The order of exposure of tau to signal transduction kinases alters the generation of "AD-like" phosphoepitopes.

1. The individual and sequential influence of protein kinase C (PKC), protein kinase A (PKA) and mitogen-activated protein kinase (MAP kinase) on human brain tau was examined. 2. A range of PKC concentrations generated certain phosphoepitopes common with paired helical filaments. These epitopes were masked by higher PKC concentrations, suggesting the presence of multiple tau phosphorylation sites for which PKC exhibited differing affinities and/or conformational alterations in tau induced by sequential PKC-mediated phosphorylation. 3. Prior phosphorylation by PKC enhanced the nature and extent of AD-like tau antigenicity generated by subsequent incubation with MAP kinase yet inhibited that generated by subsequent incubation with PKA. 4. Dephosphorylation of tau prior to incubation with kinases significantly altered the influence of individual and multiple kinase incubation on tau antigenicity in a site-specific manner, indicating that prior in situ phosphorylation events markedly influenced subsequent cell-free phosphorylation. 5. In addition to considerations of the potential impact of tau phosphorylation by individual kinases, these findings extend previous studies which indicate that tau antigenicity, and, presumably, its behavior in situ, is influenced by the sequential and convergent influences of multiple kinases.