Familial FTDP-17 missense mutations inhibit microtubule assembly-promoting activity of tau by increasing phosphorylation at Ser202 in vitro

Abstract

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 approximately 60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as approximately 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 approximately 60-kDa bands to approximately 64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser(202), and Ser(202) 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 Ser(202) phosphorylation and mobility shift to a approximately 68-kDa band. Furthermore, Ser(202) 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 Ser(202), inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser(202) phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.

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.

FIGURE 2.

SDS-PAGE of phosphorylated tau(WT) and FTDP-17 tau mutants. Indicated tau species (5 μg each) phosphorylated by Cdk5 under identical conditions for 60 min (lanes 2–5) 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.

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 μg 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-GSK3β was used as a positive control on lane 1 of pT212 blot. Recombinant GST-GSK3β was purified as described (30, 53).

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 μg 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.

FIGURE 5.

Effect of Ser²⁰² phosphorylation on SDS-gel mobility of tau. Tau(WT) and tau(S202A) phosphorylated by Cdk5 using [γ³²P]ATP for the indicated time points were analyzed by Western blot (IB). The blot was subsequently autoradiographed to monitor radioactivity in each band.

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.

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.

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.

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.

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.