Myosin Phosphorylation in Smooth Muscle

It has been established for several years that the major mechanism for regulation of contraction in smooth muscle is myosin phosphorylation (Hart-shorne, 1987). Phosphorylation of the two 20,000-dalton light chains of myosin (LC20) activates the actin-dependent ATPase activity of myosin and this initiates the contractile response. Dephosphorylated myosin is associated with relaxed muscle. In this scheme there are two key enzymes the myosin light chain kinase (MLCK) and the myosin light chain phosphatase (MLCP). Obviously a balance of these two activities determines the level of myosin phosphorylation. 

Much more information is available for MLCK. Several cDNAs have been isolated and cloned and some of the functional regions of the molecule have been identified. This is not the case for MLCP. Until recently there was no consensus about the identity of the phosphatase involved in myosin dephosphorylation and since there are no strict sequence requirements (in terms of primary structure) for most phosphatases, there was the possibility that several phosphatases could be involved. 

An impetus to smooth muscle phosphatase research was provided in the last few years. With the increased application of fluorescent dyes to measure internal Ca2+ concentration it was realized that the force/Ca2+ ratio could vary for different methods of 

Protein phosphatases (PP) are divided into phos-phoserine/threonine (SerZThr)-specific and phospho-tyrosine (Tyr)-specific enzymes based on the distinct specificity toward the phosphorylated residues. Smooth muscle myosin is phosphorylated at Ser 19 

Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 

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Pfitzer G (2001) Invited review regulation of myosin phosphorylation in smooth muscle. J Appl Physiol... 

The identification and development of specific inhibitors of MLCK could provide valuable experimental tools for exploring physiological functions of MLCK and myosin phosphorylation in smooth muscle and nonmuscle cells. Although inhibitors that act by binding calmodulin have been used experimentally, their usefulness is limited because they inhibit other calmodulin-dependent enzymes in tissues and cells (Asano and Stull, 1985 Nakanishi et al., 1992). Therefore, attempts have been made to develop novel reagents that inhibit MLCK activity directly. ML-9 [l-(5-chloronaphthalenesulfonyl)-lH-hexahydro-l, 4-diazepine] inhibits purified smooth muscle MLCK competitively with respect to ATP (Saitoh et al., 1987). Predictably, it inhibits RLC phosphorylation in ac-tomyosin, skinned fibers, and smooth muscle strips (Ishikawa et al., 1988). However, ML-9 also inhibits other protein kinases, so its specificity under different experimental conditions needs to be established. 

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