Hierarchical phosphorylation of recombinant tau by the paired-helical filament-associated protein kinase is dependent on cyclic AMP-dependent proteinkinase

Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's di sease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of e xogenous MgCl2 and ATP. PHF kinase activity is shown to be present in immun oaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome , and Pick's disease but not from normal brain homogenates. Although initia l studies failed to show that the kinase was able to induce the phosphoryla tion of tau, additional studies presented in this article show that only cy clic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mappin g, and site-directed mutagenesis have identified the PHF kinase phosphoryla tion sites as amino acids Thr(361) and Ser(412) in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Se-356 and Ser(409) in htau40. A dditional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau in to exogenously added AD-derived PHFs, providing evidence that certain uniqu e phosphorylations of tau may play a role in the pathogenesis of neurofibri llary pathology in AD.