Abassi Y A, Carroll D J, Giusti A F, Belton R J, Foltz K R
Department of Molecular, Cellular and Developmental Biology and the Marine Science Institute, University of California at Santa Barbara, Santa Barbara, California 93106, USA.
Dev Biol. 2000 Feb 15;218(2):206-19. doi: 10.1006/dbio.1999.9582.
The initiation of Ca(2+) release from internal stores in the egg is a hallmark of egg activation. In sea urchins, PLCgamma activity is necessary for the production of IP(3), which leads to the initial rise in Ca(2+). To examine the possible function of a tyrosine kinase in activating PLCgamma at fertilization, sea urchin eggs were treated with the specific Src kinase inhibitor PP1 or microinjected with recombinant Src-family SH2-domain proteins, which act as dominant interfering inhibitors of Src-family kinase function. Both modes of inhibiting Src-family kinases resulted in a specific and dose-dependent delay in the onset of Ca(2+) release from the endoplasmic reticulum at fertilization. The rise in cytoplasmic pH at fertilization also was inhibited by microinjection of Src-family SH2-domain proteins. Further, an antibody directed against Src-type kinases recognized a protein of ca. M(r) 57K that was enriched in the membrane fraction of eggs. The kinase activity of this protein was stimulated rapidly and transiently at fertilization, as measured by autophosphorylation and by phosphorylation of an exogenous substrate. Together, these data indicate that a Src-type tyrosine kinase is necessary for the initiation of Ca(2+) release from the egg ER at fertilization and identify a Src-type p57 protein as a candidate in the signaling pathway leading to this Ca(2+) release.
卵子内部储存库中Ca(2+)释放的启动是卵子激活的一个标志。在海胆中，PLCγ活性对于IP(3)的产生是必需的，而IP(3)会导致Ca(2+)的初始升高。为了研究酪氨酸激酶在受精时激活PLCγ的可能功能，用特异性Src激酶抑制剂PP1处理海胆卵子，或向其中显微注射重组Src家族SH2结构域蛋白，这些蛋白可作为Src家族激酶功能的显性干扰抑制剂。抑制Src家族激酶的这两种方式都导致受精时内质网Ca(2+)释放起始出现特异性的剂量依赖性延迟。显微注射Src家族SH2结构域蛋白也会抑制受精时细胞质pH的升高。此外，一种针对Src型激酶的抗体识别出一种约M(r) 57K的蛋白，该蛋白在卵子的膜部分富集。通过自身磷酸化和对外源底物的磷酸化测定，该蛋白的激酶活性在受精时迅速且短暂地受到刺激。这些数据共同表明，Src型酪氨酸激酶对于受精时卵子内质网Ca(2+)释放的启动是必需的，并确定一种Src型p57蛋白是导致这种Ca(2+)释放的信号通路中的一个候选蛋白。
