Myosin phosphatase autoinhibition in gastrointestinal smooth muscle contraction

描述（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、括约肌功能障碍、运动异常等病理中起关键作用。SM是由一个复杂的信号通路网络严格调节的，该信号通路通过肌球蛋白磷酸化调节收缩性。肌球蛋白轻链磷酸酶（MLCP）是这些信号通路的主要下游靶点，但其抑制和激活的分子机制尚不清楚，这是我们研究的重点。RhoA GTPase、rho激酶（ROCK）等激酶的效应物使MLCP靶向亚基（MYPT1）在Thr696和Thr853位点磷酸化，抑制MLCP，而cAMP/cGMP信号和环核苷酸靶点端粒蛋白逆转抑制作用，使GI SM松弛。我们的合作为MLCP抑制MYPT1磷酸化的机制建立了一个新的模型。在该模型中，包含Thr696或Thr853位点磷酸化的MYPT1片段直接结合MLCP的活性位点，导致MLCP的自抑制。在Aim 1中，我们将确定包括Thr696 （AI-1）或Thr853 （AI-2）在内的MYPT1自抑制（AI）结构域在调节肠道SM音调中的结构/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Ca2+敏化导致松弛。环核苷酸在GI SM中被认为是重要的生理松弛介质。激活MLCP的一个主要环核苷酸靶点是末端蛋白，它在GI SM中表达最高。与野生型相比，基因敲除小鼠的pCa-force关系左移。在Aim 2a中，我们将确定端粒蛋白诱导的MLCP活性激活的分子机制。表面等离子体共振、等温量热法、gst下拉法、邻近连接法（PLA）和FRET生物传感器将用于测试两种末端蛋白诱导的MLCP激活的分子模型。功能分析将利用基因表达蛋白-/-和WT小鼠的GI SM。在Aim 2b中，我们将验证环核苷酸诱导的不同胃肠道平滑肌中MLCP的去自抑制是由多种途径介导的，但主要是由不同的途径来放松Ca2+敏化力。我们将通过对固定核苷酸的光解，确定非端粒介导的环核苷酸自身抑制的衰减是否通过Epac激活Rap1下调RhoA活性，通过MYPT1的Ser695磷酸化或RhoA的抑制性磷酸化发生。利用我们的体内和体外数据进行模拟和拟合，我们期望建立这些信号通路的贡献的大小和层次，并得出环核苷酸诱导的GI SM松弛的新的机制计算模型。我们预计眼底和回肠SM将由不同的通路主导，反映其不同的功能作用。这一发现将为靶向治疗带来新的见解。

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.