Function of cyclin regulatory factors in cell cycle control.

有丝分裂周期蛋白的降解在有丝分裂的细胞周期进程中起着至关重要的作用。有丝分裂周期蛋白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周期蛋白的n端结构域。以2杂交筛选的cyclin A1的n端为诱饵，我们鉴定出一种爪蟾蛋白XDRP1，它的n端含有一个泛素样结构域，其c -末端50个残基与S.cerevisiae Dsk2具有显著的同源性。XDRP1是爪蟾细胞中的核磷酸化蛋白，其磷酸化是由细胞周期蛋白a依赖性激酶介导的。XDRP1与爪蟾细胞的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。XDRP1的n端泛素样结构域，而不是c端dsk2样结构域，是与细胞周期蛋白A相互作用所必需的。XDRP1需要细胞周期蛋白A1的130-160残基才能有效结合，而不包括细胞周期蛋白A的破坏盒。将细菌表达的XDRP1蛋白添加到蛙卵提取物中，可以抑制Ca2+诱导的细胞周期蛋白A的降解，但对细胞周期蛋白b没有作用。这些结果表明XDRP1是周期蛋白a降解的负调控因子，XDRP1在其n端含有泛素样结构域，并与S.cerevisiae Dsk2具有显著的同源性。基于以上证据，我们研究了XDRP1/Dsk2在酵母系统中泛素依赖性蛋白水解中的功能。当在酵母细胞中过表达时，Dks2引起多泛素化积累。酵母中DSK2毒性的抑制因子被鉴定为蛋白酶体亚基的突变；核心亚基为PRE2，调控亚基为RPN1，表明Dsk2与蛋白酶体存在遗传互作。结果表明，Dsk2在泛素-蛋白酶体降解途径中具有调控作用。

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.