Limited proteolysis of smooth muscle myosin light chain kinase.
Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D005895:Gizzard, Avian / Q000201:enzymology; D008970:Molecular Weight; D009219:Myosin-Light-Chain Kinase; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002147:Calmodulin; D010446:Peptide Fragments; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin