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 light chain 2 (MLC2) in adult rat heart cells has been investigated. PKC-mediated phosphorylation of MLC2 in adult rat cardiac myofibrils in vitro occurs with a stoichiometry (0.7 mol of phosphate/mol of protein) similar to that mediated by myosin light chain kinase (MLCK). Two-dimensional tryptic phosphopeptide mapping of MLC2 following phosphorylation by PKC or MLCK in vitro yields the same major phosphopeptides for each protein kinase. These sites are also 32P-labelled in situ when isolated cardiomyocytes are incubated with [32P]P(i). 32P labelling of MLC2 in cardiomyocytes is increased by 5-fold in 10 min upon incubation with the phosphatase inhibitor calyculin A, demonstrating the existence of a rapidly turning over component of MLC2 phosphorylation in these cells. 32P label is completely removed from MLC2 when myocytes are exposed to 2,3-butanedione monoxime, an inhibitor of cardiac contraction known to desensitize the myofilaments to activation by Ca2+. 32P labelling of MLC2 is also decreased by 50-100% following exposure to the PKC-selective inhibitors calphostin C and chelerythrine, suggesting that PKC, and not MLCK, is primarily responsible for incorporation of rapidly turning over phosphate into MLC2 in situ. Taken together, these 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.
Venema
R C
RC
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322.
Raynor
R L
RL
Noland
T A
TA
Kuo
J F
JF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008387:Marine Toxins; D009206:Myocardium / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; C004717:diacetylmonoxime; C059041:calyculin A; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D014357:Trypsin; D009218:Myosins; D003931:Diacetyl; D002118:Calcium