Dual Effects of PKNα and Protein Kinase C on Phosphorylation of Tau Protein by Glycogen Synthase Kinase-3β

Abstract

We analyzed the effects of PKNα and protein kinase C (PKC) on phosphorylation of tau protein by glycogen synthase kinase (GSK)-3β using monoclonal antibodies (AT8, AT180, and AT270). These antibodies are highly specific for phosphorylated tau in Alzheimer paired helical filaments, and recognize phosphorylated Ser202/Thr205, Thr231, and Thr181 of tau protein, respectively. Immunoblot analysis demonstrated that PKNα and PKC did not directly phosphorylate their sites, whereas GSK-3β efficiently did so. Incubating GSK-3β with PKNα or PKC subtypes inhibited subsequent GSK-3β-induced AT8 and AT270 immunoreactivity. However, the constitutive active form of the GSK-3β(S9A) mutant was almost totally inert to each enzyme. Incubating tau with PKNα increased the GSK-3β-induced AT180 immunoreactivity, which was further enhanced when the S9A mutant was used instead of the wild type GSK-3β. These results suggest that PKNα and PKC directly inhibit GSK-3β activity at least in part by phosphorylating Ser9 of GSK-3β, and that they indirectly suppress GSK-3β-stimulated phosphorylation of tau at amino acids Ser202/Thr205 and Thr181, but enhanced phosphorylation at Thr231 through phosphorylation at other sites of tau.