Activation of the Bur1-Bur2 Cyclin-Dependent Kinase Complex by Cak1
Activation of the Bur1-Bur2 Cyclin-Dependent Kinase Complex by Cak1
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DOI：
10.1128/mcb.22.19.6750-6758.2002
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发表时间：
2002-10
影响因子：
5.3
通讯作者：
Shengli Yao;G. Prelich
中科院分区：
文献类型：
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作者：
Shengli Yao;G. Prelich
摘要细胞周期蛋白依赖性蛋白激酶(CDK)最初被认为是真核细胞周期进程的调节因子，但后来发现几种CDK在转录调控中发挥重要作用。虽然参与细胞周期进程的CDK的调控机制已经被很好地记录下来，但对于参与转录的CDK是如何调控的却知之甚少。在酿酒酵母中，Bur1和Bur2组成一个CDK复合体，参与转录调控，可能是通过其对RNA聚合酶II最大亚基的羧基末端结构域(CTD)的磷酸化而介导的。为了研究Bur1在体内的调控，我们寻找了Bur1温度敏感突变的高拷贝数抑制子，鉴定了单个基因CAK1。已知Cak1通过磷酸化保守的T-环区中的苏氨酸来激活酵母中的另外两个CDK。Bur1在其T环中也有保守的苏氨酸，因此是Cak1的潜在直接靶点。其他试验建立了Cak1与Bur1-Bur2 CDK复合体之间的直接功能相互作用：Bur1在体内被磷酸化，保守的Bur1 T-loop苏氨酸和Cak1都是体内磷酸化和Bur1功能所必需的，以及重组Cak1在体外刺激纯化的Bur1-Bur2复合体的CTD激酶活性。因此，遗传和生化证据都表明，Cak1是Bur1激酶的生理调节因子。
ABSTRACT Cyclin-dependent kinases (Cdks) were originally identified as regulators of eukaryotic cell cycle progression, but several Cdks were subsequently shown to perform important roles as transcriptional regulators. While the mechanisms regulating the Cdks involved in cell cycle progression are well documented, much less is known regarding how the Cdks that are involved in transcription are regulated. In Saccharomyces cerevisiae, Bur1 and Bur2 comprise a Cdk complex that is involved in transcriptional regulation, presumably mediated by its phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. To investigate the regulation of Bur1 in vivo, we searched for high-copy-number suppressors of a bur1 temperature-sensitive mutation, identifying a single gene, CAK1. Cak1 is known to activate two other Cdks in yeast by phosphorylating a threonine within their conserved T-loop domains. Bur1 also has the conserved threonine within its T loop and is therefore a potential direct target of Cak1. Additional tests establish a direct functional interaction between Cak1 and the Bur1-Bur2 Cdk complex: Bur1 is phosphorylated in vivo, both the conserved Bur1 T-loop threonine and Cak1 are required for phosphorylation and Bur1 function in vivo, and recombinant Cak1 stimulates CTD kinase activity of the purified Bur1-Bur2 complex in vitro. Thus, both genetic and biochemical evidence demonstrate that Cak1 is a physiological regulator of the Bur1 kinase.