CALCIUM AND CALMODULIN IN SMOOTH MUSCLE CONTRACTION

钙调素（CaM）是一种普遍存在的钙结合蛋白， 成为钙信号的通用调节器。 作为 胞质Ca升高，Ca与CaM结合产生结构变化 它可以结合并激活三十多种不同的 靶蛋白。 钙调素直接参与钙依赖性 调节平滑肌收缩，腺和 神经分泌、DNA复制、基因表达和细胞 师. 目前的研究将利用我们的新的生物学 选择性报告CaM结合的活性荧光CaM 其靶蛋白。 我们将确定并讲述案件 钙调素与其靶蛋白结合的依赖性 肌肉的钙依赖性，其激活。 使用 荧光停流技术，我们将确定的速率 的钙依赖性协会和解离的钙调素与其 靶蛋白，包括肌球蛋白轻链激酶（MLCK）和 caldesmon。 将这些速率与来自于细胞的钙的解离速率进行比较。 CaM-靶蛋白复合物。 这些研究将提供 与Ca瞬变相关的时间信息（例如那些 肌肉）与钙调素-靶蛋白结合率的关系， 分离 大多数外部刺激通过细胞内信号通路影响细胞内事件。 细胞激酶的激活和特异性 底物蛋白 最近，有证据表明， 酪氨酸激酶（包括胰岛素受体和癌基因 产物P60-src）能够磷酸化酪氨酸残基 在钙调素的钙结合位点III和IV。 我们将检验 这种磷酸化作用对钙调素钙（和药物）结合的影响 性质和钙依赖性的钙调素相互作用和 激活其靶蛋白。 如果磷酸化钙调素 显示改变的钙依赖性激活MLCK，它将是 检查其调节钙依赖性张力的能力， 冠状动脉被化学腐蚀 因为钙调素调节 细胞代谢和生长的许多方面， 这些酪氨酸激酶可能发挥关键作用， 胰岛素的作用机制和细胞转化 编码酪氨酸激酶的致癌病毒。

Calmodulin (CaM) is a ubiquitous Ca binding protein that has emerged as a universal regulator of the calcium signal. As cytosolic Ca rises, Ca binds to CaM producing structural changes which allow it to bind and activate more than thirty different target proteins. CaM is directly involved in the Ca dependent regulation of smooth muscle contraction, glandular and neurosecretion, DNA replication, gene expression and cell division. The present studies will utilize our new biologically active fluorescent CaM's which selectively report CaM binding to its target proteins. We will determine and relate the Ca dependence of CaM binding to its target proteins in smooth muscle to the Ca dependence of their activation. Using fluorescence stopped-flow techniques we will determine the rates of the Ca dependent association and dissociation of CaM with its target proteins, including myosin light chain kinase (MLCK) and caldesmon. These rates will be compared to the off-rates of calcium from CaM-target protein complexes. These studies will provide temporal information relating Ca transients (such as those muscle) with the rate of CaM-target protein association and disassociation. Most external stimuli influence intracellular events by the activation of cellular kinases and the phosphorylation of specific substrate proteins. Recently, it has been shown that several tyrosine kinases (including, the insulin receptor and the oncogene product, P60-src) are capable of phosphorylating tyrosine residues in Ca binding sites III and IV of CaM. We will examine the effect of this phosphorylation on CaM's calcium (and drug) binding properties and on the Ca dependence of CaM interaction with and activation of its target proteins. If phosphorylated CaM exhibition altered Ca dependent activation of MLCK, it will be examined for its ability to regulate Ca dependent tension in chemically skinned coronary arteries. Because CaM regulates many aspects of cell metabolism and growth its phosphorylation by these tyrosine kinases might play a pivotal role in the mechanism of action of Insulin and in the transformation of cells by oncogenic viruses which code for tyrosine kinases.