CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1
CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1
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DOI：
10.1101/gad.1968210
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发表时间：
2010-10-15
影响因子：
10.5
通讯作者：
Greenleaf, Arno L.
中科院分区：
文献类型：
--
作者：
Bartkowiak, Bartlomiej;Liu, Pengda;Greenleaf, Arno L.
果蝇含有一种（dCDK 12）蛋白，人类含有两种（hCDK 12和hCDK 13）蛋白，它们是酵母Ctk 1最接近的进化亲属，酵母Ctk 1是酿酒酵母中主要延伸期C末端重复结构域（CTD）激酶CTDK-I的催化亚基。然而，到目前为止，CDK 12和CDK 13都没有被证明是真正的CTD激酶。使用果蝇，我们证明，dCDK 12（CG 7597）是一个转录相关的CTD激酶，直向同源的yCtk 1。荧光显微镜显示，dCDK 12在甲醛固定的多线染色体上的分布与超磷酸化RNA聚合酶II（RNAPII）的分布几乎相同，但与P-TEFb（dCDK 9 + dCyclin T）的分布不同。染色质免疫沉淀（ChIP）实验证实dCDK 12存在于活性果蝇基因的转录区域上。与P-TEFb相比，dCDK 12的量在基因的5'端较低，而在基因的中部和39端较高（均归一化为RNAPII）。适当地，从核提取物中纯化的果蝇dCDK 12在体外表现出CTD激酶活性。有趣的是，我们发现，细胞周期蛋白K与纯化的dCDK 12，暗示它作为这个CTD激酶的细胞周期蛋白亚基。最重要的是，我们证明了S2细胞中dCDK 12的RNAi敲低改变了CTD的磷酸化状态，降低了其Ser 2磷酸化水平。类似地，在人HeLa细胞中，我们表明从核提取物纯化的hCDK 13在体外显示CTD激酶活性，如预期的那样。此外，我们发现，嵌合（酵母/人类）版本的Ctk 1含有激酶同源结构域的hCDK 12/13（或hCDK 9）在酵母细胞中的功能（也在体外），使用这个系统，我们表明，bur 1（ts）突变体被拯救更有效地由hCDK 9嵌合体比hCDK 13嵌合体，表明以下的直系关系：Bur 1 CDK 9和Ctk 1 CDK 12/13。最后，我们表明，siRNA敲低HeLa细胞中的hCDK 12导致CTD磷酸化状态的改变。我们的研究结果表明，后生动物CDK 12和CDK 13是CTD激酶，CDK 12是酵母Ctk 1的直链。
Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyper-phosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 39 end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 CDK9 and Ctk1 CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.