Runft Linda L, Carroll David J, Gillett Jessica, Giusti Andrew F, O'Neill Forest J, Foltz Kathy R
Department of Molecular, Cellular & Developmental Biology and the Marine Science Institute, University of California-Santa Barbara, Santa Barbara, CA 93106-9610, USA.
Dev Biol. 2004 May 1;269(1):220-36. doi: 10.1016/j.ydbio.2004.01.031.
At fertilization, eggs undergo a cytoplasmic free Ca2+ rise, which is necessary for stimulating embryogenesis. In starfish eggs, studies using inhibitors designed against vertebrate proteins have shown that this Ca2+ rise requires an egg Src family kinase (SFK) that directly or indirectly activates phospholipase C-gamma (PLC-gamma) to produce IP3, which triggers Ca2+ release from the egg's endoplasmic reticulum (ER) [reviewed in Semin. Cell Dev. Biol. 12 (2001) 45]. To examine in more detail the endogenous factors in starfish eggs that are required for Ca2+ release at fertilization, an oocyte cDNA encoding PLC-gamma was isolated from the starfish Asterina miniata. This cDNA, designated AmPLC-gamma, encodes a protein with 49% identity to mammalian PLC-gamma1. A 58-kDa Src family kinase interacted with recombinant AmPLC-gamma Src homology 2 (SH2) domains in a specific, fertilization-responsive manner. Immunoprecipitations of sea urchin egg PLC-gamma using an affinity-purified antibody directed against AmPLC-gamma revealed fertilization-dependent phosphorylation of PLC-gamma. Injecting starfish eggs with the tandem SH2 domains of AmPLC-gamma (which inhibits PLC-gamma activation) specifically inhibited Ca2+ release at fertilization. These results indicate that an endogenous starfish egg PLC-gamma interacts with an egg SFK and mediates Ca2+ release at fertilization via a PLC-gamma SH2 domain-mediated mechanism.
在受精过程中，卵子会经历细胞质游离钙离子浓度升高，这对于刺激胚胎发育是必需的。在海星卵子中，使用针对脊椎动物蛋白设计的抑制剂进行的研究表明，这种钙离子浓度升高需要一种卵子Src家族激酶（SFK），它直接或间接激活磷脂酶C-γ（PLC-γ）以产生IP3，IP3触发卵子内质网（ER）释放钙离子[见《细胞与发育生物学综述》12卷（2001年）第45页]。为了更详细地研究海星卵子受精时钙离子释放所需的内源性因子，从微小海星（Asterina miniata）中分离出一个编码PLC-γ的卵母细胞cDNA。这个cDNA，命名为AmPLC-γ，编码一种与哺乳动物PLC-γ1有49%同源性的蛋白质。一种58 kDa的Src家族激酶以一种特定的、对受精有反应的方式与重组的AmPLC-γSrc同源2（SH2）结构域相互作用。使用针对AmPLC-γ的亲和纯化抗体对海胆卵子PLC-γ进行免疫沉淀，结果显示PLC-γ存在受精依赖性磷酸化。向海星卵子注射AmPLC-γ的串联SH2结构域（可抑制PLC-γ激活）能特异性抑制受精时的钙离子释放。这些结果表明，内源性海星卵子PLC-γ与卵子SFK相互作用，并通过PLC-γ SH2结构域介导的机制在受精时介导钙离子释放。
