinations.
207	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
208	13430 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	13430 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
209	VOLUME 284 · NUMBER 20 · MAY 15, 2009-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	VOLUME 284 · NUMBER >>>ER 20 · MAY 15, 2009
210	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
211	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
212	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
213	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
214	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
215	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
216	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
217	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
218	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
219	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
220	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
221	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
222	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
223	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
224	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
225	Microtubule nucleation-promoting activity of phosphorylated WT is 6 min, whereas that of phosphorylated WT(S202A) is 5 min  (supplemental Table S3). This means that blocking Ser202 phosphorylation increases the nucleation activity of phosphorylated WT by 16.6%. This, in turn, indicates that 16.6% of the microtubule nucleation-promoting activity of tau(WT) is inhibited by Ser202 phosphorylation. As shown in Fig. 10B, Ser202 phosphorylation inhibits the microtubule nucleationpromoting activity of G272V, P301L, V337M, and R406W by 22.2, 18.2, 27.3, and 25.0%, respectively. Ser202 phosphorylation inhibits the microtubule polymerization-promoting activity of WT by 37.5% and that of G272V, P301L, V337M, and R406W by 45.0, 63.6, 44.2, and 70.0%, respectively. Similarly, Ser202 phosphorylation inhibits the microtubule formation-promoting activity of WT by 40.6%, which is increased in G272V, P301L, V337M, and R406W mutants to 53.8, 71.4, 65.2, and 88.9%, respectively. Thus, compared with the WT, Ser202 phosphorylation inhibits the microtubule nucleation-promoting activity of G272V, P301L, V337M, and R406W 1.34-, 1.10-, 1.64-, and 1.50-fold more, respectively, microtubule polymerization-promoting activity 1.32-, 1.75-, 1.60-, and 2.20-fold more, respectively, and microtubule formation-promoting activity 1.32-, 1.75-, 1.6-, and 2.20-fold more, respectively (Fig. 10C). These data indicate that phosphorylation at Ser202 has a more profound inhibitory effect on the microtubule assembly-promoting activity of FTDP-17 mutants than on that of the WT.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Microtubule nucleati>>>n on that of the WT.
226	DISCUSSION-->id=6, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->[]--->title	DISCUSSION>>>DISCUSSION
227	The presence of 64- and 68-kDa tau bands is a characteristic feature of the AD brain, and studies suggest that the appearance of these species correlates with the disease progression (12, 13). Because these tau species are formed due to abnormal tau phosphorylation, tau sites that are responsible for causing their formation are suggested to be involved in the development of AD pathology in brain (12, 13).-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	The presence of 64- >>>y in brain (12, 13).
228	Cdk5 is one of the kinases suggested to phosphorylate tau in the AD brain (27, 39). In vitro, Cdk5 phosphorylates tau on several sites that are phosphorylated in PHFs including Ser202, Thr231, and Ser396 (27). In this study, nonphosphorylated tau migrated as a 60-kDa band on SDS-gel. Upon phosphorylation by Cdk5, tau migrated as 60-, 64-, and 68-kDa bands (Fig. 4). These data indicate that phosphorylation by Cdk5 cause mobility shift of tau to 64- and 68-kDa bands. Although phosphorylation at Ser396/404 also promoted tau mobility shift to a 64-kDa band, only Ser202-phosphorylated tau displayed both 64- and 68-kDa bands, and phosphorylation at Ser202 correlated with their formation (Figs. 3 and 4). Furthermore, blocking Thr231 or Ser396 did not affect tau mobility shift to 64- or 68-kDa bands. Blocking Ser202 phosphorylation blocked mobility shift to a 68-kDa band (Fig. 7). Our data indicate that tau mobility shift to a 64-kDa band can occur without Ser202 phosphorylation. However, mobility shift to a 68-kDa band in response to Cdk5 phosphorylation-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Cdk5 is one of the k>>>Cdk5 phosphorylation
229	requires Ser202 phosphorylation.-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	requires Ser202 phos>>>202 phosphorylation.
230	As shown in Fig. 3, the 68-kDa band formed by Cdk5 phosphorylation is phosphorylated on Ser202 but not at Ser396/404, Thr231, Ser235,orThr212. This indicates that the 68-kDa band is formed as a result of phosphorylation at Ser202 but not at Ser396/404, Thr231, Ser235,orThr212. Similarly, the 64-kDa band is phosphorylated at Ser202 and Ser396/404 but not at Thr231, Ser235, and Thr212, indicating that the 64-kDa band is formed due to phosphorylation at Ser202 and Ser396/404 and not at Thr231, Ser235,orThr212. Furthermore, the Ser202-phosphorylated tau first migrates as a 60-kDa band (Fig. 4A, lane 2). With the increase in phosphorylation time, 64-kDa band followed by 68-kDa band appear. These data suggest that Ser202phosphorylated tau migrates as a 60-kDa band and that phosphorylation on this site alone does not cause a tau band shift. The mobility shift of tau may, therefore, occur by a sequential mechanism. First-step phosphorylation at Ser202 may allow Cdk5 to perform second-step phosphorylation on new sites that are not accessible in non-Ser202-phosphorylated tau. Firstplus second-step-phosphorylated tau may then migrate as a 64-kDa band. The 64-kDa phosphorylated tau may then undergo third-step phosphorylation on additional sites and become hyperphosphorylated. The hyperphosphorylated tau may then migrate as a 68-kDa band. Note that in this mechanism, the 68-kDa band will be expected to contain phosphate on all the sites, including Ser202, that are involved in the mobility shift from 60- to 64-kDa and then to 68-kDa bands. As shown in Fig. 3, the 68-kDa band is phosphorylated at Ser202 but not at Ser396/404, Ser235, Thr231,orThr212. This observation suggests that phosphorylation on the new, additional sites that occurs after phosphorylation at Ser202, and results in a 60 ­ 68-kDa band shift, has to occur at sites other than Ser396/404, Ser235, Thr231, and Thr212. In fact, in addition to the sites mentioned above, PHF-tau is phosphorylated on a number of proline-directed sites, including Ser199, Thr181, Thr217, and Ser222, which are potential targets of Cdk5 (8).-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	As shown in Fig. 3, >>>targets of Cdk5 (8).
231	Our data have demonstrated that Thr231 phosphorylation did not have a significant effect on microtubule nucleation or formation-promoting activities and only slightly affected the microtubule polymerization-promoting activity of tau (Fig. 8A). Likewise, Ser396 phosphorylation inhibited microtubule polymerization and formation-promoting activities but did not affect microtubule nucleation-promoting activity (Fig. 8A and supplemental Table S2). Ser202 phosphorylation, on the other hand, not only inhibited microtubule nucleation-promoting activity, but it inhibited microtubule polymerization and formation-promoting activities 2- and 1.5-fold more than Ser396 phosphorylation (Fig. 8B). Our results indicate that Ser202 phosphorylation significantly inhibits the microtubule assembly-promoting activity of tau in vitro. It should be noted that under our experimental conditions, Cdk5 may have phosphoysis of the brain using AT8 monoclonal antibody (43, 44). The AT8 epitope first appears in the pre-AD brain areas that do not show any brain degeneration and are devoid of NFTs. But slowly as the disease progresses and the brain begins to degenerate, the intensity of AT8 reactivity increases (43­ 45). Among several tau phosphorylation-sensitive antibodies tested, AD brains stain strongest with AT8 (46). When purified PHF-tau is Western blotted, 60-, 64-, and 68-kDa tau bands cross-react with AT8 antibody (47). AT8 immunoreactivity in the brain is regarded as the abnormal cytoskeletal change that occurs during the AD development (43, 44). In vitro AT8 specifically recognizes tau phosphorylated at Ser202 and/or Ser205 (48). However, mass spectrometric studies have determined that PHF-tau is phosphorylated at Ser202 but not at Ser205 (8, 9). Thus, AT8 immunoreactivity in the brain represents tau phosphorylated at Ser202. Moreover, among several sites tested, only phosphorylation at Ser202 correlates with mobility shift of tau to 64- and 68-kDa bands upon Cdk5 phosphorylation in vitro (Figs. 3 and 4). Also, all pathogenic FTDP-17 tau missense mutations that accelerate NFT pathology in the brain promote phosphorylation at Ser202 (Figs. 3 and 4). Taken together, these observations suggest that Ser202 phosphorylation is the major pathological event in the brain leading to brain degeneration in AD.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Our data have demons>>> degeneration in AD.
232	rylated tau at Ser202 to a higher extent than at Ser396 and Thr231.-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	rylated tau at Ser20>>>t Ser396 and Thr231.
233	The observed difference in the ability of each of the above sites to inhibit tau microtubule assembly-promoting activity may be due, in part, to the differences in extent of phosphorylation.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	The observed differe>>> of phosphorylation.
234	The pathological significance of Ser202 phosphorylation in the brain is not very clear. Studies suggest that mild memory impairment is the earliest clinical feature of AD and is associated with subtle cytoskeletal alterations in pre-tangle neurons. This alteration can be detected by immunohistochemical anal--->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	The pathological sig>>>ohistochemical anal-
235	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
236	MAY 15, 2009 · VOLUME 284 · NUMBER 20-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	MAY 15, 2009 · VOLUM>>>LUME 284 · NUMBER 20
237	JOURNAL OF BIOLOGICAL CHEMISTRY 13431-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 13431
238	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
239	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
240	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
241	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
242	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
243	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
244	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
245	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
246	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
247	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
248	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
249	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
250	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
251	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
252	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
253	Exonic and intronic mutations have been discovered in the familial type of FTDP-17. Although intronic mutations interfere with pre-mRNA splicing leading to an increase in fourrepeat tau in the brain, the mechanism by which exonic mutations promote NFT pathology is an area of current research in neurobiology. Studies using antibodies directed against tau phosphorylated at various sites have determined that V337M FTDP-17 brains stain most intensely with AT8 (Ser202), less-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Exonic and intronic >>>h AT8 (Ser202), less
254	intensely with PHF-1 (Ser396/404), AT100 (Thr212/Ser214),-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	intensely with PHF-1>>>100 (Thr212/Ser214),
255	AT180 (Thr231/Ser235), AT270 (Ser181), and 12E8 (Ser262), indicating that V337M in the brain is most strongly phosphorylated-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	AT180 (Thr231/Ser235>>>ongly phosphorylated
256	at Ser202 and less strongly at Ser396/404, Thr212/214, Thr231/-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->parr	at Ser202 and less s>>> Thr212/214, Thr231/
257	Ser235, Ser181, and Ser262 (46). Likewise, P301L in the brain is most extensively phosphorylated at Ser202, Ser396/404, and Thr212/Ser214 and less extensively at Thr231/Ser235 and Ser262 (49, 50). G272V in the brain is most extensively phosphorylated at Ser202, less extensively at Ser396/404 and Thr231/Ser235, and not at all at Ser262 (17). Finally, R406W in the brain is most strongly phosphorylated at Ser202, Ser396/404, and Thr212/Ser214 (50). Thus, among all of the different sites examined, all FTDP-17 tau mutants in the brain are most extensively phosphorylated at Ser202 recognized by AT8 antibody. In addition, PHF-tau isolated from all FTDP-17 mutant brains migrates as 60-, 64-, and 68-kDa bands on SDS-gel (11, 17, 46, 49, 50), and in vitro Ser202 phosphorylation promotes tau mobility shift to 64- and 68-kDa bands (Fig. 4). These studies indicate that all missense FTDP-17 mutations promote tau phosphorylation at Ser202 in the brain.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Ser235, Ser181, and >>>Ser202 in the brain.
258	In this study, we examined four FTDP-17 tau mutations, G272V, P301L, V337M, and R406W, and found that these mutations, by promoting phosphorylation at some sites and inhibiting at some sites, do not significantly affect the total amount of phosphate incorporated into the tau molecule. Although phosphorylation at Thr231 is not influenced by any mutation, phosphorylation at Ser396/404 is promoted by G272V and P301L but inhibited is by R406W (Fig. 3). Ser235 phosphorylation is promoted by V337M, G272V, and P301L but is inhibited by R406W. Phosphorylation at Ser202, on the other hand, is promoted by all of the FTDP-17 missense mutations, and by enhancing phosphorylation at Ser202 each mutation promoted a tau mobility shift to 64- and 68-kDa bands (Figs. 3, 4, and 9). Furthermore, compared with the phosphorylated WT, each phosphorylated FTDP-17 mutant displayed reduced microtubule assembly-promoting activity (Fig. 10). When Ser202 phosphorylation was blocked, each phosphorylated mutant recovered more relative amounts of microtubule assembly-promoting activity than the phosphorylated WT (Fig. 10A and  supplemental Table S3). Moreover, compared with the WT, G272V is 175% more phosphorylated at Ser202 (Fig. 3) and causes 1.4 times less microtubule formation than the phosphorylated WT  (supplemental Table S3). Likewise, P301L, V337M, and R406W are phosphorylated 225, 219, and 259% more than the WT at Ser202. The amount of microtubules formed in the presence of phosphorylated P301L, V337M, and R406W is 1.7-, 1.5-, and 2.2-fold less than formed in the presence of phosphorylated WT. Thus there is a correlation between the amount of Ser202 phosphorylation and the amount of loss in the microtubule formation-promoting activity of various FTDP-17 tau mutants. Our data indicate that FTDP-17 missense mutations inhibit tau microtubule assembly-promoting activity by promoting tau phosphorylation at Ser202, suggesting that FTDP-17 mutations may accelerate NFT pathology by increasing Ser202 phosphorylation in the brain.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	In this study, we ex>>>lation in the brain.
259	Previous studies have shown that 60-, 64-, and 68-kDa tau bands of AD brain are also immunoreactive to PHF-1 antibody specific for Ser396/404-phosphorylated tau (51). This observation suggests that Ser396/404 phosphorylation may also promote the formation of 60-, 64-, and 68-kDa tau bands in the brain. However, phosphorylation by Cdk5 at Ser396/404 does not cause tau mobility shift to the 68-kDa band (Fig. 3). Our data indicate that Cdk5 phosphorylation alone is not sufficient for the Ser396/404-phosphorylated 68-kDa mobility shift. It is possible that for the formation of the Ser396/404-phosphorylated 68kDa tau band, in addition to Cdk5, phosphorylation by another or other brain kinases may be required. Alternatively, the Ser396/404-phosphorylated 68-kDa band in the brain may result from phosphorylation by kinases other than Cdk5.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	Previous studies hav>>>ses other than Cdk5.
260	In this study we used the longest human tau isoform, which migrates as a 60-kDa band on SDS-gels. We showed that upon Cdk5 phosphorylation, this isoform migrates as 60-, 64-, and 68-kDa bands on an SDS-gel. We also demonstrated that various FTDP-17 mutations enhance the mobility shift of this isoform from 60- to 64-kDa and 68-kDa bands upon Cdk5 phosphorylation. Because PHF-tau isolated from AD brain migrates as 60-, 64-, and 68-kDa bands on an SDS-gel (4, 5, 11), we argued that Cdk5 phosphorylation converts tau to a PHF-like state. However, in adult human brain there are six tau isoforms, and all are present in PHFs (52). Migration of PHF-tau as 60-, 64-, and 68-kDa bands may also be due in part to the difference in the sizes of various tau isoforms present in PHFs. More studies will be required to determine how Cdk5 phosphorylation affects the SDS-gel mobility of tau isoforms not analyzed in this study.-->id=11, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=1193--->[]--->parr	In this study we use>>>lyzed in this study.
261	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
262	13432 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	13432 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY
263	VOLUME 284 · NUMBER 20 · MAY 15, 2009-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	VOLUME 284 · NUMBER >>>ER 20 · MAY 15, 2009
264	at-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[u'a']--->note	at>>>at
265	Centro-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Centro>>>Centro
266	Nacional-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Nacional>>>Nacional
267	de-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	de>>>de
268	Investigaciones-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Investigaciones>>>Investigaciones
269	Oncológicas,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Oncológicas,>>>Oncológicas,
270	on-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	on>>>on
271	May-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	May>>>May
272	28,-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	28,>>>28,
273	2010-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	2010>>>2010
274	www.jbc.org-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	www.jbc.org>>>www.jbc.org
275	Downloaded-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	Downloaded>>>Downloaded
276	from-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->note	from>>>from
277	http://www.jbc.org/content/suppl/2009/03/20/M901095200.DC1.html-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	http://www.jbc.org/c>>>/M901095200.DC1.html
278	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at:
279	Acknowledgment--We thank Dr. Peter Davies of Albert Einstein College of Medicine (Bronx, NY) for providing plasmids containing various FTDP-17 tau mutants and PHF1, TG3, and MC6 antibodies.-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->parr	Acknowledgment--We t>>> and MC6 antibodies.
280	REFERENCES-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->parr	REFERENCES>>>REFERENCES
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327	53. Yuan, Z., Agarwal-Mawal, A., and Paudel, H. K. (2004) J. Biol. Chem. 279, 26105­26114-->id=5, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=397--->[]--->parr	53. Yuan, Z., Agarwa>>>em. 279, 26105­26114
328	Phosphorylation of FTDP-17 Tau Mutants-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note	Phosphorylation of F>>> FTDP-17 Tau Mutants
329	MAY 15, 2009 · VOLUME 284 · NUMBER 20-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->['U']--->note	MAY 15, 2009 · VOLUM>>>LUME 284 · NUMBER 20
330	JOURNAL OF BIOLOGICAL CHEMISTRY 13433-->id=3, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=63--->[]--->note	JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 13433
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345	Supplemental Material can be found at:-->id=7, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=69--->[]--->note	Supplemental Materia>>>ial can be found at: