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 Ca(2+)-dependent proteinases (calpains) in processing of various muscle PKC isozymes and to obtain a mechanistic description of the processing of PKCs by examining the temporal relationships between phorbol ester-dependent translocation of muscle PKCs and calpains between cytosolic and membrane compartments. Using six isoform (alpha, beta, gamma, delta, epsilon, zeta)-specific polyclonal antibodies, PKC alpha, delta and zeta were detected in rat skeletal muscle and in L8 myoblasts and myotubes. PKC alpha and zeta were primarily localized in the cytosolic fraction of L8 myotubes whereas PKC delta was more abundant in the membrane fraction. Phorbol ester (TPA) caused rapid depletion of myotube PKC alpha and PKC alpha and PKC delta isoforms from the cytosolic compartment and rapid appearance of these isoforms in the membrane fraction. However, long-term exposure of myotubes to TPA eventually caused down-regulation of PKCs in the membrane compartment. Down-regulation of PKCs in the membrane fraction was partially blocked by calpain inhibitor II. However, the rapid TPA-dependent cytosolic depletion of PKCs was unaffected by calpain inhibitor. This suggests that calpains may be responsible for membrane-associated down-regulation of PKCs but not for cytosolic depletion. In the final study we assessed the effects of phorbol ester on compartmentation of m-calpain with PKCs in muscle 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 membranes where processing of PKCs may occur via the limited proteolysis exerted by calpains.
Hong
D H
DH
Department of Animal Sciences, Oregon State University, Corvallis 97331-6702, USA.
Huan
J
J
Ou
B R
BR
Yeh
J Y
JY
Saido
T C
TC
Cheeke
P R
PR
Forsberg
N E
NE
D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000276:immunology / Q000494:pharmacology*; D002478:Cells, Cultured; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D010703:Phorbol Esters; D011493:Protein Kinase C; D010450:Endopeptidases; D002154:Calpain