CALCIUM AND CALMODULIN IN SMOOTH MUSCLE CONTRACTION

钙和钙调蛋白在平滑肌收缩中的作用

• 批准号: 3232126
• 负责人: J DAVID JOHNSON
• 金额: $ 7.99万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3232126
• 关键词: allosteric site; calcium channel blockers; calcium transporting ATPase; calmodulin; cell cell interaction; chemical binding; coronary artery; coronary vasodilator; fluorescent dye /probe; muscle contraction; myosins; pharmacology; proteolysis; smooth muscle; swine; troponin

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.

钙调蛋白 (CaM) 是一种普遍存在的 Ca 结合蛋白，具有 成为钙信号的通用调节剂。 作为 胞质 Ca 升高，Ca 与 CaM 结合产生结构变化 这使得它能够结合并激活三十多种不同的 目标蛋白质。 CaM 直接参与 Ca 依赖性 调节平滑肌收缩、腺体和 神经分泌、DNA复制、基因表达和细胞 分配。 目前的研究将利用我们的新生物学 活性荧光 CaM，选择性报告 CaM 结合 它的目标蛋白。 我们将确定并关联 Ca 光滑细胞中 CaM 与其靶蛋白结合的依赖性 肌肉对其激活的 Ca 依赖性。 使用 荧光停流技术我们将确定速率 CaM 的 Ca 依赖性结合和解离及其 靶蛋白，包括肌球蛋白轻链激酶 (MLCK) 和 卡尔德斯蒙。 这些速率将与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 CaM 靶蛋白关联率以及 分离。 大多数外部刺激通过以下方式影响细胞内事件： 细胞激酶的激活和特定的磷酸化 底物蛋白。 近日，有数据显示，多 酪氨酸激酶（包括胰岛素受体和癌基因 产品，P60-src）能够磷酸化酪氨酸残基 位于 CaM 的 Ca 结合位点 III 和 IV 中。 我们来检验一下效果 这种磷酸化对 CaM 钙（和药物）结合的影响 性质以及 CaM 与 和 相互作用的 Ca 依赖性 激活其靶蛋白。 如果磷酸化 CaM 展览改变了 MLCK 的 Ca 依赖性激活，这将是 检查其调节钙依赖性张力的能力 化学剥皮的冠状动脉。 因为 CaM 调节 细胞代谢和生长的许多方面及其磷酸化 这些酪氨酸激酶可能在 胰岛素的作用机制和细胞转化 由编码酪氨酸激酶的致癌病毒引起。