Abstract

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 fas. 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 (PKC?, -?I, -?II, -?, -?, -?, and -?) 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 PKC? levels to only 40%, and did not prevent CREB phosphorylation upon myotube depolarization. Immunocytochemical analysis revealed selective and rapid PKC? 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 PKC?, -?, and -?, CREB phosphorylation also depended on PKC?. These results strongly implicate nuclear PKC? translocation in CREB phosphorylation induced by skeletal muscle membrane depolarization.