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), prote in kinase A (PKA) and mitogen-activated protein kinase (MAP kinase) on huma n brain tau was examined. 2. A range of PKC concentrations generated certain phosphoepitopes common w ith paired helical filaments. These epitopes were masked by higher PKC conc entrations, suggesting the presence of multiple tau phosphorylation sites f or which PKC exhibited differing affinities and/or conformational alteratio ns in tau induced by sequential PKC-mediated phosphorylation. 3. Prior phosphorylation by PKC enhanced the nature and extent of AD-like t au antigenicity generated by subsequent incubation with MAP kinase yet inhi bited 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 a ntigenicity in a site-specific manner, indicating that prior in situ phosph orylation events markedly influenced subsequent cell-free phosphorylation. 5. In addition to considerations of the potential impact of tau phosphoryla tion by individual kinases, these findings extend previous studies which in dicate that tau antigenicity, and, presumably, its behavior in situ is infl uenced by the sequential and convergent influences of multiple kinases.