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: 7582294
• 负责人: David John Hartshorne
• 金额: $ 28.24万
• 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/7582294
• 关键词: Address; Affect; Alzheimer' s Disease; Area; Binding; Biological Assay; Blood Platelets; Blood Vessels; Calmodulin; Calpain; Cataract; Cell physiology; Cells; Chronic; Consensus Sequence; Crystallins; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Detection; Enzymes; Event; Genes; Goals; Homeostasis; In Vitro; Incubated; Ionophores; Learning; Life; Location; MAPK1 gene; MAPK3 gene; Mass Spectrum Analysis; Measures; Monitor; Muscle; Muscular Dystrophies; Mutate; Mutation; Myocardial Infarction; Normal Cell; Okadaic Acid; Pathology; Peptide Hydrolases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Property; Protein Kinase; Protein Kinase C; Proteins; Proteolysis; Rattus; Regulation; Regulation of Proteolysis; Research; Research Personnel; Rest; Role; Screening procedure; Site; Site-Directed Mutagenesis; Small Interfering RNA; Spinal cord injury; Stroke; Testing; Tissues; base; calpastatin; calyculin A; citrate carrier; enzyme activity; extracellular; in vitro Assay; in vivo; inhibitor/antagonist; kinase inhibitor; m-calpain; muscle form; phosphatase inhibitor; programs; response

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.Dis- ruption of the gene encoding the 28-kDa subunit common to u- and m-calpain is embryonically lethal. Inappropriate calpain degradation is implicated in many tissue pathologies ranging from loss of muscle mass in the muscular dystrophies, to crystallin 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/ cal- pastatin interaction. Changes in calpain phosphorylation will be compared with changes in catalytic prop- erties 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.

本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）的活性如何 在活细胞中调节。钙蛋白酶是在每种脊椎动物细胞中发现的钙依赖性蛋白酶。 编码u-和m-钙蛋白酶共有的28-kDa亚基的基因的破裂是胚胎致死的。 不适当的钙蛋白酶降解与许多组织病理学有关， 在肌营养不良症，晶体蛋白降解和白内障形成，阿尔茨海默病， 阻塞血管附近缺血区域的组织损伤（中风或心肌梗死）， 脊髓损伤等。这些病理中的钙蛋白酶活性由升高的细胞内[Ca 2 +]触发; 然而，在体外测定中钙蛋白酶的Ca 2+需要量为3-50 μ M（u-钙蛋白酶）或300-500 μ M（m-钙蛋白酶）。 钙蛋白酶），远高于细胞内游离[Ca 2 +]。因此，细胞具有 降低钙蛋白酶活性所需的[Ca 2 +]的机制。这种机制显然在某种程度上改变了 在局部缺血时激活钙蛋白酶同样的机制必须调节钙蛋白酶活性 在正常细胞功能中。我们已经发现u-和m-钙蛋白酶在多个位点磷酸化， 去磷酸化钙蛋白酶是无蛋白水解活性的。本申请中的研究验证了这一假设 磷酸化调节细胞中钙蛋白酶的活性。有五个目标。1.测量磷酸化 从已经与磷酸酶/激酶孵育的细胞中分离的钙蛋白酶的状态和活性 抑制剂来改变这些细胞中钙蛋白酶磷酸化的水平。2.确定磷酸化状态 以及从已经被处理以激活钙蛋白酶的细胞中分离的钙蛋白酶的活性。3.确定 通过选择的激酶进行的体外磷酸化对已经被 去磷酸化4.使用特定磷酸化位点的定点突变，根据以下条件选择 目的1和2的结果，以及大鼠m-钙蛋白酶的表达，以确定这种突变对 m-钙蛋白酶的活性。5.确定u-或m-钙蛋白酶磷酸化对钙蛋白酶/钙蛋白酶的影响。 pastatin相互作用钙蛋白酶磷酸化的变化将与催化蛋白酶的变化进行比较。 和钙蛋白酶的Ca 2+需求。这些研究使用了质谱和Western分析 以监测钙蛋白酶磷酸化和酶测定以监测钙蛋白酶的催化性质。