The structure and substrate specificity of human Cdk12/Cyclin K

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.

Figure 1. Structure of the human Cdk12/CycK complex.

(a) Overall structure assembly of Cdk12 (blue) and CycK (red). The bound nucleotide and the phospho-threonine residue in the activation segment are highlighted. (b) Close-up of the kinase active site showing the ADP nucleotide, AlF₃, Mg ions and water molecules. The final 2F_o−F_c electron density is displayed at 1σ. (c) Close-up of pT893 in the Cdk12 T-loop segment and side chain contacts. R773 of the P₇₆₈ITAIRE sequence forms electrostatic contacts with E108 of the K₁₀₅VEE motif in CycK but is not directly contacting pT893. The other two canonical Cdk arginines, R858 and R882, form ionic interactions with the phosphate group. (d) Stereo image of the coordination of pT893 in the Cdk12 T-loop segment. The final 2F_o−F_c electron density is displayed at 1σ.

Figure 2. The C-terminal helix associates with the Cdk12 kinase core domain.

(a) Close-up of DCHEL sequence interactions with the kinase core domain and the bound nucleotide. A network of weak water-mediated contacts is formed between E1041 and L1042 of the C-terminal helix and the hydroxyl groups of the ATP ribose that is stabilized by I733, E735 and T737 of the glycine-rich loop. (b) D1038 interacts with residues of the N- and C-terminal kinase lobe, while H1040 directly contacts the adenine base of the bound nucleotide. (c) Sequence alignment of the transcription-associated kinases Cdk12 and Cdk9 from human and their respective orthologous from S. cerevisiae and S. pombe compared with Cdk2. A C-terminal HE-motif followed by a polybasic region is conserved in the CTD kinases. Residues of the N- and C-terminal lobe that interact with the C-terminal helix are shaded grey. (d) Surface representations of Cdk12 (this study) and Cdk2 (3QHR; ref. 32) show the different accessibilities of the ATP ribose in the transcription-associated kinase and the cell cycle kinase. While a lysine residues (K89) in Cdk2 points towards the hydroxyl groups of the ATP ribose, this position is engaged with a small glycine in CTD kinases. (e) The activity of Cdk12 decreases upon truncation of the C-terminal helix. Four different length versions of Cdk12 were tested for their activity with two different kinase substrates, consensus CTD and pS7-CTD, using the same CycK (1–267) construct. Truncation of the polybasic cluster in Cdk12 from residue 1,063 to 1,044 led to a significant loss in activity for the pS7-CTD substrate. Data are the mean±s.d. of three independent experiments.

Figure 3. Cdk12 activity on the CTD requires Ser7 prephosphorylation.

(a) Design of CTD substrate peptides used for kinetic and ESI-MS analyses. Peptides contained three consensus hepta-repeats with either no modification (cons. CTD_[3]) or with phosphorylation marks continuously set at one residue of the heptad sequence as shown for Ser7 (pS7-CTD_[3]). The same design principle was used for CTD peptides phosphorylated at Tyr1 (pY1-CTD_[3]), Ser2 (pS2-CTD_[3]), Thr4 (pT4-CTD_[3]) and Ser5 (pS5-CTD_[3]) or a peptide that contained a lysine at position 7 (K7-CTD_[3]). (b) Activity of Cdk12/CycK for various CTD substrates. Of the seven peptides tested, Cdk12 showed the highest activity for continuous Ser7 prephosphorylation. Minor activities were detected for Ser2 and Thr4 prephosphorylated peptides. Remarkably, no activity was seen for cons. CTD_[3] or the K7-peptide, which is in contrast to P-TEFb. In addition, Cdk12 exhibited no activity on the pSer5 peptide. (c) ESI-MS analyses of CTD peptides before and after 4 h of incubation with 2 μM Cdk12/CycK. Whereas no phosphorylation occurred for the consensus peptide, the pS7-CTD_[3] substrate got readily phosphorylated up to three times. (d) Time course of Cdk12/CycK mediated CTD phosphorylation. The prephosphorylated Ser7 peptide got phosphorylated over time to saturation, while the non-phosphorylated peptide showed low susceptibility as a Cdk12 substrate. Data in panels b and d are the mean±s.d. of three independent experiments.

Figure 4. Substrate preferences of Cdk12 phosphorylation.

(a) Activity of Cdk12 on CTD peptide substrates with serine-to-alanine mutations either at position 2 or at position 5 in combination with Ser7 pre-phosphorylations. Only minor activity was displayed on the three non-phosphorylated peptides cons. CTD, S2A-CTD and S5A-CTD. The pS7-CTD substrate instead was readily phosphorylated followed by the S2A-pS7-CTD peptide. The S5A-pS7-CTD peptide exhibited almost no susceptibility as a kinase substrate, suggesting that Cdk12 phosphorylates Ser5 in the context of Ser7 prephosphorylation. Cartoons displaying the peptide templates are shown right. (b) Priming and directionality of Cdk12 phosphorylation. Serine 2, 5 and 7 phosphorylation marks were set either at the N terminus or at the C terminus of triple hepta-repeats. While the activity of Cdk12 towards these peptides was overall very weak, the substrate with the pS7 mark set at the C terminus gained highest recognition. (c) In contrast, Cdk9 exhibits high activity towards C-terminally phosphorylated peptides at either position 5 or position 7, suggesting the priming of the kinase by these prephosphorylations. All data are reported as the mean±s.d. from three independent experiments.

Figure 5. Cdk12/CycK is a promiscuous CTD kinase.

Immunoprecipitated Pol IIA with the antibody 1C7 from HeLa whole-cell extract was used as substrate for Cdk12 and Cdk9, respectively, and subjected to western blot analysis using mAbs specific for Rpb1 (Pol 3.3. and 1C7, the latter recognizes non-modified CTD) or CTD phosphorylations pSer2 (3E10), pSer5 (3E8), pSer7 (4E12) and pThr4 (6D7). IgG2a is an isotype control. The hyper- (II0) and hypophosphorylated forms (IIA) of Pol II are indicated. The Pol IIA form corresponds to an aberrant molecular mass of 250 kDa. (a) Comparison of Cdk12 and Cdk9 CTD substrate specificities. Incubation of Rpb1 with Cdk12 results in phosphorylation of Ser5 and to a small extent Ser7, which decreased upon C-terminal truncation of the kinase. Cdk9 showed similar substrate specificity albeit at a significantly higher enzymatic activity. (b) Cdk12 (696–1,082) phosphorylates Pol IIA at Ser5 residues, and to a lesser extent at Ser7 residues, in a concentration-dependent manner. (c) T-loop phosphorylation enhances the activity of Cdk12/CycK for Ser5 phosphorylation. Incubation of the RNAPII substrate with phosphorylated Cdk12 (lane 3), non-phosphorylated Cdk12 (lane 4) or Cdk9 (lane 5) showed a significantly enhanced Ser5 phosphorylation for the activated Cdk12 kinase. (d) Quantitative ELISA data for Cdk12 and Cdk9 kinase activity on a CTD peptide. A CTD consensus peptide of two hepta-repeats was used as kinase substrate. Error bars show s.d. of three independent experiments. (e) In vitro kinase assays with Cdk12 and Cdk9 and GST-tagged full-length human CTD as a substrate. Flag-tagged kinases were immunoprecipitated from HCT116 cells and compared with recombinant Cdk12 (696–1,082)/CycK (1–267). Recombinant and flag-tagged Cdk12 phosphorylate Ser2 and Ser5 to the same extent (lanes 2 and 4). Flag-tagged Cdk9 strongly phosphorylates Ser5 and to lesser extent Ser7 and Ser2 (lane 5). EV indicates the empty vector control. Exposure times are indicated on the left. (f) Display of the protein input for the in vitro kinase assays by western blot analysis. Equal amounts of proteins were used in in vitro kinase assays as shown by probing western blots with anti-flag (upper panel) and anti-CycK (middle panel) antibodies. Cdk12 purifications are free of Cdk9 contaminants as shown by western blot analysis with an anti-Cdk9 antibody (lower panel).

Figure 6. Inhibition of CTD kinases Cdk12 and Cdk9 by small molecule inhibitors.

(a) A panel of eleven small molecule inhibitors was tested at 10- and 100-fold molar excess against the kinase activity of Cdk12/CycK. Flavopiridol showed the highest efficacy for Cdk12 inhibition followed by CR8. (b) The same panel of inhibitors was analysed for Cdk9/CycT1 inhibition. Strongest inhibition was indeed achieved with flavopiridol, whose potency to inhibit Cdk9 turned out to be at least one order of magnitude higher than the closest followers, CR8 and Staurosporin. (c) Concentration series of flavopiridol for Cdk12/CycK and Cdk9/CycT1 at 0.2 μM kinase concentration. The IC₅₀ values were determined to be 1.37 μM against Cdk12 and 0.144 μM against Cdk9 using pS7-CTD₃ as substrate. All data were reported as the mean±s.d. from three independent experiments.

Figure 7. 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 k_BT (red) to +5 k_BT (blue) for all three Cdk/cyclin complexes.