Signal transduction mechanisms to myosin phosphatase

描述(申请人提供)：许多细胞表面受体的激活启动了不同的细胞运动，如细胞迁移、细胞-基质黏附和收缩。这些运动响应细胞内钙离子浓度的升高和钙/钙调蛋白(CaM)依赖的肌球蛋白轻链激酶(MLCK)的激活。MLCK使肌球蛋白调节轻链亚基(RLC)磷酸化，使肌球蛋白与肌动蛋白细丝结合进行收缩。可以通过信号通路抑制MLCP活性，从而增加RLC的磷酸化(钙敏化)。MLCP亚基MYPT1和抑制蛋白CPI-17可能被不同的钙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基因消融的方法来确定MLCK在小鼠钙依赖信号转导中的作用，我们提出了类似的方法来解开与MLCP和钙增敏机制相关的整合信号通路。具体目的1：肌球蛋白轻链磷酸酶(MYPT1)的调节亚单位对于有效的RLC磷酸化信号是必需的吗？我们将通过他莫昔芬控制的Cre在平滑肌细胞中的特异性表达，在含有Floated MYPT1等位基因的成年小鼠中敲除MYPT1。大体病理将评估时相(回肠)和紧张期(气管)的平滑肌，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)引起的副交感神经反应将引起细胞内[Ca~(2+)]i、RLC磷酸化和收缩的短暂变化。我们将通过测量对照组织中的MYPT1 Thr696和Thr853的磷酸化以及CPI-17的磷酸化来评估钙敏化指数。我们将检验MYPT1对于RLC去磷酸化是必需的这一假设。我们还将确定CPI-17的磷酸化与MYPT1的磷酸化在M受体诱导的钙敏化反应中的相对重要性。具体目标2：MYPT1如何调节MLCP活性以维持钙敏化过程中RLC的磷酸化？我们将分别或同时突变MYPT1中的两个调节磷酸化位点(Thr696Ala和Thr853Ala)，并测量生化和细胞反应，如目标1所述。因此，我们将检验MYPT1 Thr696和Thr696磷酸化对钙敏感性反应都很重要的假设。具体目标3：钙非依赖性ZIPK是否影响钙敏化的MYPT1和CPI-17的磷酸化？含有ZIPK基因的小鼠将用于成年动物的基因消融，因为这种激酶没有选择性的药物抑制剂。我们将检验这一假设，即作为钙敏化过程的一个组成部分，钙非依赖性激酶ZIPK使MYPT1和CPI-17磷酸化。

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.