Cyclin-dependent protein kinase 5 primes microtubule-associated protein tau site-specifically for glycogen synthase kinase 3β

In the preceding paper, we showed that GSK3 beta phosphorylates tau at S-202, T-321, S-396, and S-400 in vivo. Phosphorylation of S-202 occurs without priming. Phosphorylation of T-231, on the other hand, requires priming phosphorylation of S-235. Similarly, priming phosphorylation of S-404 is essential for the sequential phosphorylation of S-400 and S-396 by GSK3 beta. The priming kinase that phosphorylates tau at S-235 and S-404 in the brain is not known. In this study, we find that in HEK-293 cells cotransfected with tau, GSK3 beta, and Cdk5, Cdk5 phosphorylates tau at S-202, S-235, and S-404. S-235 phosphorylation enhances GSK3 beta-catalyzed T-231 phosphorylation. Similarly, Cdk5 by phosphorylating S-404 stimulates phosphorylation of S-400 and S-396 by GSK3 beta. These data indicate that Cdk5 primes tau for GSK3 beta in intact cells. To evaluate if Cdk5 primes tau for GSK3 beta in mammalian brain, we examined localizations of Cdk5, tau, and GSK3 beta in rat brain. We also analyzed the interaction of Cdk5 with tau and GSK3 beta in brain microtubules. We found that Cdk5, GSK3 beta, and tau are virtually colocalized in rat brain cortex. When bovine brain microtubules are analyzed by FPLC gel filtration, Cdk5, GSK3 beta, and tau coelute within an similar to 450 kDa complex. From the fractions containing the similar to 450 kDa complex, tau, Cdk5, and GSK3 beta co-immunoprecipitate with each other. In HEK-293 cells transfected with tau, Cdk5, and GSK3 beta in different combinations, tau binds to Cdk5 in a manner independent of GSK3 beta and to GSK3 beta in a manner independent of Cdk5. However, Cdk5 and GSK3 beta bind to each other only in the presence of tau, suggesting that tau connects Cdk5 and GSK3 beta. Our results suggest that in the brain, tau, Cdk5, and GSK3 beta are components of an similar to 450 kDa complex. Within the complex, Cdk5 phosphorylates tau at S-235 and primes it for phosphorylation of T-231 by GSK3 beta. Similarly, Cdk5 by phosphorylating tau at S-404 primes tau for a sequential phosphorylation of S-400 and S-396 by GSK3 beta.