ROLE AND REGULATION OF CPLA2 IN ANOXIC CELL INJURY

本研究的目的是探索激活机制 大分子量胞质型磷脂酶A2（cPLA 2）。我们的假设是 cPLA 2的完全激活需要两个 磷酸化/去磷酸化步骤，除了增加 [Ca2+]：丝氨酸-505和苏氨酸-60。Three-60已经被控制住了 在cPLA 2的钙脂质结合结构域（CaLB）内，并且是保守的 在所有响应[Ca 2 +]的蛋白质中。我们的具体目标 1.确定cPLA 2是否在能量引起的细胞损伤中起作用 通过比较过表达cPLA 2的LLC-Pk 1细胞和 通过使用寡脱氧核苷酸抑制cPLA 2表达。2、检查 磷酸化在酶活化和易位中的作用。3.到 确定cPLA 2的磷酸化位点。4.评估是否 p42 MAP激酶与cPLA 2在细胞内相关， 非激酶蛋白与cPLA 2相互作用。这些最后的实验 使用一种新型的双杂交交互系统。环境很理想 成功完成这项提案。Bonventre医生很好 受人尊敬的研究者，具有PLA 2、细胞 损伤和信号转导。所描述的方法已经在使用中 在他的实验室里，涉及PLA 2的多个项目正在进行中， 他的实验室。

The research proposal is designed to explore the mechanisms of activation of the large molecular weight cytosolic PLA2 (cPLA2). Our hypothesis is that complete activation of cPLA2 requires two phosphorylation/dephosphorylation steps in addition to an increase in [Ca2+]: at serine-505 and at threonine-60. Threonine-60 is contained within the calcium lipid binding domain (CaLB) of cPLA2 and is conserved in all proteins that translocate in response to [Ca2+]. Our Specific Aims are 1.To determine if cPLA2 plays a role in cell injury due to energy depletion by comparing injury in LLC-Pk1 cells which overexpress cPLA2 and by inhibiting cPLA2 expression using oligodeoxynucleotides. 2 To examine the role of phosphorylation in enzyme activation and translocation. 3. To determine the site of phosphorylation of cPLA2. 4. TO evaluate whether p42MAP Kinase associates with cPLA2 intracellularly and whether any other non-kinase proteins interact with cPLA2. These last experiments will be done using a novel two hybrid interaction system. The environment is ideal for the successful completion of this proposal. Dr. Bonventre is very well respected established investigator with areas of expertise in PLA2, cell injury and signal transduction. The methods described are already in use in his laboratory and multiple projects involving PLA2 are in progress in his laboratory.