Abstract
We analyzed the effects of PKNalpha and protein kinase C (PKC) on phosphorylation of tau protein by glycogen synthase kinase (GSK)-3beta 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 PKNalpha and PKC did not directly phosphorylate their sites, whereas GSK-3beta efficiently did so. Incubating GSK-3beta with PKNalpha or PKC subtypes inhibited subsequent GSK-3beta-induced AT8 and AT270 immunoreactivity. However, the constitutive active form of the GSK-3beta(S9A) mutant was almost totally inert to each enzyme. Incubating tau with PKNalpha increased the GSK-3beta-induced AT180 immunoreactivity, which was further enhanced when the S9A mutant was used instead of the wild type GSK-3beta. These results suggest that PKNalpha and PKC directly inhibit GSK-3beta activity at least in part by phosphorylating Ser9 of GSK-3beta, and that they indirectly suppress GSK-3beta-stimulated phosphorylation of tau at amino acids Ser202/Thr205 and Thr181, but enhanced phosphorylation at Thr231 through phosphorylation at other sites of tau.
Copyright 2000 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Antibodies, Monoclonal / immunology
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Bacterial Proteins*
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Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
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Calcium-Calmodulin-Dependent Protein Kinases / genetics
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
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Epitopes / immunology
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Glycogen Synthase Kinase 3
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Glycogen Synthase Kinases
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Humans
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Molecular Sequence Data
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Mutation / genetics
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Phosphoproteins / chemistry
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Phosphoproteins / immunology
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Phosphoproteins / metabolism
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Phosphorylation
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Phosphoserine / immunology
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Phosphoserine / metabolism
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Phosphotyrosine / immunology
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Phosphotyrosine / metabolism
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Protein Kinase C / chemistry
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Protein Kinase C / metabolism*
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Protein Kinases / chemistry
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Protein Kinases / genetics
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Protein Kinases / metabolism*
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Rats
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / immunology
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Recombinant Fusion Proteins / metabolism
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tau Proteins / chemistry
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tau Proteins / immunology
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tau Proteins / metabolism*
Substances
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Antibodies, Monoclonal
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Bacterial Proteins
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Epitopes
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Phosphoproteins
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Recombinant Fusion Proteins
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tau Proteins
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Phosphoserine
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Phosphotyrosine
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Protein Kinases
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PknA protein, Nostoc sp. PCC 7120
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Glycogen Synthase Kinases
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Protein Kinase C
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Calcium-Calmodulin-Dependent Protein Kinases
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Glycogen Synthase Kinase 3