Phosphorylation of P400 protein by cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II.
Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 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 P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Yamamoto
H
H
Department of Pharmacology, Kumamoto University Medical School, Japan.
Maeda
N
N
Niinobe
M
M
Miyamoto
E
E
Mikoshiba
K
K
D000818:Animals; D001921:Brain / Q000201:enzymology*; D015220:Calcium Channels; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002531:Cerebellum / Q000201:enzymology; D000242:Cyclic AMP / Q000494:pharmacology*; D004027:Diencephalon / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D013687:Telencephalon / Q000201:enzymology; D013049:Spectrin
D015220:Calcium Channels; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002352:Carrier Proteins; D000242:Cyclic AMP; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D011494:Protein Kinases; D018160:Receptors, Cytoplasmic and Nuclear; D013049:Spectrin; C025389:Itpr1 protein, mouse; C488910:Itpr1 protein, rat