Depolarization of skeletal muscle cells induces phosphorylation of cAMP response element binding protein via calcium and protein kinase Cα

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, -theta, and -zeta) in rat primary myotubes. The PKC inhibitors bisindolymaleimide I and Go6976, 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.