REGULATION OF PHOSPHOLIPASE A2 NUCLEAR TARGETING

胞浆磷脂酶A2（cPLA 2）是介导激动剂诱导的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，也可以转化为多种有效的脂质代谢物，类花生酸。 这通过脂氧合酶和环氧合酶途径发生，用于产生白三烯和白藜芦醇。 花生四烯酸的这些含氧代谢产物在正常的体内平衡和介导炎症反应中起着至关重要的作用。 花生四烯酸的细胞内水平受到严格控制，cPLA 2受到复杂的激活机制的影响，这些机制尚不清楚。cPLA 2通过磷酸化和细胞内钙水平进行后调节。 激动剂诱导的cPLA 2在Ser 505上的丝氨酸磷酸化通过丝裂原活化蛋白激酶增强cPLA 2活性。 细胞内钙的增加通过诱导cPLA 2从细胞质移位到核膜/内质网（ER）来触发cPLA 2活化，其中5-脂氧合酶和环氧合酶也位于核膜/内质网中。钙诱导的cPLA 2与膜的结合由cPLA 2氨基末端上的钙依赖性磷脂结合结构域（CaLB或C2结构域）介导。 尽管CaLB结构域在膜结合中的作用已经确定，但cPLA 2特异性靶向核膜/ER所涉及的机制尚不清楚，对其的阐明是本提案的主要目标。 该提案的具体目的包括定义C2结构域和C2结构域下游区域（包括cPLA 2的plekstrin同源结构域）在调节核靶向中的作用，并鉴定结合cPLA 2的蛋白质并确定其在调节cPLA 2活性和易位中的作用。 已经使用生物化学方法和筛选噬菌体展示文库鉴定了潜在的cPLA 2结合蛋白。 将使用定点诱变鉴定在调节核靶向中重要的cPLA 2结构域。 突变对激动剂诱导的花生四烯酸释放和cPLA 2易位至细胞核的影响将通过使用杆状病毒在哺乳动物细胞和昆虫细胞中表达来评价。 还将评价纯化的突变体构建体对酶活性和脂质囊泡结合的影响。 了解cPLA 2的调节将提供更深入的了解参与脂质介质生产的信号转导机制，并导致炎症过程的药理学调节的潜在方法。

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.