The binding of smooth muscle myosin light chain kinase and phosphatases to actin and myosin.
Contractile activity in smooth muscle cells is regulated by phosphorylation-dephosphorylation of the 20,000-Da light chain of myosin. In an attempt to better understand the localization in muscle of the enzymes which catalyze the phosphorylation-dephosphorylation process, we measured the binding constants of turkey gizzard smooth muscle myosin light chain (MLC) kinase and smooth muscle phosphatases (SMP) to myosin and actin under identical conditions by a sedimentation method. We have observed that MLC kinase binds strongly to both actin and myosin. When tropomyosin is complexed to actin, the affinity of MLC kinase to actin increases 2-3-fold. The presence of calcium-calmodulin weakens the binding of MLC kinase to actin, actin-tropomyosin, and myosin by about 3-fold. Increasing the ionic strength of the binding assay also decreases the binding of MLC kinase to myosin and actin-tropomyosin. MLC kinase is observed to bind to rod subfragment, a fragment of myosin which does not contain the phosphorylatable light chain suggesting that the kinase also binds to domains of the myosin other than the 20,000-Da light chain. Of the phosphatases tested, only SMP-III and -IV bind strongly to unphosphorylated myosin. When the myosin is thiophosphorylated , the binding constants of SMP-III and -IV increase dramatically. SMP-I and -II do not bind to unphosphorylated and thiophosphorylated myosin. However, the free catalytic subunit of SMP-I binds weakly to thiophosphorylated myosin. None of the phosphatases binds to actin. Our study suggests that in muscle, the myosin phosphatase is localized in the thick filament while the MLC kinase may be associated with the thick filaments, thin filaments, or even both.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D015879:Myosin Subfragments / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D014335:Tropomyosin; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins