Phosphorylation, calcium and smooth muscle contraction

描述（由申请人提供）：人们普遍认为[Ca2+]的增加通过激活肌球蛋白轻链（MLC）激酶和20kda MLC的磷酸化来启动收缩。我们以前曾提出，存在第二个平行的、独立的途径来调节收缩。在这项支持更新的提案中，我们将继续研究这一途径的机制，通过验证磷酸化对caldesmon的去抑制作用允许构成型活性肌球蛋白交叉桥与肌动蛋白相互作用并产生力的假设。我们的另一种假设是，caldesmon在信号传输中很重要，使磷酸化的交叉桥能够协同打开未磷酸化的交叉桥。为了验证这些假设，我们将利用反义寡核苷酸（ON）方法产生caldesmon缺陷维管组织。我们将使用完整剥皮的猪颈动脉条作为模型。我们将在含有内源性caldesmon的对照条和缺乏caldesmon的对照条中测定Ca2+对力的依赖性、过桥循环率和附着、激酶活性、蛋白质含量和蛋白质磷酸化。一旦我们确定了caldesmon如何调节平滑肌，我们就能确定caldesmon是如何被调节的。本建议将实现下列具体目标：利用ONs抑制caldesmon的合成，并利用酵母双杂交筛选鉴定caldesmon的潜在调节因子。2. 为了验证caldesmon对组成活性交叉桥的抑制假说，或者caldesmon通过磷酸化交叉桥调节非磷酸化交叉桥的协同激活。这将通过确定caldesmon缺陷组织中活跃的交叉桥循环和附着是否可以量化来实现，并确定caldesmon去除对曲线力/MLC磷酸化关系的影响。和3。确定负责磷酸化和解除caldesmon抑制的生理相关激酶的身份。这将使用32p标记与药理学试剂和特异性抗磷酸desmon抗体一起进行。我们还将利用tof质谱法鉴定caldesmon的磷酸肽序列。我们认为，这一建议的结果将为以下问题提供相当明确的答案：caldesmon在收缩中重要吗？如果是这样，caldesmon是如何调节平滑肌的，又是如何调节caldesmon的。

DESCRIPTION (provided by applicant): It is widely accepted that an increase in [Ca2+] initiates contraction by activation of myosin light chain (MLC) kinase and phosphorylation of the 20 kDa MLC. We have previously suggested that a second parallel, independent pathway exists for the regulation of contraction. In this proposal for renewal of support, we will continue our studies on the mechanism(s) of this pathway by testing the hypothesis that disinhibition of caldesmon by phosphorylation allows constitutively active myosin crossbridges to interact with actin and produce force. Our alternate hypothesis is that caldesmon is important in the transmission of the signal that allows phosphorylated crossbridges to cooperatively turn on unphosphorylated crossbridges. To test these hypotheses, we will utilize an antisense oligonucleotide (ON) approach to produce a caldesmon-deficient vascular tissue. We will use, as our model, intact and skinned strips of swine carotid artery. We will determine the Ca2+ dependence of force, crossbridge cycling rate and attachment, kinase activities, protein content, and protein phosphorylation in control strips containing endogenous caldesmon and in caldesmon-deficient strips. Once we determine how caldesmon regulates smooth muscle, we will determine how caldesmon is regulated. The following specific aims will be pursued in this proposal: 1. To use ONs to inhibit the synthesis of caldesmon and to identify potential regulators of caldesmon using the yeast two-hybrid screen. 2. To test the hypotheses that caldesmon tonically inhibits constitutively active crossbridges or alternatively, that caldesmon regulates the cooperative activation of non-phosphorylated crossbridges by phosphorylated crossbridges. This will be performed by determining if active crossbridge cycling and attachment can be quantified in caldesmon deficient-tissues and to determine the effect of caldesmon removal on the curvilinear force/MLC phosphorylation relationship. And 3. To determine the identity of the physiologically relevant kinase that is responsible for phosphorylation and therefore disinhibition of caldesmon. This will be performed using 32p labeling in conjunction with pharmacological agents and specific anti-phosphocaldesmon antibodies. We will also utilize TOF-mass spectrometry to identify phosphopeptide sequences of caldesmon. We believe the results of this proposal will provide reasonably definitive answers to the questions: Is caldesmon important in contraction? And if so, how does caldesmon regulate smooth muscle and how is caldesmon regulated.