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The structure and substrate specificity of human Cdk12/Cyclin K.

Bösken CA, Farnung L, Hintermair C, Merzel Schachter M, Vogel-Bachmayr K, Blazek D, Anand K, Fisher RP, Eick D, Geyer M - Nat Commun (2014)

Bottom Line: Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9.T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase.These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

View Article: PubMed Central - PubMed

Affiliation: 1] Group Physical Biochemistry, Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, Bonn 53175, Germany [2] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund 44227, Germany.

ABSTRACT
Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

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Electrostatic surface display of transcription and cell cycle associated Cdk/cyclin pairs.Basic patches at the surface of Cdk12/CycK (4NST, this study) and Cdk9/CycT1 (3BLQ,ref. 25) surrounding the active site of the kinase may accomplish the recognition of the highly negatively charged RNA Pol II CTD. In contrast, the cell cycle regulating Cdk2/CycA (1JST, ref. 58) heterodimer is largely negatively charged, in line with the recognition specificity for an SPx(K/R) motif for the kinase domain and an RxL motif at the cyclin domain. Note that the polybasic cluster at the C terminus of the Cdk12 kinase domain is not contained in this display. Electrostatic surface charge is shown from −5 kBT (red) to +5 kBT (blue) for all three Cdk/cyclin complexes.
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f7: Electrostatic surface display of transcription and cell cycle associated Cdk/cyclin pairs.Basic patches at the surface of Cdk12/CycK (4NST, this study) and Cdk9/CycT1 (3BLQ,ref. 25) surrounding the active site of the kinase may accomplish the recognition of the highly negatively charged RNA Pol II CTD. In contrast, the cell cycle regulating Cdk2/CycA (1JST, ref. 58) heterodimer is largely negatively charged, in line with the recognition specificity for an SPx(K/R) motif for the kinase domain and an RxL motif at the cyclin domain. Note that the polybasic cluster at the C terminus of the Cdk12 kinase domain is not contained in this display. Electrostatic surface charge is shown from −5 kBT (red) to +5 kBT (blue) for all three Cdk/cyclin complexes.

Mentions: Experiments using either recombinant or immunoprecipitated P-TEFb confirm Cdk9 as a Ser5 CTD kinase as recently described34. A similar observation has been made by in vivo live imaging experiments of RNA Pol II transcription factories in primary cells, where Cdk9 foci colocalized with pSer5 but not pSer2 marks43. Chromatin immunoprecipitation assays coupled with deep sequencing (ChIP-seq) showed that the genome-wide occupancy of Cdk9 is similar to RNA Pol II Ser5 phosphorylation, with the highest enrichment in the 5′-end of genes43. The various substrate specificities are partly contained in the electrostatic surface characteristics of the Cdk–cyclin complexes that allow association with the negatively charged CTD substrate when phosphorylated (or primed) at neighbouring repeats. Different basic surface patches are indeed seen for the catalytic sites of Cdk12/CycK compared with Cdk9/CycT1 that could account for the kinase phosphorylation activities (Fig. 7). The cell cycle regulating Cdk2/CycA complex instead exhibits a largely acidic surface at the kinase active site, underlining its preference for the SPx(K/R) and RxL substrate recognition motifs26.


The structure and substrate specificity of human Cdk12/Cyclin K.

Bösken CA, Farnung L, Hintermair C, Merzel Schachter M, Vogel-Bachmayr K, Blazek D, Anand K, Fisher RP, Eick D, Geyer M - Nat Commun (2014)

Electrostatic surface display of transcription and cell cycle associated Cdk/cyclin pairs.Basic patches at the surface of Cdk12/CycK (4NST, this study) and Cdk9/CycT1 (3BLQ,ref. 25) surrounding the active site of the kinase may accomplish the recognition of the highly negatively charged RNA Pol II CTD. In contrast, the cell cycle regulating Cdk2/CycA (1JST, ref. 58) heterodimer is largely negatively charged, in line with the recognition specificity for an SPx(K/R) motif for the kinase domain and an RxL motif at the cyclin domain. Note that the polybasic cluster at the C terminus of the Cdk12 kinase domain is not contained in this display. Electrostatic surface charge is shown from −5 kBT (red) to +5 kBT (blue) for all three Cdk/cyclin complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3973122&req=5

f7: Electrostatic surface display of transcription and cell cycle associated Cdk/cyclin pairs.Basic patches at the surface of Cdk12/CycK (4NST, this study) and Cdk9/CycT1 (3BLQ,ref. 25) surrounding the active site of the kinase may accomplish the recognition of the highly negatively charged RNA Pol II CTD. In contrast, the cell cycle regulating Cdk2/CycA (1JST, ref. 58) heterodimer is largely negatively charged, in line with the recognition specificity for an SPx(K/R) motif for the kinase domain and an RxL motif at the cyclin domain. Note that the polybasic cluster at the C terminus of the Cdk12 kinase domain is not contained in this display. Electrostatic surface charge is shown from −5 kBT (red) to +5 kBT (blue) for all three Cdk/cyclin complexes.
Mentions: Experiments using either recombinant or immunoprecipitated P-TEFb confirm Cdk9 as a Ser5 CTD kinase as recently described34. A similar observation has been made by in vivo live imaging experiments of RNA Pol II transcription factories in primary cells, where Cdk9 foci colocalized with pSer5 but not pSer2 marks43. Chromatin immunoprecipitation assays coupled with deep sequencing (ChIP-seq) showed that the genome-wide occupancy of Cdk9 is similar to RNA Pol II Ser5 phosphorylation, with the highest enrichment in the 5′-end of genes43. The various substrate specificities are partly contained in the electrostatic surface characteristics of the Cdk–cyclin complexes that allow association with the negatively charged CTD substrate when phosphorylated (or primed) at neighbouring repeats. Different basic surface patches are indeed seen for the catalytic sites of Cdk12/CycK compared with Cdk9/CycT1 that could account for the kinase phosphorylation activities (Fig. 7). The cell cycle regulating Cdk2/CycA complex instead exhibits a largely acidic surface at the kinase active site, underlining its preference for the SPx(K/R) and RxL substrate recognition motifs26.

Bottom Line: Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9.T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase.These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

View Article: PubMed Central - PubMed

Affiliation: 1] Group Physical Biochemistry, Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, Bonn 53175, Germany [2] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund 44227, Germany.

ABSTRACT
Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

Show MeSH