Analysis of a protein kinase which is activated upon fertilization

1. Src 2为编码非洲爪蟾Src家族蛋白酪氨酸激酶Xyk的基因的鉴定我们发现Xyk是由非洲爪蟾src 2编码的。氨基末端的豆蔻酰化也得到了证实.受精后卵蛋白的酪氨酸磷酸化Xyk在受精后1分钟内被激活。同时，在随后的几分钟内发现许多蛋白质被酪氨酸磷酸化. Src家族蛋白激酶在爪蟾卵激活中的作用抑制Xyk活性的抑制剂，如染料木素、除草霉素A和PP2，在抑制后均能抑制爪蟾卵的激活.磷脂酶Cγ是Xyk下游靶蛋白的鉴定受精后发现磷脂酶Cγ与Xyk结合，在复合物中，磷脂酶Cγ被酪氨酸磷酸化并上调。Src抑制剂PP2.5可抑制磷脂酶Cγ的结合和活化。H_2O_2对卵的孤雌激活作用用H_2O_2对非洲爪蟾卵进行短暂的处理，可以激活卵，就像受精一样。卵的激活包括Xyk蛋白表达上调、磷脂酶Cγ磷酸化和激活、IP_3产生增加和钙瞬变。这一结果表明H_2O_2处理可以作为孤雌激活的一种方法。

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.