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.

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

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.

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.

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.

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.

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).

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.

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

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.

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.

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.

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.

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.

Effect of Ser202 Phosphorylation

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.

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 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.

Effect of Ser202 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 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.

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

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.

Effect of Ser202 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 Ser202 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 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.

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.

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).

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

requires Ser202 phosphorylation.

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).

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.

rylated tau at Ser202 to a higher extent than at Ser396 and Thr231.

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 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-

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

intensely with PHF-1 (Ser396/404), AT100 (Thr212/Ser214),

AT180 (Thr231/Ser235), AT270 (Ser181), and 12E8 (Ser262), indicating that V337M in the brain is most strongly phosphorylated

at Ser202 and less strongly at Ser396/404, Thr212/214, Thr231/

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.

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.

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.

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.

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.

REFERENCES

1. Iqbal, K., Alonso Adel, C., Chen, S., Chohan, M. O., El-Akkad, E., Gong, C. X., Khatoon, S., Li, B., Liu, F., Rahman, A., Tanimukai, H., and GrundkeIqbal, I. (2005) Biochim. Biophys. Acta 1739, 198 ­210

2. Lee, V. M., Goedert, M., and Trojanowski, J. Q. (2001) Annu. Rev. Neurosci. 24, 1121­1159

3. Avila, J., Lucas, J. J., Perez, M., and Hernandez, F. (2004) Physiol. Rev. 84, 361­384

4. Lee, V. M., Balin, B. J., Otvos, L., Jr., and Trojanowski, J. Q. (1991) Science 251, 675­ 678

5. Greenberg, S. G., and Davies, P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5827­5831

6. Bramblett, G. T., Goedert, M., Jakes, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M. (1993) Neuron 10, 1089 ­1099

7. Alonso, A. C., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5562­5566

8. Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Yoshida, H., Titani, K., and Ihara, Y. (1995) J. Biol. Chem. 270, 823­ 829

9. Hanger, D. P., Betts, J. C., Loviny, T. L., Blackstock, W. P., and Anderton, B. H. (1998) J. Neurochem. 71, 2465­2476

10. Biernat, J., Gustke, N., Drewes, G., Mandelkow, E. M., and Mandelkow, E. (1993) Neuron 11, 153­163

11. Crowther, R. A., and Goedert, M. (2000) J. Struct. Biol. 130, 271­279

12. Flament, S., Delacourte, A., Delaere, P., Duyckaerts, C., and Hauw, J. J. (1990) Acta Neuropathol. 80, 212­215

13. Flament, S., and Delacourte, A. (1989) FEBS Lett. 247, 213­216

14. Steiner, B., Mandelkow, E. M., Biernat, J., Gustke, N., Meyer, H. E., Schmidt, B., Mieskes, G., Soling, H. D., Drechsel, D., Kirschner, M. W., Goedert, M., and Mandelkow, E. (1990) EMBO J. 9, 3539 ­3544

15. Hutton, M., Lendon, C. L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A., Hackett, J., Adamson, J., Lincoln, S., Dickson, D., Davies, P., Petersen, R. C., Stevens, M., de Graaff, E., Wauters, E., van Baren, J., Hillebrand, M., Joosse, M., Kwon, J. M., Nowotny, P., Che, L. K., Norton, J., Morris, J. C., Reed, L. A., Trojanowski, J., Basun, H., Lannfelt, L., Neystat, M., Fahn, S., Dark, F., Tannenberg, T., Dodd, P. R., Hayward, N., Kwok, J. B., Schofield, P. R., Andreadis, A., Snowden, J., Craufurd, D., Neary, D., Owen, F., Oostra, B. A., Hardy, J., Goate, A., van Swieten, J., Mann, D., Lynch, T., and Heutink, P. (1998) Nature 393, 702­705

16. Arima, K., Kowalska, A., Hasegawa, M., Mukoyama, M., Watanabe, R., Kawai, M., Takahashi, K., Iwatsubo, T., Tabira, T., and Sunohara, N. (2000) Neurology 54, 1787­1795

17. Bronner, I. F., ter Meulen, B. C., Azmani, A., Severijnen, L. A., Willemsen, R., Kamphorst, W., Ravid, R., Heutink, P., and van Swieten, J. C. (2005) Brain 128, 2645­2653

18. Clark, L. N., Poorkaj, P., Wszolek, Z., Geschwind, D. H., Nasreddine, Z. S., Miller, B., Li, D., Payami, H., Awert, F., Markopoulou, K., Andreadis, A., D'Souza, I., Lee, V. M., Reed, L., Trojanowski, J. Q., Zhukareva, V., Bird, T., Schellenberg, G., and Wilhelmsen, K. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13103­13107

19. Matsumura, N., Yamazaki, T., and Ihara, Y. (1999) Am. J. Pathol. 154, 1649 ­1656

20. Vogelsberg-Ragaglia, V., Bruce, J., Richter-Landsberg, C., Zhang, B., Hong, M., Trojanowski, J. Q., and Lee, V. M. (2000) Mol. Biol. Cell 11, 4093­ 4104 21. Sahara, N., Tomiyama, T., and Mori, H. (2000) J. Neurosci. Res. 60, 380 ­387

22. Perez, M., Lim, F., Arrasate, M., and Avila, J. (2000) J. Neurochem. 74, 2583­2589 23. Mack, T. G., Dayanandan, R., Van Slegtenhorst, M., Whone, A., Hutton, M., Lovestone, S., and Anderton, B. H. (2001) Neuroscience 108, 701­712 24. Krishnamurthy, P. K., and Johnson, G. V. (2004) J. Biol. Chem. 279, 7893­7900

25. Connell, J. W., Gibb, G. M., Betts, J. C., Blackstock, W. P., Gallo, J., Lovestone, S., Hutton, M., and Anderton, B. H. (2001) FEBS Lett. 493, 40­44 26. Alonso Adel, C., Mederlyova, A., Novak, M., Grundke-Iqbal, I., and Iqbal, K. (2004) J. Biol. Chem. 279, 34873­34881

27. Paudel, H. K., Lew, J., Ali, Z., and Wang, J. H. (1993) J. Biol. Chem. 268, 23512­23518

28. Sobue, K., Agarwal-Mawal, A., Li, W., Sun, W., Miura, Y., and Paudel, H. K. (2000) J. Biol. Chem. 275, 16673­16680

29. Li, T., and Paudel, H. K. (2006) Biochemistry 45, 3125­3133

30. Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933­11940 31. Li, T., Hawkes, C., Qureshi, H. Y., Kar, S., and Paudel, H. K. (2006) Biochemistry 45, 3134 ­3145

32. Agarwal-Mawal, A., and Paudel, H. K. (2001) J. Biol. Chem. 276, 23712­23718

33. Li, T., Chalifour, L. E., and Paudel, H. K. (2007) J. Biol. Chem. 282, 6619 ­ 6628

34. Hashiguchi, M., Sobue, K., and Paudel, H. K. (2000) J. Biol. Chem. 275, 25247­25254

35. Gustke, N., Trinczek, B., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (1994) Biochemistry 33, 9511­9522

36. Gelfand, V. I., and Bershadsky, A. D. (1991) Annu. Rev. Cell Biol. 7, 93­116 37. Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., Morris, J. C., Wilhelmsen, K. C., Schellenberg, G. D., Trojanowski, J. Q., and Lee, V. M. (1998) Science 282, 1914 ­1917

38. Hasegawa, M., Smith, M. J., and Goedert, M. (1998) FEBS Lett. 437, 207­210

39. Paudel, H. K. (1997) J. Biol. Chem. 272, 28328 ­28334

40. Lu, P. J., Wulf, G., Zhou, X. Z., Davies, P., and Lu, K. P. (1999) Nature 399, 784 ­788

41. Scott, C. W., Spreen, R. C., Herman, J. L., Chow, F. P., Davison, M. D., Young, J., and Caputo, C. B. (1993) J. Biol. Chem. 268, 1166 ­1173

42. Jicha, G. A., Weaver, C., Lane, E., Vianna, C., Kress, Y., Rockwood, J., and Davies, P. (1999) J. Neurosci. 19, 7486 ­7494

43. Braak, E., Braak, H., and Mandelkow, E. M. (1994) Acta Neuropathol. 87, 554 ­567

44. Braak, H., and Braak, E. (1995) Neurobiol. Aging 16, 271­278; discussion 278 ­284

45. Mesulam, M., Shaw, P., Mash, D., and Weintraub, S. (2004) Ann. Neurol. 55, 815­ 828

46. Spillantini, M. G., Crowther, R. A., and Goedert, M. (1996) Acta Neuropathol. 92, 42­ 48

47. Goedert, M., Jakes, R., Crowther, R. A., Six, J., Lubke, U., Vandermeeren, M., Cras, P., Trojanowski, J. Q., and Lee, V. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5066 ­5070

48. Goedert, M., Jakes, R., and Vanmechelen, E. (1995) Neurosci. Lett. 189, 167­169

49. Spillantini, M. G., Crowther, R. A., Kamphorst, W., Heutink, P., and van Swieten, J. C. (1998) Am. J. Pathol. 153, 1359 ­1363

50. van Swieten, J. C., Stevens, M., Rosso, S. M., Rizzu, P., Joosse, M., de Koning, I., Kamphorst, W., Ravid, R., Spillantini, M. G., Niermeijer, and Heutink, P. (1999) Ann. Neurol. 46, 617­ 626

51. Yang, L. S., Gordon-Krajcer, W., and Ksiezak-Reding, H. (1997) J. Neurochem. 69, 1548 ­1558

52. Goedert, M., Spillantini, M. G., Cairns, N. J., and Crowther, R. A. (1992) Neuron 8, 159 ­168

53. Yuan, Z., Agarwal-Mawal, A., and Paudel, H. K. (2004) J. Biol. Chem. 279, 26105­26114