Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C.
The phosphorylation of synthetic peptides derived from the NH2-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1-13) (MLC(1-13)). Kinetic analysis of MLC(1-13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1-13) had a V/K of 2.4 min-1.mg-1, whereas the V/K of MLC(3-13) was 3.0 min-1.mg-1. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent Vmax.Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent Km increased 7-fold and the Vmax decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain peptides can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.
Hassell
T C
TC
Department of Biochemistry, University of North Texas, Denton.
Magnino
P E
PE
Masaracchia
R A
RA
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism
D004075:Diglycerides; D009842:Oligopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium