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