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: 7213465
• 负责人: DARREL E GOLL
• 金额: $ 28.81万
• 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/7213465
• 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; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Detection; Disruption; Endopeptidases; Enzymes; Event; Genes; Goals; Homeostasis; In Vitro; Incubated; Ionophores; Learning; Life; Localized; Location; MAPK1 gene; MAPK3 gene; Mass Spectrum Analysis; Measures; Monitor; Muscle; Muscular Dystrophies; Mutate; Mutation; Myocardial Infarction; Normal Cell; Numbers; Okadaic Acid; Pathology; Peptide Hydrolases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Property; Protein Kinase; Protein Kinase C; Proteins; Proteolysis; Range; 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; phosphatase inhibitor; programs; response

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（由申请人提供）：本研究的长期目标是了解活细胞中“无处不在的钙蛋白酶”u-和m-钙蛋白酶的活性是如何被调节的。钙蛋白酶是一种Ca2+依赖性蛋白酶，存在于所有脊椎动物细胞中。编码u-和m-钙蛋白酶共有的28-kDa亚基的基因的破坏是胚胎致命的。钙蛋白酶的不适当降解与许多组织病理有关，包括肌肉萎缩症引起的肌肉质量损失、晶体降解和白内障形成、阿尔茨海默病、血管阻塞附近缺血区域的组织损伤（中风或心肌梗死）、创伤性脊髓损伤等。这些病理中的钙蛋白酶活性是由升高的细胞内[Ca2+]触发的；然而，在体外实验中，钙蛋白酶的Ca2+需求量为3-50 μ m （u-calpain）或300-500 μ m (m- calpain)，远高于细胞内游离[Ca2+]，甚至在缺血区域附近。因此，细胞具有降低钙蛋白酶活性所需的[Ca2+]的机制。这一机制明显改变，在缺血事件中激活脑钙。在正常的细胞功能过程中，钙蛋白酶的活动也有相同的调节机制。我们发现u-和m-calpain在多个位点被磷酸化，而去磷酸化的calpain是无蛋白水解活性的。本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. 测量从磷酸酶/激酶抑制剂孵育的细胞中分离的钙蛋白酶的磷酸化状态和活性，以改变这些细胞中钙蛋白酶的磷酸化水平。2. 确定从细胞中分离的钙蛋白酶的磷酸化状态和活性，这些细胞已被处理以激活钙蛋白酶。3. 确定选定的激酶体外磷酸化对已去磷酸化的u-和m-钙蛋白酶活性的影响。4. 根据目的1和2的结果和大鼠m-calpain的表达，使用特定磷酸化位点的定点突变来确定这种突变对m-calpain活性的影响。5. 确定u-或m-calpain磷酸化对calpain/ calpastatin相互作用的影响。钙蛋白酶磷酸化的变化将与钙蛋白酶的催化性能和Ca2+需求的变化进行比较。本研究使用质谱法和Western分析法来监测钙蛋白酶的磷酸化和酶分析来监测钙蛋白酶的催化性能。