Function of hybrid human-yeast cyclin-dependent kinases in Saccharomyces cerevisiae.
Function of hybrid human-yeast cyclin-dependent kinases in Saccharomyces cerevisiae.
复制标题
酿酒酵母中杂交人酵母细胞周期蛋白依赖性激酶的功能。
DOI：
10.1007/s004380050878
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Bitter,GA
中科院分区：
文献类型：
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作者：
Bitter,GA
现在已经确定，真核细胞周期的进程是由细胞周期蛋白依赖性激酶（CDK）活性的振荡控制的。然而，在许多情况下，CDK的生理底物是未知的。SaccharomycesParaePHO 5基因编码一种分泌型酸性磷酸酶，这种磷酸酶在磷饥饿时被诱导。PHO 5基因由PHO 4p转录因子激活，而PHO 4p转录因子本身通过PHO 80和PHO 85产物的磷酸化负调控。Pho 80 p和Pho 85 p分别与细胞周期蛋白和CDK同源，并且Pho 80 p/Pho 85 p异源二聚体满足细胞周期蛋白/CDK的生物化学定义。在本研究中，几个报告基因表达inS.从启动子被激活的转录因子Pho 4p，从而产生的酵母菌株表现出可量化的表型，反映了一个特定的细胞周期蛋白/CDK的活性。用E. coli neo和酵母LEU 2基因。构建了酵母PHO 80和PHO 85基因的染色体断裂，并定义了质粒携带基因互补的条件。互补在Pho 80 p和Pho 85 p的表达水平非常低的情况下实现。Pho 80 p的高水平表达导致异常的PHO 5启动子调节，其特征在于在低磷酸盐培养基中不能去抑制。构建了编码杂合CDK的基因，其中Pho 85 p的区域被人Cdk 2的同源区域取代，并通过互补pho 85染色体基因破坏来测试在链球菌中的功能。其中超过三分之二的分子来自人Cdk 2的杂交蛋白保留了PHO 85 p的功能，相对于PHO 5启动子的高磷酸盐抑制。杂合蛋白需要PHO 80基因产物来实现这一功能。在Pho 85 p和人Cdk 2中完全保守的九肽序列内缺失单个氨基酸的杂合体人-酵母CDK保留了全部功能。这些结果表明，在CDK的保守结构的背景下，可以引入相当大的一级序列变异性而不损失CDK的细胞周期蛋白依赖性功能。
It is now well established that progression through the eukaryotic cell cycle is controlled by oscillations in the activity of cyclin- dependent kinases (CDKs). In many cases, however, the physiological substrate(s) of CDKs are unknown. TheSaccharomyces cerevisiaePHO5gene encodes a secreted acid phosphatase which is induced in response to phosphate starvation. ThePHO5gene is activated by the Pho4p transcription factor, which itself is negatively regulated through phosphorylation by the products ofPHO80andPHO85. Pho80p and Pho85p are homologous to cyclins and CDKs, respectively, and the Pho80p/Pho85p heterodimer satisfies the biochemical definition of a cyclin/CDK. In the present study, several reporter genes were expressed inS. cerevisiaefrom promoters which are activated by the transcription factor Pho4p, thereby generating yeast strains which exhibit quantifiable phenotypes that reflect the activity of a specific cyclin/CDK. Positive genetic selections for inhibition of cyclin/CDK function were characterized using theE. coli neoand yeastLEU2genes. Chromosomal disruptions of the yeastPHO80andPHO85genes were constructed and conditions for complementation by plasmid-borne genes were defined. Complementation is achieved at very low levels of expression of both Pho80p and Pho85p. High-level expression of Pho80p results in aberrantPHO5promoter regulation, characterized by failure to derepress in low-phosphate medium. Genes encoding hybrid CDKs in which regions of Pho85p were replaced with the homologous region of human Cdk2 were constructed, and tested for function inS. cerevisiaeby complementation of thepho85chromosomal gene disruption. Hybrid proteins in which more than two-thirds of the molecule were derived from human Cdk2 retained Pho85p function with respect to high-phosphate repression of thePHO5promoter. The hybrid proteins require thePHO80gene product for this function. A hybrid human-yeast CDK in which a single amino acid is deleted, within a nonapeptide sequence which is perfectly conserved in Pho85p and human Cdk2, retains full function. These results demonstrate that, within the context of the conserved structure of CDKs, considerable primary sequence variability can be introduced without loss of the cyclin-dependent function of the CDK.