PHOSPHORYLATION OF CALPAINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项研究的长期目标是了解在活细胞中如何调节普遍存在的钙蛋白酶？？和m-钙蛋白酶的活性。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β-和m-钙蛋白酶共有的28 kDa亚基的基因的破坏在胚胎上是致命的。钙蛋白酶的不适当降解与许多组织病理有关，从肌营养不良的肌肉质量丧失，到晶体蛋白降解和白内障形成，到阿尔茨海默病？S病，到血管阻塞(中风或心肌梗死)附近的缺血区的组织损伤，到创伤性脊髓损伤等。在这些病理中，钙蛋白酶的活性是由细胞内[Ca~(2+)]升高触发的；然而，体外分析表明，Calain对钙的需求量为3-50M(？-钙痛)或300-500M(间-钙痛)，远远高于细胞内游离[Ca+]i，甚至在缺血区附近。因此，细胞有一种机制来减少钙蛋白酶活性所需的[钙]。这种机制显然是以一种在缺血事件中激活钙调蛋白的方式改变的。在正常的细胞功能过程中，同样的机制也必须调节钙蛋白酶的活性。我们已经发现，β-和m-calain在多个位点都被磷酸化。我们目前的研究项目的目标是确定β-和m-calain上的磷酸化位点，并确定它们对Calain活性的调节作用。

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.