Regulatory Mechanism of M-phase by MPF,M-phase Promoting Factor

MPF、M相促进因子对M相的调节机制

• 批准号: 03405004
• 负责人: KISHIMOTO Takeo
• 金额: $ 14.02万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03405004/
• 关键词: M-phase; MPF; cdc2 kinase; cdc2; cyclin B complex; sucl-affinity chromatography; nuclear transport; chromosome condensation; mitotic apparatus; ヒストンH1; ヒトデ卵; アフリカツメガエル卵; 微小管; 中間径繊維; MAP; suc1産物; ビメンチンキナーゼ; MTOC; suc1遺伝子

MPF (M-phase promoting factor), which is composed of the p34^<cdc2>/cyclin B complex, cdc2 kinase, governs M-phase in all eukaryotic cells. In this project, we studied how M-phase is executed by cdc2 kinase, and obtained following results.1.We developed a new method to purify cdc2 kinase at high performance by modifying p13^<sucl>-affinity chromatography. This enabled us to analyze the function of cdc2 kinase in inducing various M-phase events.2.At G2/M-phase border, the inactive form of p34^<cdc2>/cyclin B complex localizes in the cytoplasm. The complex is transformed to active form in the cytoplasm at the transition to M-phase. Thereafter, a part of the complex moves into nucleus, while other associates with mitotic apparatus. These specific localizations of the complex might support its specific function to execute M-phase within a cell.3.In contrast to the previous belief that histone H1 phosphorylation by cdc2 kinase plays a key role in chromosome condensation, condensation occurs normally even in the chromatins lacking histone H1. This suggests the presence of an unknown major target of cdc2 kinase for chromosome condensation other than histone H1.4.The p34^<cdc2/>cyclin B complex associates with a microtubule via the binding between cyclin B component and the prolin-rich region of MAP4. In such a complex, cdc2 kinase phosphorylates MAP4 to decrease its microtubule-stabilizing ability, resulting in increased dynamic instability of an individual microtubule. This effect of cdc2 kinase appears to contribute to form metaphase spindle.

MPF（M期促进因子）由p34 β<cdc2>/细胞周期蛋白B复合物、cdc 2激酶组成，在所有真核细胞中控制M期。本课题主要研究了cdc 2激酶如何进入M期，并取得了以下研究成果：1.建立了一种新的纯化cdc 2激酶的方法，即改进的p13^<sucl>-亲和层析法。2.在G2/M期交界处，p34^ /cyclin B复合物的失活形式<cdc2>定位于细胞质中。该复合物在向M期过渡时在细胞质中转化为活性形式。随后，一部分复合体进入细胞核，另一部分则与有丝分裂器结合。这些特异性定位可能支持其在细胞内执行M期的特异性功能。3.与以前认为的cdc 2激酶磷酸化组蛋白H1在染色体浓缩中起关键作用相反，即使在缺乏组蛋白H1的染色质中也正常发生浓缩。这表明除了组蛋白H1.4外，cdc 2激酶还存在一个未知的主要作用靶点。p34 → cyclin B复合物通过cyclin B组分与MAP 4富含脯氨酸的区域结合而与微管结合。在这样的复合物中，cdc 2激酶磷酸化MAP 4以降低其微管稳定能力，导致单个微管的动态不稳定性增加。cdc 2激酶的这种作用似乎有助于形成中期纺锤体。

项目成果

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Kusubata, M.,et al.：“在体外，P13 suc1 抑制 p34 cdc2 激酶对中间丝蛋白的催化功能。”

Ohta,K.: "Increase of microtubule nucleating activity of centrosome in cellーfree extracts from Xenopus eggs and its regulation by protein phosphorylation." Proc.Natl.Acad.Sci.USA.89. (1992)

Ohta, K.：“非洲爪蟾卵无细胞提取物中中心体微管成核活性的增加及其通过蛋白质磷酸化的调节。”（Proc.Natl.Acad.Sci.USA.89）。

Ando,S.,et al.: "Phosphorylation of synthetic vimentin peptides by cdc2 kinase." Biochem.Biophys.Res.Commun.195. 837-843 (1993)

Ando,S.,et al.：“cdc2 激酶对合成波形蛋白肽进行磷酸化。”

Hisanaga,S.,et al.: "Phosphorylation of neurofilament H subunit at the tail domain by CDC2..." J.Biol.Chem.266. 21798-21803 (1991)

Hisanaga,S.,et al.：“CDC2 尾部结构域神经丝 H 亚基的磷酸化……”J.Biol.Chem.266。