Phosphorylation, calcium and smooth muscle contraction

描述（由申请人提供）：人们普遍认为，[Ca 2 +]增加通过激活肌球蛋白轻链（MLC）激酶和20 kDa MLC的磷酸化启动收缩。我们以前曾提出，第二个平行的，独立的途径存在的收缩的调节。在这一支持更新的建议，我们将继续我们的研究机制（S）的这一途径，通过测试的假设，钙调蛋白的磷酸化解除抑制，允许组成型活性肌球蛋白横桥与肌动蛋白相互作用，并产生力。我们的另一种假设是，钙调蛋白是重要的信号，允许磷酸化的横桥合作打开非磷酸化的横桥的传输。为了验证这些假设，我们将利用反义寡核苷酸（ON）的方法来产生钙调素缺陷的血管组织。我们将使用完整的猪颈动脉条作为模型。我们将确定力的Ca 2+依赖性，跨桥循环速率和附着，激酶活性，蛋白质含量，和蛋白质磷酸化在含有内源性钙调素的控制条和钙调素缺乏条。一旦我们确定了钙调素如何调节平滑肌，我们将确定钙调素是如何调节的。本提案将追求以下具体目标：1.利用ONs抑制caldesmon的合成，并利用酵母双杂交筛选caldesmon的潜在调控因子。2.为了测试假设，caldesmon紧张性抑制组成性活性的横桥，或者，caldesmon调节非磷酸化的横桥的磷酸化的横桥的合作激活。这将通过确定是否可以在钙调素缺乏的组织中量化主动横桥循环和附着来进行，并确定钙调素去除对曲线力/MLC磷酸化关系的影响。和3.确定负责磷酸化并因此解除钙调蛋白抑制的生理相关激酶的身份。这将使用32 p标记结合药理学试剂和特异性抗磷酸钙调蛋白抗体进行。我们也将利用飞行时间质谱鉴定钙调素的磷酸肽序列。我们相信，这一建议的结果将提供合理的明确答案的问题：是caldesmon重要的收缩？如果是这样，那么钙调素是如何调节平滑肌的，钙调素又是如何调节的。

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.