SEQUENTIAL PHOSPHORYLATION OF TAU BY GLYCOGEN-SYNTHASE KINASE-3-BETA AND PROTEIN-KINASE-A AT THR212 AND SER214 GENERATES THE ALZHEIMER-SPECIFIC EPITOPE OF ANTIBODY AT100 AND REQUIRES A PAIRED-HELICAL-FILAMENT-LIKE CONFORMATION

AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Hen we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), nex t of Thr212 by glycogen synthase kinase (GSK)-3 beta (a proline-direct ed kinase), then of Ser14 by protein kinase A (PKA). Conversely, if Se r214 is phosphorylated first it protects Thr212 and the Ser-Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformati on of tau induced by polyanions such as heparin, RNA or poly(Glu), con ditions which also favor the formation of paired helical filaments. Th e Alzheimer-like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK-3 beta and PKA, which can be inhibited by their specific inhib itors LiCl and R-II, respectively. A cellular model displaying the rea ction with AT100 is presented by Sf9 insect cells transfected with Tau . Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.