Activation of many cell surface receptors initiates diverse cellular movements such as cell migration, cell- matrix adhesion and contraction. These movements respond to increased cytosolic Ca2+ concentrations ([Ca2+]i) and activation of Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). MLCK phosphorylates myosin regulatory light chain subunit (RLC), allowing myosin to bind actin filaments for contraction. Signaling pathways are proposed for inhibition of MLCP activity which increases RLC phosphorylation (Ca2+-sensitization). MLCP subunit MYPT1 and the inhibitor protein CPI-17 may be phosphorylated by different Ca2+-independent kinases. Based on our recent successes in using molecular transgenic and conditional gene ablation approaches to establish MLCK's role in Ca2+-dependent signaling in mice, we propose similar approaches to unravel integrative signaling pathways in relation to MLCP and Ca2+- sensitization mechanisms.
Specific Aim 1 : Is the regulatory subunit of myosin light chain phosphatase (MYPT1) necessary for effective signaling to RLC phosphorylation? We will knock out MYPT1 in adult mice containing floxed MYPT1 alleles by tamoxifen-controlled Cre expression specifically in smooth muscle cells. Gross pathology will be assessed in phasic (ileum) and tonic (trachea) smooth muscles, including histological analyses and expression of contractile and signaling proteins. Carbachol and electric field stimulation (EFS) eliciting responses from parasympathetic nerves will induce temporal cellular changes in [Ca2+]i, RLC phosphorylation and contraction. We will assess Ca2+-sensitization indices by measuring MYPT1 Thr696 and Thr853 phosphorylation in control tissues in addition to CPI-17 phosphorylation. We will test the hypothesis that MYPT1 is necessary for robust RLC dephosphorylation. We will also determine the relative importance of CPI-17 phosphorylation compared to MYPT1 phosphorylation in the Ca2+-sensitization response induced by muscarinic M3 receptors.
Specific Aim 2 : How does MYPT1 regulate MLCP activity to sustain RLC phosphorylation during Ca2+-sensitization? We will mutate the two regulatory phosphorylation sites in MYPT1 (Thr696Ala and Thr853Ala) individually or together, and measure biochemical and cellular responses as described in Aim 1. We will thus test the hypothesis that both MYPT1 Thr696 and Thr696 phosphorylation are important for Ca2+-sensitization responses.
Specific Aim 3 : Does Ca2+-independent ZIPK affect Ca2+- sensitizing MYPT1 and CPI-17 phosphorylation? Mice containing floxed genes for ZIPK will be used for gene ablation in adult animals because there are no selective pharmacological inhibitors for this kinase. We will test the hypothesis that the Ca2+-independent kinase ZIPK phosphorylates MYPT1 and CPI-17 as an integral component of the Ca2+-sensitization process.
The calcium sensitization pathway involving inhibition of myosin phosphatase may be enhanced in diseases involving smooth muscles but sufficient knowledge about the most important cell based mechanisms or development of selective pharmacological agents are needed to provide perspectives for clinical strategies. The involved signal transduction pathways offer novel targets for treatment of diverse diseases including hyperactive smooth muscle in intestinal inflammation and asthma as well as systemic hypertension and other diseases.
