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。这些运动响应于增加的细胞质Ca2+浓度（[Ca2+]i）和Ca2+/钙调素（CaM）依赖性肌球蛋白轻链激酶（MLCK）的激活。MLCK磷酸化肌凝蛋白调控轻链亚基（RLC），允许肌凝蛋白结合肌动蛋白丝收缩。提出了抑制MLCP活性的信号通路，从而增加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+独立激酶磷酸化。基于我们最近成功地使用分子转基因和条件基因消融方法来确定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揭示与MLCP和Ca2+致敏机制相关的综合信号通路。特异性目标1：肌球蛋白轻链磷酸酶（MYPT1）的调控亚基对RLC磷酸化的有效信号传导是必要的吗？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，敲除含有floxed MYPT1等位基因的成年小鼠的MYPT1。将评估相（回肠）和强直（气管）平滑肌的大体病理，包括组织学分析和收缩和信号蛋白的表达。碳水化合物和电场刺激（EFS）引起副交感神经的反应将诱导[Ca2+]i， RLC磷酸化和收缩的颞细胞变化。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+敏化指数。我们将验证MYPT1是RLC去磷酸化所必需的假设。我们还将确定在毒蕈碱M3受体诱导的Ca2+敏化反应中，CPI-17磷酸化与MYPT1磷酸化的相对重要性。特异性目标2：在Ca2+敏化过程中，MYPT1如何调节MLCP活性以维持RLC磷酸化？我们将单独或共同突变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Aim 1所述。因此，我们将验证MYPT1 Thr696和Thr696磷酸化对Ca2+敏化反应都很重要的假设。特异性目标3:Ca2+非依赖性ZIPK是否影响Ca2+敏化MYPT1和CPI-17磷酸化？由于没有针对这种激酶的选择性药理学抑制剂，因此含有ZIPK固定基因的小鼠将用于成年动物的基因消融。我们将测试Ca2+独立激酶ZIPK磷酸化MYPT1和CPI-17作为Ca2+敏化过程的一个组成部分的假设。

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.