Role of Phosphorylation in Regulating Calpain Activity

这项研究的长期目标是了解“无处不在的钙蛋白酶”，u-和m-钙蛋白酶是如何活动的。 在活细胞中被调节。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。 编码u-和m-calain共有的28 kDa亚基的基因的中断在胚胎上是致命的。 不适当的钙蛋白酶降解与许多组织病理有关，包括肌肉质量的丧失 在肌肉营养不良、晶体蛋白降解和白内障形成、阿尔茨海默病、 血管阻塞(中风或心肌梗死)附近缺血区的组织损伤，至创伤性 脊髓损伤等。在这些病理中，钙蛋白酶活性是由升高的细胞内[Ca~(2+)]触发的； 然而，在体外实验中，钙蛋白酶的钙需要量为3-50微米(u-钙蛋白酶)或300-500微米(m-钙蛋白酶)。 Calain)，远高于细胞内游离[Ca~(2+)]，甚至在缺血区附近。因此，细胞有一个 降低钙蛋白酶活性所需[Ca~(2+)]的机制这一机制显然在某种程度上被改变了 这会在缺血事件中激活钙调蛋白。同样的机制也必须调节钙蛋白酶的活性 在正常的细胞功能过程中。我们已经发现u-和m-calain在多个位置都被磷酸化， 这种去磷酸化的钙蛋白在蛋白水解性上是不活跃的。本应用程序中的研究验证了该假说 这种磷酸化可以调节细胞中的钙蛋白酶活性。有五个目标。1.测量磷酸化程度 与磷酸酶/激酶共同孵育的细胞中钙激活蛋白的状态和活性 用来改变这些细胞中钙蛋白磷酸化水平的抑制剂。2.确定磷酸化状态 以及从细胞中分离出来的钙蛋白酶的活性，这些细胞已经被处理来激活钙蛋白酶。3.确定 选择性蛋白水解酶的体外磷酸化对已被激活的u-和m-calain活性的影响 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 结果在AIMS 1和2中，以及大鼠m-calain的表达，以确定这些突变对 M-钙蛋白酶的活性。5.确定u-或m-calain的磷酸化对calain/calin-1的影响 糊精相互作用。Calain磷酸化的变化将与催化属性的变化相比较- ERTES与钙调蛋白的钙需求。这些研究使用了质谱仪和蛋白质分析。 监测钙蛋白酶的磷酸化和酶分析，以监测钙蛋白酶的催化性质。

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.