REGULATION OF PHOSPHOLIPASE A2 NUCLEAR TARGETING

胞质磷脂酶A2 （cPLA2）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，即类二十烷酸。这是通过脂肪加氧酶和环加氧酶途径产生白三烯和前列腺素。这些花生四烯酸的氧合代谢物在正常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，cPLA2受到复杂的激活机制的影响，而这些机制尚不清楚。cPLA2受磷酸化和细胞内钙水平的翻译后调控。有丝分裂原活化蛋白激酶诱导cPLA2丝氨酸磷酸化Ser505增强cPLA2活性。细胞内钙的增加通过诱导cPLA2从细胞质溶胶转运到核膜/内质网（ER）而触发cPLA2活化，其中5-脂加氧酶和环加氧酶也被定位。钙诱导的cPLA2与膜的结合是由cPLA2氨基端钙依赖性磷脂结合域（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，阐明这些机制是本研究的主要目的。该提案的具体目标包括确定C2结构域和C2结构域下游区域（包括cPLA2的plekstrin同源结构域）在调节核靶向中的作用，鉴定结合cPLA2的蛋白并确定其在调节cPLA2活性和易位中的作用。利用生化方法和噬菌体展示文库筛选鉴定了潜在的cPLA2结合蛋白。cPLA2的结构域在调节核靶向中是重要的，将通过位点定向诱变来确定。突变对激动剂诱导的花生四烯酸释放和cPLA2向细胞核转运的影响将通过在哺乳动物细胞和用杆状病毒在昆虫细胞中的表达来评估。纯化的突变体结构也将评估对酶活性和脂质囊泡结合的影响。了解cPLA2的调控将有助于深入了解脂质介质产生的信号转导机制，并为炎症过程的药理调节提供潜在的方法。

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.