CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1

Department of Biochemistry, Duke Center for RNA Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Genes & development (Impact Factor: 10.8). 10/2010; 24(20):2303-16. DOI: 10.1101/gad.1968210
Source: PubMed


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 hyperphosphorylated 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 3' 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.

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Available from: Arno L Greenleaf, Jan 20, 2015
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    • "Among all members of the CDK super family, CDK12 deserves special attention because of its reported phosphorylation of the Pol II CTD on Ser2, an ability that is shared with CDK9 (Bartkowiak et al., 2010). To determine whether i-CDK9 also affects CDK12 kinase activity, we examined the abilities of affinity-purified Flag-tagged CDK12 (CDK12-F), CDK9 (CDK9-F) and their associated cyclin partners to phosphorylate GST-CTD in the presence of increasing amounts of i-CDK9. "
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    • "During the transition from initiation to early elongation, Ser2 is phosphorylated by P-TEFb (CDK9/Cyclin T) [8]. As transcription proceeds from 5′ to 3′ direction, Ser2P levels are gradually increased through the actions of P-TEFb [8] and CDK12/13 [9]; concurrently, Ser5P levels decline [2], [5], [10]. Ser2P promotes the recruitment of a histone methyltransferase, 3′-end processing factors, and transcription termination factors to the elongating Pol II [2], [3], [6], [10]. "
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