| Project/Area Number |
10680653
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | KOBE UNIVERSITY |
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Principal Investigator |
FUKAMI Yasuo Kobe University Laboratory of Molecular Biology, Associate Professor, 遺伝子実験施設, 助教授 (00156746)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Ken-ichi Kobe University Laboratory of Molecular Biology, Assistant Professor, 遺伝子実験施設, 助手 (30235337)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Fertilization / Egg activation / Protein phosphorylation / Tyrosine kinases / Signal transduction / Xenopus / Src / Phospholipase Cγ / 蛋白質チロシンリン酸化 / 卵細胞膜ラフト / 胚発生 / ジグナル伝達 / 蛋白質リン酸化 / カルシウムシグナル |
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| Research Abstract |
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 fertilization
Xyk 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 activation
All 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 Xyk
Association 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_2
A 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.
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