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Regulation of the functional activity of Anaphase Promoting Complex

后期促进复合物功能活性的调节

基本信息

批准号：
10680676
负责人：
TANAKA Hirofumi
金额：
$ 2.11万
依托单位：
Tokyo University of Pharmacy and Life Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680676/
关键词：
mammalian cells cell cycle mitosis ubiquitin proteolysis cyclin B 蛋白質分解

项目摘要

Anaphase-promoting complex (APC) functions as a cell cycle-regulated ubiquitin ligase that mediates destruction of cell cycle-regulatory factors such as cyclin B and cut2 by proteasomes dirung mitosis. APC is activated at metaphase-anapgase transition and remains active until late G1 phase. APC consists of 12 subunits at least in mammalian cells, which were recently identified. The APC activity has been thought to be regulated by cell cycle-specific phosphorylation and dephosphorylation of ithe subunits, but the precise regulatory mechanism of APC activation has remained unclear. Furthermore, the binding of APC and its secific ubiquitin conjugating enzyme (UbcH10) has not been reported, and the mechanism of substrate recognition remains unresolved. We identified a novel protein H10BH(UbcH10-binding protein with a hect-like domain) by the yeast two-hybrid system with UbcH10 as a bait. H10BH binds with UbcH10 in vitro and in vivo. Furthermore, H10BH interacts with cyclin B and Cdc27, an APC substrate and an APC component, respectively. H10BH recognizes the cyclin-box in cyclin B by its C-terminal domain.. H10BH greatly enhanced the ubiquitination of cyclin B in the presence of HeLa cell lysates. When H10BH was incubated with ubiquitin, E1, UbcH10 and ubiquitin, the self-ubiquitination of H10BH was observed. H10BH also showed a weak activity of ubiquitin-ligase for cyclin B in the absence of APC. These activities were mediated by the C-terminal hect-like domain of H10BH. Homolgy search of H10BH revealed that Saccharomyces cerevisiae genome contains a homologue of H10BH. Desruption of this gene showed that it is a essential gene for the groth of budding yeasts. These results suggests that H10BH is a novel regulator of APC or a ubiquitin-ligase itself.

后期促进复合体(APC)作为一种细胞周期调节的泛素连接酶，通过有丝分裂过程中的蛋白酶体破坏细胞周期调节因子，如细胞周期蛋白B和Cut2。APC在中期-锐尖相转变时被激活，并一直保持活性直到G1期晚期。APC至少在哺乳动物细胞中由12个亚基组成，这是最近被发现的。APC的活性被认为是通过细胞周期特异性的亚基的磷酸化和去磷酸化来调节的，但APC激活的确切调控机制尚不清楚。此外，APC与其特定的泛素结合酶(UbcH10)的结合还没有报道，底物识别的机制也没有解决。我们以UbcH10为诱饵，通过酵母双杂交系统鉴定了一个新的蛋白质H10BH(具有类似hect结构域的UbcH10结合蛋白)。H10BH在体内外与UbcH10结合。此外，H10BH分别与APC底物Cyclin B和APC组分Cdc27相互作用。H10BH通过其C-末端结构域识别Cyclin B中的Cyclin-box。在HeLa细胞裂解物存在下，H10BH极大地促进了细胞周期蛋白B的泛素化。当H10BH与泛素、E1、UbcH10和泛素孵育时，观察到H10BH的自身泛素化。H10BH在没有APC的情况下，对细胞周期蛋白B的泛素连接酶活性也很弱。这些活性是由H10BH的C端hect样结构域介导的。H10BH的同源性搜索表明，酿酒酵母基因组中含有H10BH的同源物。该基因的克隆表明，该基因是芽殖酵母生长所必需的基因。这些结果表明，H10BH是一种新的APC调节因子或泛素连接酶本身。