Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro*

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.

Neurofibrillary tangles (NFTs) 4 and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD).
The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. [1][2][3]. 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 (1)(2)(3).
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.
Tau purified from the human brain migrates as ϳ50 -60-kDa 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 ϳ60-, 64-, and 68 kDa-bands on an SDS-gel (4,5,11). Studies have shown that ϳ64and 68-kDa tau bands (the authors have described the ϳ68-kDa tau band as an ϳ69-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 ϳ64and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The ϳ64and 68-kDa tau bands were suggested to be the pathological marker of AD (12,13). Biochemical analyses determined that ϳ64and 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 ϳ64and 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.
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 , corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays ϳ60-, 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 (15)(16)(17)(18). 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.
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 ϳ64and 68 kDa-bands. Although the mobility shift to a ϳ64-kDa band is achieved by phosphorylation at Ser 396/404 or Ser 202 , the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser 202 . We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser 202 , enhance the mobility shift to ϳ64and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser 202 phosphorylation is the major event leading to NFT pathology in AD and related tauopathies.

MATERIALS AND METHODS
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 (5Ј-AAAAAACGCCATATGGCT-GAGCCCCGC-3Ј) that contained an NdeI site and a reverse primer (5Ј-AAA AAA GGA TCC TCA CAA ACC CTG CTT GG-3Ј) 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 5Ј-CAG CGG CTA CAG CAG CCC CGG CGC CCC AGG CAC TCC CGG CAG CCG C-3Ј and 5Ј-GCG GCT GCC GGG AGT GCC TGG GGC GCC GGG GCT GCT GTA GCC GCT G-3Ј. All cDNA clones and mutations were confirmed by DNA sequencing.
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 bacterial culture. The culture containing isopropyl 1-thio-␤-D-galactopyranoside was allowed to grow for 3 h at 37°C with shaking and then was centrifuged. The pellet was suspended in Pipes buffer (100 mM Pipes (pH 6.8), 1 mM EGTA, 1 mM MgSO 4 ) 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 (ϳ1 ml; Amersham Biosciences) equilibrated previously in Pipes buffer. The flow-through containing tau was loaded onto an SP-Sepharose Fast Flow column (ϳ1 ml) equilibrated in Pipes buffer. The column was washed with ϳ20 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 Ϫ80°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 have been described previously (29,31). Polyclonal antibodies pS202 and pT212 against tau phosphorylated at Ser 202 and Thr 212 , 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).
Protein Concentrations-Tau(WT) concentration is based on its absorption at A 280 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 Bio-Rad 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 mM EDTA, 0.2 mM dithiothreitol, 0.1 M NaCl, 10 mM MgCl 2 , 0.5 mM [␥ 32 P] 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.
Microtubule Assembly Assay-The microtubule assembly was monitored by measuring the increase of A 350 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 A 350 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 MgSO 4 , 1 mM 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 A 350 is detected since initiation of the assay. The rate of polymerization is the A 350 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 A 350 reached during the assay.

Effect of Phosphorylation on Microtubule
Assembly-promoting Activity of FTDP-17 Tau Mutants-In vitro, purified tubulin polymerizes in the presence of GTP/Mg 2ϩ 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 (35)(36)(37). 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.
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 A 350 ϭ 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 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.
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 ϳ60 and 64 kDa (lane 2). Control tau(WT), incubated with all of the components of the phosphorylation mixture except Cdk5, migrated as one ϳ60-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 ϳ60to 64-kDa band on an SDS-gel. Interestingly, when phosphorylated P301L was analyzed, it displayed three bands of sizes ϳ60, 64, and 68 kDa (lane 3). Phosphorylated V337M and R406W also displayed ϳ60-, 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 ϳ60and ϳ64-kDa bands, phosphorylated tau(WT) also showed a faint ϳ68-kDa band (data not shown). All FTDP-17 mutants, on the other hand, displayed prominent ϳ60-, 64-, and 68-kDa bands. When monoclonal antibody AT8, which recognizes phosphorylated tau, was used in Western blot analysis, all ϳ60-, 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 ϳ64and 68-kDa bands and displayed a major ϳ60-kDa band (data not shown). Based on these results, we concluded that phosphorylation by Cdk5 caused a mobility shift of tau(WT) to ϳ64and 68-kDa bands and that the FTDP-17 missense mutations P301L, V337M, and R406W promoted a phosphorylation-induced mobility shift.
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, Ser 262 is located only 10 residues away from Gly 272 , 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 ϳ60and 64-kDa bands, whereas phosphorylated P301L, V337M, and R406W all showed ϳ60-, 64-, and 68-kDa bands. Phosphorylated S214A and S262A, on the other hand, migrated as ϳ60and 64-kDa bands in a manner similar to the phosphorylated WT (data not shown, but see Fig. 3A). More importantly, phosphorylated G272V displayed  MAY 15, 2009 • VOLUME 284 • NUMBER 20 ϳ60-, 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 ϳ68-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.

Phosphorylation of FTDP-17 Tau Mutants
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 ϳ1.1 mol of phosphate/mol of protein in 15 min. This value increased to ϳ2.4 in 30 min and became ϳ4.1 in 60 min. On SDS-gel, tau(WT) phosphorylated for 15 min migrated as a ϳ60-kDa band but became ϳ60and 64-kDa bands after 60 min of phosphorylation (data not shown). G272V incorporated ϳ1.5, 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 ϳ1.3, 2.5, and 4 mol of phosphate/mol of protein, respectively, whereas V337M incorporated ϳ1.2, 3.2, and 3.9 mol of phosphate/mol of protein, respectively. R406W, on the other hand, incorporated ϳ1.2, 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 ϳ60-, 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.
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 Ser 396 , Ser 404 , Ser 235 , Thr 231 , Ser 202/205 , and Thr 212 , which are potential targets of Cdk5 (27).
As shown in Fig. 3, compared with the WT, G272V and P301L are more phosphorylated and R406W is less phosphorylated at Ser 396/404 . At Ser 235 , G272V and V337M are more phosphorylated and R406W is less phosphorylated than the WT. At Thr 231 , WT and all mutants are phosphorylated to similar extents. However, at Ser 202 , G272V, P301L, V337M, and R406W are 1.8-, 2.2-. 2.1-, and 2.5-fold more phosphorylated than the WT. At Thr 212 , on the other hand, neither WT nor any of the FTDP-17 mutants was phosphorylated. Thus, at Ser 235 and Ser 396/404 , some mutations promoted and some inhibited phosphorylation. At Ser 202 , neither the S214A nor the S262A control showed any effect, whereas all FTDP-17 mutations pro- Recombinant GST-GSK3␤ was purified as described (30,53). moted phosphorylation. To substantiate these data, we analyzed all of the above phosphorylated samples by using a monoclonal AT8 antibody specific for Ser 202 -phosphorylated tau. Like pS202, AT8 antibody indicated that all FTDP-17 mutations promote tau phosphorylation at Ser 202 (data not shown).
Effect of Ser 202 Phosphorylation on Tau Structure-Tau(WT) displayed a mobility shift that correlated with phosphorylation at Ser 202 , 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 Ser 202 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 2-5). At 15 min, tau(WT) displayed mobility shift to ϳ64 kDa, which became prominent at 60 min (Fig.  5B, lanes 2-4). At 120 min, the ϳ60-kDa band of tau(WT) faded significantly with the appearance of a ϳ68-kDa band (Fig.  5B, lane 5). S202A showed mobility shift, and a ϳ64-kDa band became visible at 15 min. With increasing time, the relative amount of ϳ64-kDa band increased progressively, and that of the ϳ60-kDa band decreased. However, even with phosphorylation at 120 min the ϳ68-kDa band was not formed significantly (Fig. 5B, lane 11). Thus, unlike tau(WT), S202A failed to display the ϳ68-kDa mobility shift in response to Cdk5 phosphorylation. These data indicate that tau does not display mobility shift to the ϳ68-kDa band upon phosphorylation by Cdk5 if phosphorylation at Ser 202 is blocked.
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 Ser 202 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 Ser 202 , Ser 396/404 , Ser 235 , and Thr 231 (Fig. 6, lane 3). S202A, on the other hand, was phosphorylated on all of the above sites except Ser 202 (Fig. 6, lane 4). Thus, blocking Ser 202 phosphorylation did not prevent phosphorylation at any of the major sites known to be phosphorylated by Cdk5.

Phosphorylation of FTDP-17 Tau Mutants
PKA is a non-proline-directed kinase and phosphorylates tau at several sites including Ser 214 but not at Ser 202 (34,41,42). To gain more evidence in favor of the idea that Ser 202 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 ϳ60-, 64-, and 68-kDa bands. S202A phosphorylated by Cdk5 under identical conditions showed only ϳ60and 64-kDa bands. Both tau(WT) and S202A phosphorylated by PKA, on the other hand, showed two bands of sizes ϳ60 and 64 kDa (data not shown). Thus, Cdk5 phosphorylates tau at Ser 202 and causes mobility shift to a ϳ68-kDa band, and PKA, which does not phosphorylate Ser 202 , does not cause this shift. Based on these results, we concluded that phosphorylation at Ser 202 is a major determinant for the tau mobility shift to the ϳ68-kDa band upon Cdk5 phosphorylation.
Effect of Ser 202 Phosphorylation on Microtubule Assembly-promoting Activity of Tau-To determine why FTDP-17 mutations promote phosphorylation at Ser 202 , we examined the effect of Ser 202 phosphorylation on the microtubule assembly-promoting activity of tau. We phosphorylated WT and S202A by Cdk5. As controls, we phosphorylated T231A, S396S, and S214A. Thr 231 and Ser 396 are phosphorylated in vivo (8) and in vitro by Cdk5 (27). Ser 214 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.
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.
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 Thr 231 phosphorylation. Blocking phosphorylation at Ser 396 or Ser 202 , however, increases the microtubule assembly-promoting activity of phosphorylated tau. These  data, in turn, indicate that phosphorylation at Ser 396 or Ser 202 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 phos-phorylated S202A, nucleation time was reduced to 5 min, an increase of 16.5% nucleation promoting activity. This means that Ser 202 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 Ser 202 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 Ser 202 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 Ser 396 (supplemental Table S2 and Fig.  8B). These data show that the microtubule assembly-promoting activity of tau is inhibited more by phosphorylation at Ser 202 than by phosphorylation at Ser 396 . Taken together, these data indicate that phosphorylation at Ser 202 has the major inhibitory impact on the microtubule assembly-promoting activity of tau in vitro.
Effect of Ser 202 Phosphorylation on SDS-gel Mobility Shift of FTDP-17 Tau Mutants-To determine whether missense FTDP-17 mutations promote mobility shift by promoting phosphorylation at Ser 202 , we mutated Ser 202 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. All nonphosphorylated single and double mutants migrated as single ϳ60-kDa band on SDS-gel (Fig. 9A). Phosphorylated WT migrated as ϳ60and 64-kDa bands and a relatively weak ϳ68-kDa band (Fig. 9B, lane 3), whereas phosphorylated S202A   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 Ser 202 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 Ser 202 phosphorylation. This, in turn, is the contribution of Ser 202 phosphorylation in inhibiting the nucleation-promoting activity of tau. To determine the percent inhibition of microtubule polymerization by Ser 202 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 Ser 202 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 Ser 396 and Thr 231 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 Ser 202 , Thr 231 , and Ser 396 . Values in B are an average of three determinations. migrated as ϳ60and 64-kDa bands (Fig. 9B, lane 2). Likewise, each phosphorylated G272V, P301L, V337M, and R406W displayed prominent ϳ60-, 64-, and 68-kDa bands (Fig. 9B, lanes  4 -7). 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 ϳ60and 64-kDa bands and failed to display any significant ϳ68-kDa mobility shift (Fig. 9B, lane 8). Similarly, phosphorylated P301L/S202A, V337M/S202A, and R406W/S202A also showed only ϳ60and 64-kDa bands (Fig.  9B, lanes 9 -11). Thus, blocking Ser 202 phosphorylation blocked the phosphorylation-induced ϳ68-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 ϳ68-kDa band if phosphorylation at Ser 202 is blocked. This in turn indicates that FTDP-17 mutations promote the ϳ68-kDa band mobility shift by enhancing phosphorylation at Ser 202 .
Effect of Ser 202 Phosphorylation 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 Ser 202 to Ala did not affect the microtubule assembly-promoting activity of WT or any of the FTDP-17 mutants.
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 Ser 202 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 Ser 202 phosphorylation increased the microtubule assembly-promoting activity of all phosphorylated FTDP-17 mutants. This indicates that   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 Ser 202 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 Ser 202 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.
Ser 202 phosphorylation inhibits the microtubule assembly-promoting activity of tau(WT) and all its FTDP-17 mutants.

DISCUSSION
The presence of ϳ64and 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).
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 Ser 202 , Thr 231 , and Ser 396 (27). In this study, nonphosphorylated tau migrated as a ϳ60-kDa band on SDS-gel. Upon phosphorylation by Cdk5, tau migrated as ϳ60-, 64-, and 68-kDa bands (Fig. 4). These data indicate that phosphorylation by Cdk5 cause mobility shift of tau to ϳ64and 68-kDa bands. Although phosphorylation at Ser 396/404 also promoted tau mobility shift to a ϳ64-kDa band, only Ser 202 -phosphorylated tau displayed both ϳ64and 68-kDa bands, and phosphorylation at Ser 202 correlated with their formation (Figs. 3 and 4). Furthermore, blocking Thr 231 or Ser 396 did not affect tau mobility shift to ϳ64or 68-kDa bands. Blocking Ser 202 phosphorylation blocked mobility shift to a ϳ68-kDa band (Fig. 7). Our data indicate that tau mobility shift to a ϳ64-kDa band can occur without Ser 202 phosphorylation. However, mobility shift to a ϳ68-kDa band in response to Cdk5 phosphorylation requires Ser 202 phosphorylation.
As shown in Fig. 3, the ϳ68-kDa band formed by Cdk5 phosphorylation is phosphorylated on Ser 202 but not at Ser 396/404 , Thr 231 , Ser 235 , or Thr 212 . This indicates that the ϳ68-kDa band is formed as a result of phosphorylation at Ser 202 but not at Ser 396/404 , Thr 231 , Ser 235 , or Thr 212 . Similarly, the ϳ64-kDa band is phosphorylated at Ser 202 and Ser 396/404 but not at Thr 231 , Ser 235 , and Thr 212 , indicating that the ϳ64-kDa band is formed due to phosphorylation at Ser 202 and Ser 396/404 and not at Thr 231 , Ser 235 , or Thr 212 . Furthermore, the Ser 202 -phosphorylated tau first migrates as a ϳ60-kDa band (Fig. 4A, lane 2). With the increase in phosphorylation time, ϳ64-kDa band followed by ϳ68-kDa band appear. These data suggest that Ser 202phosphorylated tau migrates as a ϳ60-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 Ser 202 may allow Cdk5 to perform second-step phosphorylation on new sites that are not accessible in non-Ser 202 -phosphorylated tau. Firstplus second-step-phosphorylated tau may then migrate as a ϳ64-kDa band. The ϳ64-kDa phosphorylated tau may then undergo third-step phosphorylation on additional sites and become hyperphosphorylated. The hyperphosphorylated tau may then migrate as a ϳ68-kDa band. Note that in this mechanism, the ϳ68-kDa band will be expected to contain phosphate on all the sites, including Ser 202 , that are involved in the mobility shift from ϳ60to 64-kDa and then to 68-kDa bands. As shown in Fig. 3, the ϳ68-kDa band is phosphorylated at Ser 202 but not at Ser 396/404 , Ser 235 , Thr 231 , or Thr 212 . This observation suggests that phosphorylation on the new, additional sites that occurs after phosphorylation at Ser 202 , and results in a ϳ60 -68-kDa band shift, has to occur at sites other than Ser 396/404 , Ser 235 , Thr 231 , and Thr 212 . In fact, in addition to the sites mentioned above, PHF-tau is phosphorylated on a number of proline-directed sites, including Ser 199 , Thr 181 , Thr 217 , and Ser 222 , which are potential targets of Cdk5 (8).
Our data have demonstrated that Thr 231 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, Ser 396 phosphorylation inhibited microtubule polymerization and formation-promoting activities but did not affect microtubule nucleation-promoting activity ( Fig. 8A and supplemental Table S2). Ser 202 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 Ser 396 phosphorylation (Fig. 8B). Our results indicate that Ser 202 phosphorylation significantly inhibits the microtubule assembly-promoting activity of tau in vitro. It should be noted that under our experimental conditions, Cdk5 may have phosphorylated tau at Ser 202 to a higher extent than at Ser 396 and Thr 231 . 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.
The pathological significance of Ser 202 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-ysis 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)(44)(45). Among several tau phosphorylation-sensitive antibodies tested, AD brains stain strongest with AT8 (46). When purified PHF-tau is Western blotted, ϳ60-, 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 Ser 202 and/or Ser 205 (48). However, mass spectrometric studies have determined that PHF-tau is phosphorylated at Ser 202 but not at Ser 205 (8,9). Thus, AT8 immunoreactivity in the brain represents tau phosphorylated at Ser 202 . Moreover, among several sites tested, only phosphorylation at Ser 202 correlates with mobility shift of tau to ϳ64and 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 Ser 202 (Figs. 3 and 4). Taken together, these observations suggest that Ser 202 phosphorylation is the major pathological event in the brain leading to brain degeneration in AD.
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 Thr 231 is not influenced by any mutation, phosphorylation at Ser 396/404 is promoted by G272V and P301L but inhibited is by R406W (Fig. 3). Ser 235 phosphorylation is promoted by V337M, G272V, and P301L but is inhibited by R406W. Phosphorylation at Ser 202 , on the other hand, is promoted by all of the FTDP-17 missense mutations, and by enhancing phosphorylation at Ser 202 each mutation promoted a tau mobility shift to ϳ64and 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 Ser 202 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 Ser 202 (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 Ser 202 . 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 Ser 202 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 Ser 202 , suggesting that FTDP-17 mutations may accelerate NFT pathology by increasing Ser 202 phosphorylation in the brain.
Previous studies have shown that ϳ60-, 64-, and 68-kDa tau bands of AD brain are also immunoreactive to PHF-1 antibody specific for Ser 396/404 -phosphorylated tau (51). This observation suggests that Ser 396/404 phosphorylation may also promote the formation of ϳ60-, 64-, and 68-kDa tau bands in the brain. However, phosphorylation by Cdk5 at Ser 396/404 does not cause tau mobility shift to the ϳ68-kDa band (Fig. 3). Our data indicate that Cdk5 phosphorylation alone is not sufficient for the Ser 396/404 -phosphorylated ϳ68-kDa mobility shift. It is possible that for the formation of the Ser 396/404 -phosphorylated ϳ68-kDa tau band, in addition to Cdk5, phosphorylation by another or other brain kinases may be required. Alternatively, the Ser 396/404 -phosphorylated ϳ68-kDa band in the brain may result from phosphorylation by kinases other than Cdk5.
In this study we used the longest human tau isoform, which migrates as a ϳ60-kDa band on SDS-gels. We showed that upon Cdk5 phosphorylation, this isoform migrates as ϳ60-, 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 ϳ60to 64-kDa and ϳ68-kDa bands upon Cdk5 phosphorylation. Because PHF-tau isolated from AD brain migrates as ϳ60-, 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 ϳ60-, 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.