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Phosphorylation of P 400 Protein by Cyclic AMP‐Dependent Protein Kinase and Ca 2+ /Calmodulin‐Dependent Protein Kinase II
Author(s) -
Yamamoto Hideyuki,
Maeda Nobuaki,
Niinobe Michio,
Miyamoto Eishichi,
Mikoshiba Katsuhiko
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb11792.x
Subject(s) - protein kinase a , map2k7 , mitogen activated protein kinase kinase , cyclin dependent kinase 2 , protein phosphorylation , cgmp dependent protein kinase , phosphorylation , microbiology and biotechnology , biochemistry , biology , autophagy related protein 13 , casein kinase 2 , protein kinase c , map kinase kinase kinase
Purified P 400 protein was phosphorylated by both purified Ca 2+ /calmodulin‐dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP‐dependent protein kinase (A‐kinase). Because P 400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P 400 protein using crude mitochondrial and microsomal fractions (P 2 /P 3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A‐kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P 400 protein was not observed in the P 2 /P 3 fraction from mouse forebrain. Cyclic AMP and A‐kinase enhanced the phosphorylation of several proteins, including P 400 protein, suggesting that P 400 protein is one of the best substrates for A‐kinase in the P 2 / P 3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P 2 /P 3 fraction, the phosphorylation of P 400 protein was weak. Immunoprecipitation with the monoclonal antibody to P 400 protein confirmed that the P 400 protein itself was definitely phosphorylated by the catalytic subunit of A‐kinase and CaM kinase II. A‐kinase phosphorylated only the seryl residue in P 400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P 400 protein, which migrated at the same position on sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis as that in the P 2 /P 3 fraction. Incubation of the cultured cerebellar cells with [ 32 P]orthophosphate resulted in the labeling of P 400 protein. These results suggest that P 400 protein is one of the best substrates for A‐kinase and CaM kinase II in cerebellum and that the function of P 400 protein is regulated by cyclic AMP‐and Ca 2+ /calmodulin‐dependent phosphorylation.