Ca(2+)-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells.
Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca2+ concentration required for half-maximal phosphorylation was 500 nM for MLCK and 250 nM for myosin light chain. Addition of KN-62 or a synthetic peptide CK II, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase II activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodamine-calmodulin in intact tracheal smooth muscle cells.
Tansey
M G
MG
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
Luby-Phelps
K
K
Kamm
K E
KE
Stull
J T
JT
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D012235:Rhodamines; D014132:Trachea / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism
D002147:Calmodulin; D005456:Fluorescent Dyes; D007546:Isoquinolines; D010446:Peptide Fragments; D010879:Piperazines; D012235:Rhodamines; C009434:tetramethylrhodamine isothiocyanate; D004533:Egtazic Acid; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium