The structure and substrate specificity of human Cdk12/Cyclin K

Bösken, Christian A.; Farnung, Lucas; Hintermair, Corinna; Schachter, Miriam Merzel; Vogel‐Bachmayr, Karin; Blažek, Dalibor; Anand, K.; Fisher, Robert P; Eick, Dirk; Geyer, Matthias

doi:10.1038/ncomms4505

Cited by 151 publications

(201 citation statements)

References 59 publications

Supporting

Mentioning

183

Contrasting

Order By: Relevance

“…2C, we detected low levels of Ser2P and Ser5 phosphorylation by CDK12 and CDK13. These results are consistent with recent studies that observed low levels of CTD phosphorylation implemented by CDK12 (33).…”

Section: Resultssupporting

confidence: 83%

“…Interestingly, while the manuscript was under preparation, it was reported that CDK12's ability to phosphorylate a peptide containing only canonical repeats was stimulated by preexisting Ser7P but was still not as active as CDK9, suggesting that there may be some functional differences between the mammalian and Drosophila CDK12 homologs (33). Although our knockdowns of CDK12, CDK13, and CCNK were not sufficient to decrease Ser2-CTD phosphorylation, the levels of knockdown were sufficient for observation of numerous defects in gene expression, including DNA damage response genes and genes for RNA processing factors.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Human Cyclin-Dependent Kinase 12 (CDK12) and CDK13 Complexes in C-Terminal Domain Phosphorylation, Gene Transcription, and RNA Processing

Liang

Gào

Gilmore

et al. 2015

Molecular and Cellular Biology

154

182

View full text Add to dashboard Cite

c Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo. CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression.T he largest subunit of RNA polymerase II (Pol II), Rpb1, contains a C-terminal domain (CTD) consisting of 52 heptad repeats of the YSPTSPS consensus sequence in humans (1). The CTD is phosphorylated within these repeats, including at serines 2, 5, and 7 (Ser2, Ser5, and Ser7, respectively) (2). The CTD serves as a phosphorylation-regulated platform for the recruitment of transcription factors, RNA processing factors, and chromatin modifiers, which affect mRNA synthesis, cotranscriptional processing, and histone modifications during the transcription cycle (3).The CTD undergoes a cycle of phosphorylation and dephosphorylation during the transcription cycle of initiation, elongation, and termination (4). During transcription initiation and early transcription, Ser5 of the CTD is phosphorylated by the cyclin-dependent kinase 7 (CDK7) subunit of the basal transcription factor TFIIH (2, 5). The positive transcription elongation factor, P-TEFb (comprised of CDK9 and cyclin T), regulates transcription elongation through phosphorylation of the CTD at Ser2 (6). P-TEFb also phosphorylates negative elongation factor (NELF) (7) and DRB sensitivity-inducing factor (DSIF) (8) during the transition to productive elongation.In the budding yeast Saccharomyces cerevisiae, there are two complexes for CTD Ser2 phosphorylation: the Bur1/Bur2 complex and the Ctk1/Ctk2/Ctk3 (CTDK) complex. The Bur complex implements CTD phosphorylation early in the transcription cycle, while the Ctk complex implements CTD phosphorylation during the elongation phase of RNA Pol II (9). CDK9 was long considered to be the only CTD Ser2 kinase in metazoans, but recently the Drosophila dCdk12/dCyclin K complex was shown to be the major CTD Ser2 kinase implementing Ser2 phosphorylation during the elongation stage, analogous to the S. cerevisiae Ctk1/2 complex (10). In ...

show abstract

Section: Resultssupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Characterization of Human Cyclin-Dependent Kinase 12 (CDK12) and CDK13 Complexes in C-Terminal Domain Phosphorylation, Gene Transcription, and RNA Processing

Liang

Gào

Gilmore

et al. 2015

Molecular and Cellular Biology

154

182

View full text Add to dashboard Cite

show abstract

“…2E). To further probe the effect of CDK inhibition on the survival of MLL-AF9;NRAS G12D cells, we tested the effect of flavopiridol, a small-molecule inhibitor of CDKs 1, 2, 4, 6, 7, 9, and 12 (33,34) and the more selective CDK 4/6 inhibitor palbociclib (35). As shown in Fig.…”

Section: Panobinostat and Dinaciclib Induce Death Of Mll-af9 Tumor Cementioning

confidence: 99%

The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia

et al. 2016

View full text Add to dashboard Cite

Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclibinduced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins. Cancer Res; 76(5); 1158-69. Ó2015 AACR.

show abstract

“…12 CDK12, a pol II CTD kinase implicated in 3 0 end formation of mRNAs 31 is a likely additional candidate kinase regulating the poly(A)-associated checkpoint as it is inhibited in vitro by DRB and Flavopidirol 29 perhaps unsurprisingly given that the active sites of CDK9 and CDK12 are strikingly similar. However, additional/alternative kinases cannot be ruled out.…”

Section: Outstanding Questionsmentioning

confidence: 99%

“…As KM05283, DRB, and Flavopiridol can inhibit kinases other than CDK9, [28][29][30] it is possible that more than one kinase is involved in poly(A)-associated checkpoint control. In addition, a relatively low level of Flavopiridol (0.2mM) specifically inhibits transcription downstream of the early elongation checkpoint without affecting transcription downstream of the poly(A) site, 13 suggesting either that a different set of kinases/targets are operating at each checkpoint or that transcription through the EEC requires a higher level of target phosphorylation.…”

Section: Outstanding Questionsmentioning

confidence: 99%