The long-term objective of the proposed research is to determine the molecular mechanism by which receptor activation increases the contractile Ca2+ sensitivity (Ca2+ sensitization) and to use this information to explore the pathological mechanism of smooth muscle abnormality such as hypertension and asthma. Regulation of the reversible Ser19 phosphorylation of 20 kDa myosin light chain (MLC) primarily governs the extent of vascular smooth muscle contraction. Although a rise in [Ca2+]i acts as the main triggering mechanism for MLC phosphorylation by activating Ca2+/calmodulin-dependent MLC kinase, G protein-coupled receptor agonists exert their effect in large part by dynamically changing the Ca2+ sensitivity of MLC phosphorylation and contraction. In this application, the molecular mechanism of a novel Ca2+ sensitizing pathway through smooth muscle-specific phospho-protein CPI-17 and increases its inhibitory potency to MLC phosphatase 1000-fold, resulting in the potent Ca2+ sensitization of MLC phosphorylation and contraction. The Ca2+ sensitizing but not Ca2+ augmenting agonist stimulation increases the phosphorylation of in-situ CPI-17 in intact and permeabilized smooth muscles, suggesting that CPI-17 has a physiological role in the CA2+ sensitization. Expression of CPI-17 differs among smooth muscle tissues and its phosphorylation levels vary with agonists. Several kinases can phosphorylate CPI-17 at Thr38 in vitro while very little is known about its in-situ kinases and phosphatase(s). The following aims shall be pursued (1) whether CPI-17 has an important role in the G protein-mediated Ca2+ sensitization in arterial smooth muscle, and which agonist in different type of smooth muscle tissue is mainly utilizing CPI-17 signaling pathway, (2) whether phosphorylated CPI-17 is translocated to thick filaments associated with MCL phosphatase, (3) whether another Ca2+ sensitizing pathway through phosphorylation of myosin targeting subunit of MLC phosphatase has a physiological significance, (4) whether in situ CPI-17 phosphatase is the MLC phosphatase itself, (5) which type of kinase mainly phosphorylates in-situ CPI-17 at Thr38 under different conditions.
