Role of Phosphorylation in Regulating Calpain Activity

磷酸化在调节钙蛋白酶活性中的作用

• 批准号: 7090374
• 负责人: DARREL E GOLL
• 金额: $ 28.75万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7090374
• 关键词: active sites; animal tissue; antibody; calcium flux; calmodulin; calpain; chickens; enzyme activity; enzyme structure; gene expression; intermolecular interaction; isozymes; kinase inhibitor; laboratory mouse; mass spectrometry; mitogen activated protein kinase; monoclonal antibody; phosphatase inhibitor; phosphoprotein phosphatase; phosphorylation; phosphotransferases; protease inhibitor; protein kinase; protein structure function; site directed mutagenesis; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The long-term goal of this research is to learn how activity of the "ubiquitous calpains", u- and m-calpain, is regulated in living cells. The calpains are Ca2+-dependent proteases found in every vertebrate cell. Disruption of the gene encoding the 28-kDa subunit common to u- and m-calpain is embryonical lethal. Inappropriate calpain degradation is implicated in many tissue pathologies ranging from loss of muscle mass in the muscular dystrophies, to crystalline degradation and cataract formation, to Alzheimer's disease, to tissue damage in ischemic areas near a blocked blood vessel (stroke or myocardial infarction), to traumatic spinal cord injury, etc. Calpain activity in these pathologies is triggered by elevated, intracellular [Ca2+]; however, Ca2+ requirement of the calpains in in vitro assays is 3-50 uM (u-calpain) or 300-500 uM (m- calpain), much higher than intracellular free [Ca2+] is, even near ischemic areas. Cells, therefore, have a mechanism to reduce the [Ca2+] required for calpain activity. This mechanism evidently is altered in a way that activates the calpains during ischemic events. The same mechanism must regulate calpain activity during normal cell function. We have found that both u- and m-calpain are phosphorylated at multiple sites, and that dephosphorylated calpain is proteolytically inactive. Studies in this application test the hypothesis that phosphorylation regulates calpain activity in cells. There are five objectives. 1. Measure phosphorylation status and activities of calpains isolated from cells that have been incubated with phosphatase/kinase inhibitors to alter the level of calpain phosphorylation in these cells. 2. Determine the phosphorylation status and activities of calpains isolated from cells that have been treated to activate the calpains. 3. Determine the effects of in vitro phosphorylation by selected kinases on the activities of u- and m-calpain that have been dephosphorylated. 4. Use site-directed mutation of specific phosphorylation sites, chosen on the basis of results in aims 1 and 2, and expression of rat m-calpain to determine the effects of such mutations on activities of m-calpain. 5. Determine the effects of phosphorylation of u- or m-calpain on the calpain/ calpastatin interaction. Changes in calpain phosphorylation will be compared with changes in catalytic properties and the Ca2+ requirement of the calpains. The studies use mass spectrometry and Western analysis to monitor calpain phosphorylation and enzyme assays to monitor catalytic properties of the calpains.

描述(申请人提供)：这项研究的长期目标是了解“无处不在的钙蛋白酶”，u-和m-钙蛋白酶的活性是如何在活细胞中调节的。钙蛋白酶是存在于所有脊椎动物细胞中的依赖于钙离子的蛋白酶。U-和m-calain共有的28 kDa亚基编码基因的破坏是胚胎致死的。钙蛋白酶的不适当降解与许多组织病理有关，从肌营养不良的肌肉质量丧失，到晶体降解和白内障形成，到阿尔茨海默病，到血管阻塞(中风或心肌梗死)附近的缺血区的组织损伤，到创伤性脊髓损伤等。在这些病理中，钙蛋白酶的活性是由细胞内高水平的[Ca~(2+)]触发的；然而，体外检测时对钙的需求为3-50um(u-calain)或300-500um(m-calain)，远高于细胞内游离[Ca~(2+)]，甚至在缺血区附近。因此，细胞有一种机制来减少钙蛋白酶活性所需的[钙]。这种机制显然是以一种在缺血事件中激活钙调蛋白的方式改变的。在正常的细胞功能过程中，同样的机制也必须调节钙蛋白酶的活性。我们已经发现，u-和m-calain在多个位点都被磷酸化，而去磷酸化的calain在蛋白水解性方面是不活跃的。这项应用中的研究验证了磷酸化调节细胞中钙蛋白酶活性的假设。有五个目标。1.检测从与磷酸酶/激酶抑制剂孵育的细胞中分离出来的钙蛋白酶的磷酸化状态和活性，以改变这些细胞中的钙蛋白酶的磷酸化水平。2.确定从细胞中分离出来的钙蛋白酶的磷酸化状态和活性，这些细胞已经被处理来激活钙蛋白酶。3.确定体外磷酸化对已去磷酸化的u-和m-calain活性的影响。4.利用根据AIMS 1和2的结果选择的特定磷酸化位点的定点突变和大鼠m-calain的表达来确定这些突变对m-calain活性的影响。5.确定u-或m-calain的磷酸化对calain/calastatin相互作用的影响。Calain磷酸化的变化将与其催化性质和对钙的需求的变化进行比较。这些研究使用质谱学和Western分析来监测calain的磷酸化，并使用酶分析来监测calain的催化性能。