REGULATION OF PHOSPHOLIPASE A2 NUCLEAR TARGETING

胞浆磷脂酶A2(CPLA2)是介导激动剂诱导花生四烯酸释放的关键调节酶。花生四烯酸本身是一种重要的细胞内调节剂，也可以转化为各种有效的脂类代谢物，二十烷基类化合物。这是通过脂氧合酶和环氧合酶途径来产生白三烯和前列腺素。这些花生四烯酸的氧化代谢物在正常的体内平衡和调节炎症反应中起着关键作用。细胞内花生四烯酸水平受到严格控制，cPLA2受到复杂的激活机制的影响，但人们对此知之甚少。CPLA2在翻译后受磷酸化和细胞内钙水平的调节。激动剂通过丝裂原活化蛋白激酶诱导Ser505上cPLA2的丝氨酸磷酸化增强cPLA2的活性。细胞内钙的增加通过诱导cPLA2从胞浆移位到核膜/内质网(ER)来触发cPLA2的激活，核膜/内质网也定位于5-脂氧合酶和环氧合酶。钙诱导的cPLA2与膜的结合是由cPLA2氨基末端的钙依赖的磷脂结合域(CalB或C2结构域)介导的。虽然Calb结构域在膜结合中的作用已经确定，但cPLA2特异性靶向核膜/内质网的机制尚不清楚，阐明它们是本提案的主要目的。该提案的具体目的包括确定C2结构域及其下游区域(包括cPLA2的plekstrin同源结构域)在调节核靶向中的作用，以及识别与cPLA2结合的蛋白质，并确定它们在调节cPLA2活性和易位中的作用。利用生化方法和筛选噬菌体展示文库，已经鉴定了潜在的cPLA2结合蛋白。将使用定点突变来确定在调节核靶向方面重要的cPLA2结构域。这些突变对激动剂诱导的花生四烯酸释放和cPLA2转位到细胞核的影响将通过在哺乳动物细胞和昆虫细胞中使用杆状病毒进行表达来评估。还将评估纯化的突变构建体对酶活性和脂泡结合的影响。了解cPLA2的调控将有助于更深入地了解参与脂质介质产生的信号转导机制，并为炎症过程的药物调控提供潜在的方法。

Cytosolic phospholipase A2 (cPLA2) is a key regulatory enzyme that mediates agonist-induced arachidonic acid release. Arachidonic acid itself is an important intracellular regulator and can also be converted to a variety of potent lipid metabolites, the eicosanoids. This occurs through the lipoxygenase and cyclooxygenase pathways for the production of leukotrienes, and prostaglandins. These oxygenated metabolites of arachidonic acid play crucial roles in normal homeostasis and in mediating the inflammatory response. Intracellular levels of arachidonic acid are tightly controlled and cPLA2 is subject to complex mechanisms of activation that are poorly understood. cPLA2 is post-translationally regulated by phosphorylation and by levels of intracellular calcium. Agonist-induced serine phosphorylation of cPLA2 on Ser505 by mitogen activated protein kinase enhances cPLA2 activity. An increase in intracellular calcium triggers cPLA2 activation by inducing its translocation from cytosol to the nuclear envelope/endoplasmic reticulum (ER), where 5-lipoxygenase and cyclooxygenase are also localized. Calcium-induced binding of cPLA2 to membrane is mediated by the calcium-dependent phospholipid binding domain (CaLB or C2 domain) on the amino terminus of cPLA2. Although the role of the CaLB domain in membrane binding is established, the mechanisms involved in the specific targeting of cPLA2 to nuclear membrane/ER are not known, and their elucidation is the main objective of this proposal. The specific aims of the proposal include defining the role of the C2 domain and regions downstream of the C2 domain (including the plekstrin homology domain of cPLA2) in regulating nuclear targeting, and identifying proteins that bind cPLA2 and determining their role in regulating cPLA2 activity and translocation. Potential cPLA2 binding proteins have been identified using a biochemical approach and screening a phage display library. Domains of cPLA2 that are important in regulating nuclear targeting will be identified using site-directed mutagenesis. Effects of the mutations on agonist-induced arachidonic acid release and translocation of cPLA2 to the nucleus will be evaluated by expression in mammalian cells and in insect cells using baculovirus. Purified mutant constructs will also be evaluated for effects on enzymatic activity and lipid vesicle binding. Understanding the regulation of cPLA2 will provide greater insight into the signal transduction mechanisms involved in lipid mediator production, and lead to potential methods for pharmacological modulation of the inflammatory process.