ROLE OF PROTEIN-KINASE-C IN THE PHOSPHORYLATION OF CARDIAC MYOSIN LIGHT CHAIN-2

The role of protein kinase C (PKC) in the phosphorylation of myosin li ght chain 2 (MLC2) in adult rat heart cells has been investigated. PKC -mediated phosphorylation of MLC2 in adult rat cardiac myofibrils in v itro occurs with a stoichiometry (0.7 mol of phosphate/mol of protein) similar to that mediated by myosin light chain kinase (MLCK). Two-dim ensional tryptic phosphopeptide mapping of MLC2 following phosphorylat ion by PKC or MLCK in vitro yields the same major phosphopeptides for each protein kinase. These sites are also P-32-labelled in situ when i solated cardiomyocytes are incubated with [P-32]P(i). P-32 labelling o f MLC2 in cardiomyocytes is increased by 5-fold in 10 min upon incubat ion with the phosphatase inhibitor calyculin A, demonstrating the exis tence of a rapidly turning over component of MLC2 phosphorylation in t hese cells. P-32 label is completely removed from MLC2 when myocytes a re exposed to 2,3-butanedione monoxime, an inhibitor of cardiac contra ction known to desensitize the myofilaments to activation by Ca2+. P-3 2 labelling of MLC2 is also decreased by 50-100% following exposure to the PKC-selective inhibitors calphostin C and chelerythrine, suggesti ng that PKC, and not MLCK, is primarily responsible for incorporation of rapidly turning over phosphate into MLC2 in situ. Taken together, t hese data implicate PKC in the phosphorylation of MLC2 in heart cells and support the hypothesis that phosphorylation of cardiac MLC2 has a role in determining myofibrillar Ca2+ sensitivity.