Gijón M A, Spencer D M, Kaiser A L, Leslie C C
Division of Basic Science, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
J Cell Biol. 1999 Jun 14;145(6):1219-32. doi: 10.1083/jcb.145.6.1219.
Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.
胞质型磷脂酶A2（cPLA2）介导激动剂诱导的花生四烯酸释放，这是类花生酸生成的第一步。cPLA2受磷酸化和钙的调节，钙与一个C2结构域结合并诱导其转位至膜上。研究了cPLA2磷酸化位点和C2结构域的功能作用。在表达cPLA2的Sf9昆虫细胞中，冈田酸以及钙动员激动剂A23187和CryIC毒素可诱导花生四烯酸释放以及绿色荧光蛋白（GFP）-cPLA2转位至核膜。cPLA2在Sf9细胞中的多个位点发生磷酸化；然而，只有S505磷酸化部分促进cPLA2的激活。尽管冈田酸不会增加钙，但将钙结合残基D43和D93突变可阻止花生四烯酸释放和cPLA2的转位，这表明需要一个功能性的C2结构域。然而，D93N突变体对A23187具有完全功能，而D43N突变体几乎无活性。与GFP相连的cPLA2的C2结构域在钙动员激动剂作用下转位至核膜，但在冈田酸作用下则不会。因此，当钙增加时，C2结构域对于cPLA2转位至核膜是必要且充分的；然而，在冈田酸作用下它是必需的但不充分。
