PHOSPHORYLATION OF CALPAINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 这项研究的长期目标是了解无处不在的钙蛋白酶的活性，和m-钙蛋白酶在活细胞中受到调节。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。破坏编码28-kDa亚基的基因，而m-钙蛋白酶对胚胎是致命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，到晶体蛋白降解和白内障形成，再到阿尔茨海默病？的疾病，组织损伤附近的缺血性血管（中风或心肌梗死），创伤性脊髓损伤等。钙蛋白酶活性在这些病理触发升高，细胞内[Ca 2 +]，然而，钙蛋白酶在体外试验中的Ca 2+需求是3-50？M（？-钙蛋白酶）或300-500？M（m-calpain），远高于细胞内游离[Ca ~（2+）]，即使在缺血区附近也是如此。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。我们发现，两者？和m-钙蛋白酶在多个位点被磷酸化。我们目前的研究项目的目标是确定磷酸化位点上的两个？-和m-钙蛋白酶，并测定它们对钙蛋白酶活性调节的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of this research is to learn how activity of the ubiquitous calpains, ?- 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 ?- 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 ?M (?-calpain) or 300-500 ?M (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 ?- and m-calpain are phosphorylated at multiple sites. The objective of our current research project is to identify the phosphorylated sites on both ?- and m-calpain and determine their effect on regulation of calpain activity.