Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p.
Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p.
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Cak1p 激活酵母 TFIIH Kin28p 亚基的磷酸化。
DOI：
10.1128/mcb.19.7.4774
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发表时间：
1999
影响因子：
5.3
通讯作者：
Solomon,MJ
中科院分区：
文献类型：
--
作者：
Kimmelman,J;Kaldis,P;Hengartner,CJ;Laff,GM;Koh,SS;Young,RA;Solomon,MJ
细胞周期蛋白依赖性激酶(CDK)激活蛋白激酶(CAKs)对CDKs如CDc2和CDK2进行必要的激活磷酸化。哺乳动物CAK的催化亚基MO15/CDK7也是一般转录因子TFIIH的一个亚基。然而，在萌芽酵母中，这些功能是分开的，其中Kin28p作为TFIIH的激酶亚基，而Cak1p作为CAK。我们发现，Kin28p本身就是一个CDK，它也包含一个激活Thr-162上的磷酸化位点。与野生型KIN28P相比，KIN28P的T162A型突变体的激酶活性降低了75%~80%。此外，含有Kin28T162A和TFB3条件等位基因(哺乳动物MAT1蛋白的同源基因，MO15和Cyclin H的组装因子)的细胞受到严重损害，表现出Kin28p活性的进一步显著降低。这一发现在体内支持了先前的生化观察，即MO15-Cyclin H复合体可以通过激活MO15的磷酸化或通过与MAT1结合来激活。最后，我们发现，在体内失活Cak1p后，Kin28p在Thr-162上不再被磷酸化，而Cak1p在体外可以磷酸化Thr-162上的Kin28p，并且这种磷酸化刺激了Kin28p的CTD激酶活性。因此，Kin28p与芽期酵母中的主要细胞周期CDK--CDC28p结合，成为一种生理的Cak1p底物。这些发现表明，尽管MO15和Cak1p构成不同形式的CAK，但两者都控制着细胞周期和TFIIH对RNA聚合酶II大亚基C末端结构域的磷酸化。
Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2. The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the general transcription factor TFIIH. However, these functions are split in budding yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p functions as a CAK. We show that Kin28p, which is itself a CDK, also contains a site of activating phosphorylation on Thr-162. The kinase activity of a T162A mutant of Kin28p is reduced by ∼75 to 80% compared to that of wild-type Kin28p. Moreover, cells containingkin28T162Aand a conditional allele ofTFB3(the ortholog of the mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are severely compromised and display a significant further reduction in Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1. Finally, we show that Kin28p is no longer phosphorylated on Thr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylate Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major cell cycle Cdk in budding yeast, as a physiological Cak1p substrate. These findings indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II by TFIIH.