PROTEIN-KINASE-C ISOFORMS IN MUSCLE-CELLS AND THEIR REGULATION BY PHORBOL ESTER AND CALPAIN

Objectives were to identify the PKC isoforms in cultured muscle cells, to examine roles of Ca2+-dependent proteinases (calpains) in processi ng of various muscle PKC isozymes and to obtain a mechanistic descript ion of the processing of PKCs by examining the temporal relationships between phorbol ester-dependent translocation of muscle PKCs and calpa ins between cytosolic and membrane compartments. Using six isoform (al pha, beta, gamma, delta, epsilon, zeta)-specific polyclonal antibodies , PKC alpha, delta and zeta were detected in rat skeletal muscle and i n L8 myoblasts and myotubes. PKC alpha and zeta were primarily localiz ed in the cytosolic fraction of L8 myotubes whereas PKC delta was more abundant in the membrane fraction, Phorbol ester (TPA) caused rapid d epletion of myotube PKC alpha and PKC delta isoforms from the cytosoli c compartment and rapid appearance of these isoforms in the membrane f raction. However, long-term exposure of myotubes to TPA eventually cau sed down-regulation of PKCs in the membrane compartment. Down-regulati on of PKCs in the membrane fraction was partially blocked by calpain i nhibitor II. However, the rapid TPA-dependent cytosolic depletion of P KCs was unaffected by calpain inhibitor. This suggests that calpains m ay be responsible for membrane-associated down-regulation of PKCs but not for cytosolic depletion. In the final study we assessed the effect s of phorbol ester on compartmentation of m-calpain with PKCs in muscl e cells. Like the PKCs, TPA caused rapid association of m-calpain with the membrane fraction followed by down-regulation. This demonstrates that phorbol esters cause translocation of both PKCs and calpains to m embranes where processing of PKCs may occur via the limited proteolysi s exerted by calpains.