Role of Phosphorylation in Regulating Calpain Activity

描述（由申请人提供）：本研究的长期目标是了解“普遍存在的钙蛋白酶”，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-钙蛋白酶共同的28-kDa亚基的基因的破坏是胚胎致死的。不适当的钙蛋白酶降解与许多组织病理学有关，从肌营养不良症中的肌肉质量损失，到晶体降解和白内障形成，到阿尔茨海默病，到阻塞血管附近缺血区域的组织损伤（中风或心肌梗塞）、创伤性脊髓损伤等。这些病理中的钙蛋白酶活性由升高的细胞内[Ca 2 +];然而，在体外测定中钙蛋白酶的Ca 2+需要量为3-50 μ M（μ-钙蛋白酶）或300-500 μ M（m-钙蛋白酶），远高于细胞内游离[Ca 2 +]，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[Ca 2 +]的机制。这一机制显然以在缺血事件期间激活钙蛋白酶的方式改变。在正常细胞功能期间，同样的机制必须调节钙蛋白酶活性。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蛋白水解无活性的。本申请的研究测试了磷酸化调节细胞中钙蛋白酶活性的假设。有五个目标。1.测量从已经用磷酸酶/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磷酸化的水平。2.测定从经处理以激活钙蛋白酶的细胞中分离的钙蛋白酶的磷酸化状态和活性。3.测定体外选定激酶磷酸化对已去磷酸化的u-和m-钙蛋白酶活性的影响。4.使用特定磷酸化位点的定点突变（根据目标1和2的结果选择）和大鼠m-钙蛋白酶的表达，以确定此类突变对m-钙蛋白酶活性的影响。5.确定u-或m-钙蛋白酶磷酸化对钙蛋白酶/钙蛋白酶抑制素相互作用的影响。钙蛋白酶磷酸化的变化将与催化性质和钙蛋白酶的Ca 2+需求的变化进行比较。这些研究使用质谱和Western分析来监测钙蛋白酶磷酸化，并使用酶测定来监测钙蛋白酶的催化特性。

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.