Depolarization of skeletal muscle cells induces phosphorylation of cAMP response element binding protein via calcium and protein kinase Calpha.
Membrane depolarization of skeletal muscle cells induces slow inositol trisphosphate-mediated calcium signals that regulate the activity of transcription factors such as the cAMP-response element-binding protein (CREB), jun, and fos. Here we investigated whether such signals regulate CREB phosphorylation via protein kinase C (PKC)-dependent pathways. Western blot analysis revealed the presence of seven isoforms (PKCalpha, -betaI, -betaII, -delta, -epsilon, -, and -zeta) in rat primary myotubes. The PKC inhibitors bisindolymaleimide I and Gö6976, blocked CREB phosphorylation. Chronic exposure to phorbol ester triggered complete down-regulation of several isoforms, but reduced PKCalpha levels to only 40%, and did not prevent CREB phosphorylation upon myotube depolarization. Immunocytochemical analysis revealed selective and rapid PKCalpha translocation to the nucleus following depolarization, which was blocked by 2-amino-ethoxydiphenyl borate, an inositol trisphosphate receptor inhibitor, and by the phospholipase C inhibitor U73122. In C2C12 cells, which expressed PKCalpha,-epsilon, and -zeta, CREB phosphorylation also depended on PKCalpha. These results strongly implicate nuclear PKCalpha translocation in CREB phosphorylation induced by skeletal muscle membrane depolarization.
Cárdenas
César
C
Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile.
Müller
Marioly
M
Jaimovich
Enrique
E
Pérez
Francisco
F
Buchuk
Diego
D
Quest
Andrew F G
AF
Carrasco
Maria Angélica
MA
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D015536:Down-Regulation; D004963:Estrenes / Q000494:pharmacology; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D017362:Cyclic AMP Response Element-Binding Protein; D004963:Estrenes; D010703:Phorbol Esters; D020033:Protein Isoforms; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D011188:Potassium