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Function of cyclin regulatory factors in cell cycle control.

细胞周期蛋白调节因子在细胞周期控制中的功能。

基本信息

批准号：
11694218
负责人：
KOBAYASHI Hideki
金额：
$ 3.33万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The degradation of mitotic cyclins plays an essential role in cell-cycle progression during mitosis. Mitotic cyclins A and B are degraded by ubiquitin-dependent proteolysis and its process requires the N-terminal domain of mitotic cyclins. Using the N-terminus of cyclin A1 in a 2-hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to S.cerevisiae Dsk2. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A.XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A.The addition of bacterially-expressed XDRP1 protein to frog egg extract inhibited the Ca2+-induced degradation of cyclin A, but not that of cyclin B.The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division. These results suggest that XDRP1 is a negative regulator of degradation of cyclin A.XDRP1 contains a ubiquitin-like domain in its N-terminus and shows significant homology to S.cerevisiae Dsk2. Based on above evidence, we investigate the function of XDRP1/Dsk2 on ubiquitin-dependent proteolysis in yeast system. When overexpressed in yeast cells, Dks2 caused to accumulate polyubiquitination. Supprcssors of DSK2 toxicity in yeast were identified as mutations in proteasome subunits ; PRE2 in core subunit, and RPN1 in regulatory subunit, indicating genetic interaction between Dsk2 and proteasome. The results suggest that Dsk2 have a regulatory role in ubiquitin-proteasome degradation pathway.

有丝分裂周期蛋白的降解在有丝分裂期间的细胞周期进程中起重要作用。有丝分裂细胞周期蛋白A和B通过泛素依赖的蛋白水解降解，其过程需要有丝分裂细胞周期蛋白的N-末端结构域。使用的N-末端的细胞周期蛋白A1作为诱饵，在一个2-杂交屏幕，我们确定了非洲爪蟾蛋白，XDRP 1，它包含一个泛素样结构域在其N-末端，并显示出显着的同源性在其C-末端50个残基的酿酒酵母Dsk 2。XDRP 1是非洲爪蟾细胞核中的一种磷蛋白，其磷酸化由细胞周期蛋白A依赖性激酶介导。XDRP 1在非洲爪蟾细胞中与胚胎和体细胞形式的细胞周期蛋白A（A1和A2）结合，但不与B型细胞周期蛋白结合。XDRP 1的N-末端泛素样结构域（而不是C-末端Dsk 2-like结构域）是与细胞周期蛋白A相互作用所必需的。XDRP 1需要细胞周期蛋白A 1的130-160位残基进行有效结合，而这些残基不包括细胞周期蛋白A的破坏盒。将细菌表达的XDRP 1蛋白添加到蛙卵提取物中抑制Ca 2+诱导的细胞周期蛋白A的降解，而不是细胞周期蛋白B。将XDRP 1蛋白注射到受精的非洲爪蟾卵中阻止了胚胎细胞分裂。这些结果表明，XDRP 1是细胞周期蛋白A降解的负调控因子，其N端含有泛素样结构域，与酿酒酵母Dsk 2具有明显的同源性。基于以上证据，我们研究了XDRP 1/Dsk 2在酵母系统中泛素依赖的蛋白水解中的作用。当在酵母细胞中过表达时，Dks 2引起多聚泛素化的积累。DSK 2在酵母中的毒性抑制因子被鉴定为蛋白酶体亚基的突变，核心亚基的PRE 2和调节亚基的RPN 1，表明DSK 2和蛋白酶体之间存在遗传相互作用。结果提示Dsk 2在泛素-蛋白酶体降解途径中具有调控作用。