Phosphorylation of microtubule-associated protein-2 in GH3 cells. Regulation by cAMP and by calcium.
The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.
Jefferson
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AB
Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
Schulman
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H
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006706:Homeostasis; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D010911:Pituitary Neoplasms; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*
D008869:Microtubule-Associated Proteins; D010710:Phosphates; D010748:Phosphopeptides; D005576:Colforsin; D014660:Vasoactive Intestinal Peptide; D003994:Bucladesine; D002772:Cholera Toxin; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine