Regulation of smooth muscle-myosin phosphatase 1 kinase

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DESCRIPTION (provided by applicant: Smooth muscle plays an essential role in a wide variety of physiological processes, from the regulation of blood pressure by controlling vessel diameter in the periphery, to digestive processes by controlling mechanical movement of food though the gut, to reproductive functions by controlling penile erection and delivery of the fetus at birth, to regulation of lung capacity through regulation of airway diameter. A major factor governing the contractile state of all smooth muscles is the phosphorylation level of myosin light chain (LC20). In smooth muscle, steady state phosphorylation of LC20 is dictated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase (SMPP-1M). Inhibition of SMPP-1M activity through G protein coupled receptors acting through the small GTP binding protein RhoA has been shown to bring about calcium sensitization smooth muscle. Alterations in the sensitivity of various smooth muscles to calcium is hypothesized to be an underlying cause associated with many diseases associated with smooth muscle dysfunction such as hypertension, bronco spasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Therefore signaling elements controlling myosin dephosphorylation state may represent attractive points of therapeutic intervention in a variety of human diseases associated with smooth muscle dysfunction. Recently our laboratory identified MYPT1 kinase (MYPT1 K) as a co-localizing SMPP-1M kinase that selectively phosphorylated the myosin targeting subunit (MYPT1) of the phosphatase and the inhibitor protein CPI17 to inhibit activity. Addition of constitutively active forms of MYPT1 K to permeabilized smooth muscles causes profound calcium sensitization through inhibition of SMPP-1M activity. In smooth muscle, MYPT1K is activated in response to a variety of calcium-sensitizing agonists and this activation can be blocked with the Rho kinase inhibitor Y-27632. These findings suggest MYPT1K mediates calcium-sensitizing signals from Rho kinase to SMPP-1M, however, the mechanisms by which MYPT1K is regulated in smooth muscle are unknown, since the protein kinase is not a direct substrate for Rho kinase in vitro. In this proposal we will employ a unique blend of muscle physiology, mass spectrometry, molecular biology, small interfering RNA's and small molecule chemistry to determine the molecular mechanisms by which Rho dependant signaling pathways bring about activation of MYPT1K in smooth muscle.