Regulatory Factors of Cyclin Functions in the Cell Cycle

细胞周期中细胞周期蛋白功能的调控因素

基本信息

批准号：
08044213
负责人：
KOBAYASHI Hideki
金额：
$ 6.14万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

According to the proposed research plan, Hideki Kobayashi, Kyushu University in Japan, carried out the collaboration on the cyclin functions in the cell cycle, with Dr.Tim Hunt at ICRF Clare Hall Laboratories, and Dr.Mark Carrington at Cambridge University, England. Hideki Kobayashi isolated Xenopus cyclin A-binding protein by yeast 2-hybrid screen and identified the novel protein THF4 that binds to the N-terminus of cyclin A and inhibits its degradation selectively. Tim Hunt analyzed its biochemical properties on the cell cycle in Xenopus egg extracts. Mark Carrington has isolated murine homolog of THF4 and identified its binding domain to cyclin A.In summary, the N-terminal domain of Xenopus cyclin A1 was used in a 2-hybrid screen to identify proteins that interact with cyclin A.The major interacting species was THF4, a protein that contains a ubiquitin-like domain in its N-terminus shows a significant homology in the C-terminus of S.cerevisiae Dsk2 and S.porn be dph1. THF4 persists … More in Xenopus cells as a nuclear phosphoprotein and its phosphorylation is induced by cyclin A-dependent kinase. THF4 binds to both embryonic and somatic forms of cyclin A (Al and A2) in vivo and in vitro, but not to B-type cyclins. The N-terminal u1biquitin-like domain of THF4, but not the C-temimal conserved domain, is required for interaction with cyclin A.THF4 requires residues 130-160 of cyclin Al for efficient binding, which do not include the destruction box of cyclin A.The addition of bacterially-expressed THF4 protein to_frog egg extract inhibited the Ca2+-induced degradation of cyclin A, but not that of cyclin B.The injection of THF4 protein into the fertilized egg blocked the embryonic cell division.These results suggest that THF4 is a candidate substrate for cyclin A-dependent kinase and that THF4 may act as a negative regulator of cyclin A degradation in mitosis.These results would give a new insight into cyclin proteolysis in the cell cycle This collaborative research project during three years (1996-1998) was quite successful, and we will continue the collaborative research more. Less

根据拟议的研究计划，日本九州大学的Hideki Kobayashi与ICRF Clare Hall实验室的Tim Hunt博士和英国剑桥大学的Mark Carrington博士就细胞周期中的细胞周期蛋白功能进行了合作。 Hideki Kobayashi通过酵母2-杂交筛选分离了非洲爪蟾细胞周期蛋白A结合蛋白，并鉴定了与细胞周期蛋白A N末端结合的新蛋白THF4， Tim Hunt 分析了非洲爪蟾卵提取物中其对细胞周期的生化特性，Mark Carrington 分离了 THF4 的小鼠同源物，并鉴定了其与细胞周期蛋白 A1 的结合域。总之，非洲爪蟾细胞周期蛋白 A1 的 N 末端结构域是用于 2 杂交筛选，以鉴定与细胞周期蛋白 A 相互作用的蛋白质。主要的相互作用种类是 THF4，一种在其内部包含泛素样结构域的蛋白质S.cerevisiae Dsk2 和 S.porn be dph1 的 N 末端显示出显着的同源性，THF4 作为核磷蛋白持续存在于非洲爪蟾细胞中，其磷酸化是由细胞周期蛋白 A 依赖性激酶结合诱导的。在体内和体外，细胞周期蛋白A（A1和A2）的胚胎和体细胞形式均受影响，但不包括B型细胞周期蛋白。与细胞周期蛋白 A 相互作用需要 THF4 的 N 端 u1biquitin 样结构域，而不是 C 端保守结构域。THF4 需要细胞周期蛋白 A 的残基 130-160 才能有效结合，其中不包括细胞周期蛋白 A 的破坏盒.向青蛙卵提取物中添加细菌表达的THF4蛋白可以抑制Ca2+诱导的细胞周期蛋白A的降解，但不能抑制细胞周期蛋白A的降解。细胞周期蛋白 B。将 THF4 蛋白注射到受精卵中可阻断胚胎细胞分裂。这些结果表明，THF4 是细胞周期蛋白 A 依赖性激酶的候选底物，并且 THF4 可能在有丝分裂中充当细胞周期蛋白 A 降解的负调节因子。这些结果将为细胞周期中的细胞周期蛋白蛋白水解提供新的见解。这个为期三年（1996-1998）的合作研究项目非常成功，我们将继续合作研究较少。