Intergenic complementation truncation mutants of cyclin-dependent kinase.
Intergenic complementation truncation mutants of cyclin-dependent kinase.
复制标题
细胞周期蛋白依赖性激酶的基因间互补截短突变体。
DOI：
10.1007/s004380051163
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Leong,K
中科院分区：
文献类型：
--
作者：
Bitter,GA;Tsai,MM;Putzke,AP;Leong,K
酿酒酵母PHO80和PHO85分别编码一种细胞周期蛋白和细胞周期蛋白依赖性的蛋白，通过磷酸化转录激活因子Ph4p负向调节PHO5基因的转录。细胞周期蛋白依赖性蛋白激酶(CDK)是一种高度保守的蛋白质，在物种内和物种间都是如此。以前利用由Pho4p在酵母中激活的报告基因证明，超过三分之二的Pho85p被人CDK2的同源区取代的杂交蛋白在启动子抑制方面保留了天然Pho85p的功能。在本研究中，对不同截短形式的人-酵母杂交CDK进行了功能测试。令人惊讶的是，291-残基杂合CDK的大部分C-末端区域被删除的截断仍然具有活性。编码人类CDK2蛋白的基因在151、140、130、120和90位氨基酸后终止，补充了染色体85基因的中断，其中HIS3基因插入在49密码子上。截短的CDK2蛋白含有不到60个氨基酸，不能补充ph85：：HIS3基因的中断。虽然功能上的C末端截断破坏了CDK2的ATP结合和活性部位，但这些截短的蛋白所介导的报告基因抑制显然是由于Pho4p的磷酸化，因为其中第33位的必需赖氨酸密码子被转换为精氨酸密码子的基因不能补充染色体基因的破坏。人类CDK2基因的截短是通过基因间互补发挥作用的。完整的CDK2-Pho85杂合CDK在酵母菌株中补充了PHO85突变，其中PHO85编码区从XVI染色体上缺失。然而，在这些菌株中，C端的CDK2截断是不起作用的，因此依赖于保留在突变85：：HIS3染色体座位上的ph85编码区的活性。这些遗传结果与蛋白质片段互补模型一致，在该模型中，CDK的活性部位被一分为二。
TheSaccharomyces cerevisiaegenesPHO80andPHO85encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulatePHO5gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomalpho85gene disruption in which theHIS3gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement thepho85::HIS3gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented thepho85mutation in yeast strains in which the entirePHO85coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on thepho85coding region which remained in the mutantpho85::HIS3chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.