Myosin phosphatase autoinhibition in gastrointestinal smooth muscle contraction

描述（由申请人提供）：平滑肌（SM）收缩状态的改变在胃肠道疾病（如胃、肠和括约肌功能障碍、运动异常和其他病理）中起关键作用。SM是由一个复杂的信号通路网络，通过肌球蛋白的磷酸化调节收缩性的关键调制。肌球蛋白轻链磷酸酶（MLCP）是这些信号通路的主要下游靶点，但对其抑制和激活的分子机制知之甚少，这是我们建议的重点。RhoA GT3、Rho-激酶（ROCK）和其他激酶的效应物在Thr 696和Thr 853处磷酸化MLCP靶向亚基（MYPT 1）并抑制MLCP，而cAMP/cGMP信号和环核苷酸靶点telokin逆转抑制作用，引起GI SM松弛。我们的合作已经导致了一种新的模型的MLCP活性抑制MYPT 1磷酸化的机制。在该模型中，包括在Thr 696或Thr 853处磷酸化的MYPT 1的片段直接结合MLCP的活性位点，导致MLCP的自抑制。在目的1中，我们将确定MYPT 1自抑制（AI）结构域（包括Thr 696（AI-1）或Thr 853（AI-2））在调节肠道SM张力中的结构/功能关系，以严格测试该模型。该模型将使用FRET生物传感器在活的眼底SM细胞中进行验证。第二个目标将解决cAMP/cGMP信号如何改变MLCP活性，以消除RhoA介导的Ca 2+致敏导致松弛。环核苷酸是公认的胃肠道平滑肌松弛的重要生理介质。显示激活MLCP的主要环核苷酸靶标是telokin，其在GI SM中表达最高。与野生型相比，在telokin敲除小鼠中pCa-力关系左移。在目标2a中，我们将确定telokin诱导MLCP活性活化的分子机制。将使用表面等离子体共振、等温量热法、GST-下拉、邻近连接测定（PLA）和FRET生物传感器来测试端蛋白诱导的MLCP活化的两种分子模型。功能测定将利用来自telokin -/-和WT小鼠的GI SM。在目标2b中，我们将检验以下假设：环核苷酸诱导的MLCP在不同胃肠平滑肌中的去自身抑制由多种途径介导，但由不同的途径主导，以放松Ca 2+敏化力。我们将确定使用笼状核苷酸的光解，非端蛋白介导的环核苷酸的自抑制的衰减是否通过Rap 1的Epac激活下调RhoA活性，通过MYPT 1的Ser 695的磷酸化或RhoA的抑制性磷酸化而发生。使用我们在体内和体外的数据进行模拟和拟合，我们希望建立这些信号通路的贡献的幅度和层次，并在GI SM中环核苷酸诱导的松弛的新的机械计算模型。我们预计，胃底和回肠SM将由不同的途径主导，反映了它们不同的功能作用。这些发现应该为靶向治疗带来新的见解。 公共卫生相关性：例如由平滑肌组织的异常收缩和松弛引起的疾病胃、肠和括约肌功能障碍、消化不良、肠性肠病、手术引起的肠运动性降低、高血压、脑和冠状动脉血管痉挛、勃起功能障碍和支气管哮喘等疾病是由平滑肌组织的异常收缩和松弛引起的.我们正在研究特定蛋白质的作用，这些蛋白质通过复杂的信号通路调节胃肠平滑肌细胞的收缩机制。这种收缩机制也在细胞迁移中起作用，例如在胃肠道发育和肿瘤转移期间发生。研究结果应该转化为针对这些疾病的新疗法。

DESCRIPTION (provided by applicant): Alterations in the contractile state of smooth muscle (SM) plays a key role in gastrointestinal diseases such as gastric, intestinal and sphincter dysfunction, abnormal motility and other pathologies. SM is critically modulated by a complex network of signaling pathways that regulate contractility through phosphorylation of myosin. Myosin light chain phosphatase (MLCP) is a major downstream target of these signaling pathways yet the molecular mechanisms responsible for its inhibition and activation are poorly understood and this is the focus of our proposal. The effector of RhoA GTPase, Rho-kinase (ROCK) and other kinases, phosphorylate MLCP targeting subunit (MYPT1), at Thr696 and Thr853 and inhibit MLCP, while cAMP/cGMP signals and the cyclic nucleotide target, telokin, reverse the inhibitory effect, causing GI SM relaxation. Our collaboration has lead to a novel model for the mechanism underlying the inhibition of MLCP activity upon MYPT1 phosphorylation. In the model, the segment including the phosphorylated MYPT1 at Thr696 or Thr853 directly binds to the active site of MLCP, resulting in an autoinhibition of MLCP. In Aim 1 we will determine the structure / function relationship of MYPT1 autoinhibitory (AI) domains including Thr696 (AI-1) or Thr853 (AI-2) in the regulation of gut SM tone to now rigorously test this model. The model will be validated in live fundus SM cells using FRET biosensors. The second aim will address how cAMP/cGMP signals alter MLCP activity to eliminate RhoA-mediated Ca2+ sensitization resulting in relaxation. Cyclic nucleotides are well established as physiologically important mediators of relaxation in GI SM. A major cyclic nucleotide target shown to activate MLCP is telokin, which is most highly expressed in GI SM. The pCa-force relationship is left shifted in telokin knockout mice compared to wild type. In Aim 2a we will determine the molecular mechanism(s) of telokin- induced activation of MLCP activity. Surface plasmon resonance, isothermal calorimetry, GST-pull down, proximity ligation assays (PLA) and a FRET biosensor will be used to test two molecular models of telokin- induced activation of MLCP. Functional assays will utilize GI SM from telokin -/- and WT mice. In Aim 2b we will test the hypothesis that cyclic nucleotide-induced de-autoinhibition of MLCP in different gastrointestinal smooth muscles is mediated by multiple, but dominated by different pathways to relax Ca2+ sensitized force. We will determine using photolysis of caged nucleotides, whether non-telokin mediated attenuation of the autoinhibition by cyclic nucleotides occurs through down regulation of RhoA activity by Epac activation of Rap1, through phosphorylation of Ser695 of MYPT1 or inhibitory phosphorylation of RhoA. Using our in vivo and in vitro data for simulations and fitting we expect to establish the magnitudes and hierarchy of the contribution of these signaling pathways and arrive at new mechanistic computational models of cyclic nucleotide-induced relaxation in GI SM. We expect that fundus and ileum SM will be dominated by different pathways, reflecting their different functional roles. Findings should lead to new insights for targeting therapies. PUBLIC HEALTH RELEVANCE: Diseases such as g by abnormal contraction and relaxation of smooth muscle tissues gastric, intestinal and sphincter dysfunction, dyspepsia, intestinal bowel disease, surgery- induced decreased gut motility, hypertension, cerebral and coronary vasospasm, erectile dysfunction, and bronchial asthma, among other diseases are caused by abnormal contraction and relaxation of smooth muscle tissues. We are studying the role of specific proteins, which through complex signaling pathways regulate the contractile machinery in gastrointestinal smooth muscle cells. This contractile machinery also functions in cell migration, such as occurs during development of the gastrointestinal tract and in tumor metastasis. The results of the research should translate into novel treatments for targeting these diseases.