Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites.
Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.
Hashimoto
Y
Y
Howard Hughes Medical Institute, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615.
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D002147:Calmodulin; D010748:Phosphopeptides; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase