Analysis of a protein kinase which is activated upon fertilization

1.鉴定src2为编码Xyk的基因，用氨基酸测序仪和质谱仪对纯化的Xyk蛋白-酪氨酸激酶的一级结构进行了分析。我们发现Xyk是由非洲爪蛙src2编码的。氨基末端肉豆蔻酰化反应也得到了证实。受精时鸡蛋蛋白的酪氨酸磷酸化Xyk已被证明在受精后1分钟内被激活。随之而来的是，在接下来的几分钟内，许多蛋白质被发现被酪氨酸磷酸化。Src家族蛋白激酶在非洲爪哇卵子激活中的作用金雀异黄素、除草剂A和PP2等抑制Xyk活性的药物均能抑制非洲爪哇受精后的卵子激活。经鉴定，磷脂酶Cγ是Xyk的下游靶标。受精后，磷脂酶Cγ与Xyk结合，在复合体中，磷脂酶Cγ被酪氨酸磷酸化并上调。磷脂酶Cγ的结合和激活被SRC抑制剂PP2.5抑制。H_2O_2激活非洲爪哇卵的孤雌生殖研究发现，在受精过程中，非洲爪哇卵被短暂地暴露在H_2O_2中可以激活卵子。EGG的激活包括Xyk的上调、磷脂酶Cγ的磷酸化和激活、IP_3的产生增加和钙瞬变。这一发现表明，H_2O_2处理可以作为孤雌生殖卵子的激活过程。

1. Identification of src2 as the gene encoding Xyk, a Xenopus Src-family protein-tyrosine kinase Purified Xyk has been analyzed for its primary structure by using amino acid sequencer and mass spectroscopy. We found that Xyk is encoded by the Xenopus src2. The amino-terminal myristoylation was also confirmed.2. Tyrosine-phosphorylation of egg proteins upon fertilizationXyk has been shown to be activated within 1 min after insemination. Concomitantly, many proteins were found to be tyrosine-phosphorylated in the following few minutes.3. Role of the Src-family protein kinases in Xenopus egg activationAll the inhibitors used to suppress Xyk activity, such as genistein, herbimycinA and PP2, were found to inhibit Xenopus egg activation after insemination.4. Identification of phospholipase Cγ as the downstream target of XykAssociation of phospholipase Cγ with Xyk was found after insemination, and in the complex, phospholipase Cγ was tyrosine-phosphorylated and up-regulated. The association and activation of phospholipase Cγ was inhibited by the Src inhibitor PP2.5. Parthenogenetic activation of eggs by H_2O_2A transient exposure of Xenopus eggs to H_2O_2 was found to activate the eggs just as fertilization. The egg activation included up-regulation of Xyk, phosphorylation and activation of phospholipase Cγ, increased IP_3 production, and calcium transient. This finding indicates that H_2O_2 treatment can be used as a parthenogenetic egg activation procedure.