Myosin II phosphorylation is a major regulatory mechanism in smooth muscle and non-muscle cells. In smooth muscle (arteries, veins, gastrointestinal tract, uterus, etc.) it controls the contraction-relaxation cycle and is implicated in angiogenesis. In non-muscle cells it regulates essential cell functions including: motility, dynamics of cytoskeletal structure, cell division (cytokinesis) and secretion. An alteration of myosin II phosphorylation is involved in several disorders of the cardiovascular system, e.g. hypertension, cerebral and coronary vasospasm, and of other hollow organs, e.g. bronchial asthma, preterm labor and erectile dysfunction. Myosin II phosphorylation also is important in cancer cell motility and metastasis. Thus, understanding the mechanisms regulating myosin II phosphorylation is essential in treatment of these disorders and is of considerable health benefit to our society. Two key enzymes control myosin II phosphorylation: a myosin kinase (usually myosin light chain kinase) and a myosin phosphatase (MP). At constant [Ca2+]i the major factor modulating myosin II phosphorylation is regulation of MP. Both activation (associated with increased cyclic nucleotide levels) and inhibition (implicated in many of the disorders listed above) are documented. The RhoA/Rho-kinase pair is important in inhibition. Objectives of this proposal are to establish the molecular basis for regulation of MP as centered on the myosin phosphatase target subunit (MYPT1). The intent of Specific Aim 1 is to characterize several important interactions of MYPT1, with emphasis on binding of substrates, and the molecular mechanism(s) of regulation. Biochemical analyses will use isolated MP holoenzyme and purified MYPT1 and fragments. The A7r5 cell line (derived from rat aorta) will be used in the next 2 Aims as a model system to test several hypotheses. In SA 2 the effects of cyclic nucleotides, initially cGMP and subsequently cAMP, will be investigated. The objective is to establish the molecular basis for activation of MP. The role of individual isoforms will be probed by RNA interference (siRNA). The final SA characterizes MYPT1 isoforms in A7r5 cells. Subcellular localizations of isoforms will be determined and putative roles of individual isoforms assessed by siRNA. These 3 aims use combined techniques of biochemistry, molecular biology and cell biology. If successful, they will establish an understanding at a molecular level of a process essential for function in muscle and non-muscle cells, and will facilitate pharmacological intervention. ? ? ?
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