Glycogen synthase kinase 3β phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism

Phosphorylation of tau on S-396 was suggested to be a key step in the development of neurofibrillary pathology in Alzheimer's disease brain [Bramblett, G. T., Goedert, M., Jacks, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Neuron 10, 1089-1099]. GSK3 beta phosphorylates Ser(396) of tau in the brain by a mechanism which is not clear. In this study, when HEK-293 cells were cotransfected with tau and GSK3 beta, GSK3 beta co-immunoprecipitated with tau and phosphorylated tau on S-202. T-231, S-396, and S-400 but not on S-262, S-235, and S-404. Blocking phosphorylation on T-231, S-235, S-396, S-400, or S-404 did not prevent the subsequent phosphorylation on S-202 by GSK3 beta. These data suggest that GSK3 beta directly phosphorylates tau on S202 (without requiring prephosphorylation). However, preventing phosphorylation on S-235, S-400, and S-404 prevented GSK3 beta-dependent phosphorylation of T-231, S-396, and S-400, respectively. This indicates that phosphorylation of T-231, S-396, and S-400 by GSK3 beta depends on a previous phosphorylation of S-235, S-400, and S-404, respectively. To examine S-396 phosphorylation, we analyzed phosphorylation of S-396, S-400, and S-404. Blocking phosphorylation of S-404 prevented the subsequent GSK3 beta-dependent phosphorylation of both S-400 and S-396. When phosphorylation of S-404 was allowed but S-400 blocked, GSK3 beta failed to phosphorylate S-396. Thus, GSK3 beta phosphorylates S-396 by a two-step mechanism. In the first step, GSK3 beta phosphorylates S-400 of previously S-404-phosphorylated tau. This event primes tau for second-step phosphorylation of S-396 by GSK3 beta. We conclude that GSK3 beta phosphorylates tau directly at S-202 but requires the previous phosphorylation on S-235 to phosphorylate T-231. Phosphorylation of S-396, on the other hand, occurs sequentially. Once a priming kinase phosphorylates S-404, GSK3 beta sequentially phosphorylates S-400 and then S-396.