Phosphatase myosin

Smooth muscle myosin phosphatase contains tree subunits, a 110-130 kDa myosin phosphatase targeting and regulatory subunit (MYPT1), a 37 kDa catalytic subunit (PP-1C) and a 20 kDa subunit of unknown function. 

A subfamily of Rho proteins, the Rnd family of small GTPases, are always GTP-bound and seem to be regulated by expression and localization rather than by nucleotide exchange and hydrolysis. Many Rho GTPase effectors have been identified, including protein and lipid kinases, phospholipase D and numerous adaptor proteins. One of the best characterized effector of RhoA is Rho kinase, which phosphorylates and inactivates myosin phosphatase thereby RhoA causes activation of actomyosin complexes. Rho proteins are preferred targets of bacterial protein toxins ( bacterial toxins). 

Calcium-dependent regulation involves the calcium-calmodulin complex that activates smooth muscle MLCK, a monomer of approximately 135 kDa. Dephosphorylation is initiated by MLCP. MLCP is a complex of three proteins a 110-130 kDa myosin phosphatase targeting and regulatory subunit (MYPT1), a 37 kDa catalytic subunit (PP-1C) and a 20 kDa subunit of unknown function. In most cases, calcium-independent regulation of smooth muscle tone is achieved by inhibition of MLCP activity at constant calcium level inducing an increase in phospho-rMLC and contraction (Fig. 1). 

Finally, if the phosphorylation of myosin is the activation mechanism, then dephosphorylation is likely to be the deactivation mechanism, and so it seems. However, there are several myosin phosphatases in smooth muscle cells and they have some range of substrate specificities. Thus, there are several possible candidates for a regulatory role. 

The dephosphorylation of myosin requires the activity of myosin phosphatase. Located in cytoplasm of the smooth muscle cell, this enzyme splits the phosphate group from the myosin. Dephosphorylated myosin is inactive crossbridge cycling no longer takes place and the muscle relaxes. 

Walsh With regards to the issue of Ca2+ sensitization, could you share your thoughts on events downstream of Rho Let s say Rho kinase is activated. What is the relative importance of myosin phosphatase versus CPU 7 as a downstream target of this pathway ... 

Somlyo AR Somlyo AV, Ca sensitivity of smooth muscle and nonmuscle myosin II Modulated by G proteins, kinases, and myosin phosphatase. Physiol... 

Surks HK, Mendelsohn ME. 2003. Dimerization of cgmp-dependent protein kinase la and the myosin-binding subunit of myosin phosphatase Role of leucine zipper domains. Cell Signal 15 937-944. 

Takai, A., Bialojan, C., Troschka, M., and Ruegg, J.C. 1987. Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin. FEES Letts 217, 81-84. 

Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and 165. Rho-associated kinase (Rho-kinase). Science 1996 273 245-248. 

Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor. Structure... 

The myosin phosphatase targeting protein (MYPT) family a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type Idelta. Arch Biochem Biophys 510 147-159... 

Thus there is ample precedence for regulation of phosphatase activity. The challenge for future research is to determine if the smooth muscle myosin phosphatase is regulated, and if so, how this mechanism integrates with the overall contraction-relaxation cycle of smooth muscle. 

TABLE 1 Myosin Phosphatases from Smooth Muscle ... 

Myosin phosphatase Bovine aorta 67,000 PP2A Werth et al. (1982)... 

Myosin phosphatases are prepared from both the soluble (cytosolic) and the myofibrillar fraction of smooth muscle. If the source of phosphatase is the soluble fraction, then chromatographic procedures are usually preceded by fractionation with ammonium sulfate. Myosin phosphatase from myofibrils, crude actomyosin, or myosin is solubilized at high ionic strength by 0.6 NaCl plus detergent (Alessi et al.,... 

A systematic evaluation of phosphatase types in different smooth muscles has not been carried out. Most of the biochemical characterizations have used gizzard as a source (for practical considerations) and only a few other smooth muscles have been studied (see Table I). A larger variety of muscle have been studied using more physiological approaches, either intact or skinned fibers, and these have frequently demonstrated existence of myosin phosphatase but generally have not identified the phosphatase involved. 

P-myosin is higher than that of the isolated catalytic subunit. Thus dissociation of the trimeric phosphatase could be a regulatory mechanism. The factors involved in dissociation of PPlc are not established, although there is the suggestion that arachidonic acid is implicated. It is possible that dissociation of the trimeric phosphatase is the end result of the G-protein-linked inhibition of phosphatase activity, observed in several systems. Another possibility is that the trimeric phosphatase binds only to phosphorylated myosin and has a lower affinity for the dephosphory-lated form. Whatever the scenario imagined, it is clear that much remains to be learned about regulation of myosin phosphatase activity. 

The system was particularly difficult to use when the rate of movement was slow and required long periods of observation. It involved the use of plant actin, which, although homologous to vertebrate actin, was not identical. In addition, there were concerns that components of the dissected Nitella cytoplasm, such as its own myosins, phosphatases, kinases, or proteases, may interfere with the movement of the exogenously added myosin. 

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