ROLE AND REGULATION OF CPLA2 IN ANOXIC CELL INJURY

研究方案旨在探索激活的机制 大分子量胞质磷脂酶A2(CPLA2)。我们的假设是 CPLA2的完全激活需要两个 磷酸化/去磷酸化步骤除了增加 [Ca~(2+)]：丝氨酸-505和苏氨酸-60。苏氨酸-60含有 在cPLA2的钙脂结合结构域(Calb)内，并且是保守的 在所有对[Ca~(2+)]作出反应而移位的蛋白质中。我们的具体目标 1.确定cPLA2是否在能量引起的细胞损伤中发挥作用 比较过表达cPLA2和cPLA2的LLC-Pk1细胞的损伤耗竭 通过使用寡脱氧核苷酸抑制cPLA2的表达。2要考查 磷酸化在酶激活和易位中的作用。3.至 确定cPLA2的磷酸化位点。4.评估是否 P42MAP激酶与cPLA2在细胞内的结合以及是否有其他 非激酶蛋白与cPLA2相互作用。这些最后的实验将是 使用了一种新颖的双混合交互系统。这里的环境很理想 为顺利完成这项提案而努力。Bonventre医生身体很好 受人尊敬的具有PLA2领域专业知识的资深调查员，CELL 损伤和信号转导。所描述的方法已经在使用中 和多个涉及PLA2的项目正在进行中 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.