Although there is general agreement that myosin light chain phosphorylation by the Ca2+/calmodulin-dependent myosin light chain kinase is a primary event in the initiation of smooth muscle contraction, there are aspects of this process that are not well understood at the cellular and molecular levels. The research projects described in this application address a number of important issues dealing with the regulation of smooth muscle contractile elements in vascular and airway smooth muscles. Intramolecular (calmodulin binding domain-pseudosubstrate inhibitory region-catalytic domain) and intermolecular (catalytic domain-protein substrate binding and catalysis) events associated with activation and catalysis will be examined and related to the binding of MLCK to actin and/or myosin filaments. A combination of experimental approaches involving biochemistry and molecular biology will be used to analyze these properties of smooth muscle myosin light chain kinase. New information indicates that myosin light chain kinase and phosphatase activities may be regulated so that the ratio of these activities is not simply determined by [Ca2+]i under all conditions for tonic and phasic smooth muscles. Regulation of both kinase and phosphatase activities would contribute to reported changes in the Ca2+ sensitivity of the contractile elements. Myosin light chain kinase is phosphorylated in tracheal smooth muscle during contraction. The functional consequences of this phosphorylation and the identification of the kinase that catalyze this reaction will be determined. The protein phosphatase that dephosphorylate myosin light chain and myosin light chain kinase physiologically will also be identified, and potential mechanisms of regulation elucidated. The research proposed in this application uses integrated experimental approaches from molecular biology, biochemistry, cell biology, physiology, and pharmacology to unravel this complex system to provide an understanding of regulatory mechanisms directly involving the contractile elements. This information will be crucial in understanding derangements of smooth muscle functions caused by disease processes such as hypertension and asthma.
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