Signal transduction mechanisms to myosin phosphatase

描述（由申请人提供）：许多细胞表面受体的激活启动不同的细胞运动，如细胞迁移、细胞-基质粘附和收缩。这些运动响应于胞质Ca 2+浓度（[Ca 2 +] i）的增加和Ca 2 +/钙调蛋白（CaM）依赖性肌球蛋白轻链激酶（MLCK）的激活。MLCK磷酸化肌球蛋白调节轻链亚基（RLC），使肌球蛋白结合肌动蛋白丝进行收缩。提出了抑制MLCP活性的信号传导途径，其增加RLC磷酸化（Ca2 +-敏化）。MLCP亚基MYPT1和抑制蛋白CPI-17可能被不同的Ca2+非依赖性激酶磷酸化。基于我们最近成功地使用分子转基因和条件性基因消融方法建立MLCK在小鼠Ca2+依赖性信号传导中的作用，我们提出了类似的方法来解开与MLCP和Ca2+致敏机制相关的整合信号传导途径。具体目标1：肌球蛋白轻链磷酸酶（MYPT 1）的调节亚基是否是RLC磷酸化的有效信号所必需的？我们将敲除MYPT1在成年小鼠含有floxed MYPT1等位基因的他莫昔芬控制的Cre表达，特别是在平滑肌细胞。将在阶段性（回肠）和强直性（气管）平滑肌中评估大体病理学，包括组织学分析以及收缩和信号蛋白的表达。卡巴胆碱和电场刺激（EFS）引起副交感神经的反应将引起[Ca 2 +] i、RLC磷酸化和收缩的时间细胞变化。我们将通过测量对照组织中MYPT1 Thr696和Thr853磷酸化以及CPI-17磷酸化来评估Ca2+致敏指数。我们将检验MYPT1对于稳健的RLC去磷酸化是必需的这一假设。我们还将确定CPI-17磷酸化与MYPT1磷酸化在毒蕈碱M3受体诱导的Ca2+致敏反应中的相对重要性。具体目标2：MYPT1如何调节MLCP活性以维持钙致敏过程中的RLC磷酸化？我们将单独或一起突变MYPT1中的两个调节磷酸化位点（Thr696Ala和Thr853Ala），并测量目标1中所述的生化和细胞反应。因此，我们将测试MYPT1 Thr696和Thr696磷酸化对Ca2+致敏反应都很重要的假设。具体目标3：Ca2+非依赖性ZIPK是否影响Ca2+敏化MYPT1和CPI-17磷酸化？含有ZIPK floxed基因的小鼠将用于成年动物的基因消融，因为该激酶没有选择性药理学抑制剂。我们将测试的假设，Ca2+非依赖性激酶ZIPK磷酸化MYPT1和CPI-17作为一个不可分割的组成部分的Ca2+敏化过程。

DESCRIPTION (provided by applicant): 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.