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Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics

Abstract

Cdk7 performs two essential but distinct functions as a CDK-activating kinase (CAK) required for cell-cycle progression and as the RNA polymerase II (Pol II) CTD kinase of general transcription factor IIH. To investigate the substrate specificity underlying this dual function, we created an analog-sensitive (AS) Cdk7 able to use bulky ATP derivatives. Cdk7-AS–cyclin H–Mat1 phosphorylates 10–15 endogenous polypeptides in nuclear extracts. We identify seven of these as known and previously unknown Cdk7 substrates that define two classes: proteins such as Pol II and transcription elongation factor Spt5, recognized efficiently only by the fully activated Cdk7 complex, through sequences surrounding the site of phosphorylation; and CDKs, targeted equivalently by all active forms of Cdk7, dependent on substrate motifs remote from the phosphoacceptor residue. Thus, Cdk7 accomplishes dual functions in cell-cycle control and transcription not through promiscuity but through distinct, stringent modes of substrate recognition.

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Figure 1: Generation and characterization of Cdk7-AS.
Figure 2: Cdk7-AS is exquisitely selective in crude extracts.
Figure 3: Identification of sites uniquely phosphorylated by Cdk7 on nuclear-extract proteins.
Figure 4: Purification and identification of Cdk7 substrates in nuclear extract (NE).
Figure 5: Cdk7 is a potential Cdk11-activating kinase.
Figure 6: Cdk7 phosphorylates recombinant Spt5.
Figure 7: Alternative modes of substrate recognition.

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References

  1. Fesquet, D. et al. The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. EMBO J. 12, 3111–3121 (1993).

    Article  CAS  Google Scholar 

  2. Poon, R.Y., Yamashita, K., Adamczewski, J.P., Hunt, T. & Shuttleworth, J. The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. EMBO J. 12, 3123–3132 (1993).

    Article  CAS  Google Scholar 

  3. Solomon, M.J., Harper, J.W. & Shuttleworth, J. CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15. EMBO J. 12, 3133–3142 (1993).

    Article  CAS  Google Scholar 

  4. Fisher, R.P. & Morgan, D.O. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell 78, 713–724 (1994).

    Article  CAS  Google Scholar 

  5. Mäkelä, T.P. et al. A cyclin associated with the CDK-activating kinase MO15. Nature 371, 254–257 (1994).

    Article  Google Scholar 

  6. Devault, A. et al. MAT1 ('ménage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK. EMBO J. 14, 5027–5036 (1995).

    Article  CAS  Google Scholar 

  7. Fisher, R.P., Jin, P., Chamberlin, H.M. & Morgan, D.O. Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase. Cell 83, 47–57 (1995).

    Article  CAS  Google Scholar 

  8. Tassan, J.-P. et al. In vitro assembly of a functional human cdk7/cyclin H complex requires MAT1, a novel 36 kD RING finger protein. EMBO J. 14, 5608–5617 (1995).

    Article  CAS  Google Scholar 

  9. Roy, R. et al. The MO15 cell cycle kinase is associated with the TFIIH transcription-DNA repair factor. Cell 79, 1093–1101 (1994).

    Article  CAS  Google Scholar 

  10. Serizawa, H. et al. Association of Cdk-activating kinase subunits with transcription factor TFIIH. Nature 374, 280–282 (1995).

    Article  CAS  Google Scholar 

  11. Shiekhattar, R. et al. Cdk-activating kinase (CAK) complex is a component of human transcription factor IIH. Nature 374, 283–287 (1995).

    Article  CAS  Google Scholar 

  12. Harper, J.W. & Elledge, S.J. The role of Cdk7 in CAK function, a retro-retrospective. Genes Dev. 12, 285–289 (1998).

    Article  CAS  Google Scholar 

  13. Larochelle, S., Pandur, J., Fisher, R.P., Salz, H.K. & Suter, B. Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity. Genes Dev. 12, 370–381 (1998).

    Article  CAS  Google Scholar 

  14. Hermand, D. et al. Fission yeast Csk1 is a CAK-activating kinase (CAKAK). EMBO J. 17, 7230–7238 (1998).

    Article  CAS  Google Scholar 

  15. Lee, K.M., Saiz, J.E., Barton, W.A. & Fisher, R.P. Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases CAKs). Curr. Biol. 9, 441–444 (1999).

    Article  CAS  Google Scholar 

  16. Saiz, J.E. & Fisher, R.P. A CDK-activating kinase network is required in cell cycle control and transcription in fission yeast. Curr. Biol. 12, 1100–1105 (2002).

    Article  CAS  Google Scholar 

  17. Wallenfang, M.R. & Seydoux, G. cdk-7 is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos. Proc. Natl. Acad. Sci. USA 99, 5527–5532 (2002).

    Article  CAS  Google Scholar 

  18. Poon, R.Y. & Hunter, T. Innocent bystanders or chosen collaborators? Curr. Biol. 5, 1243–1247 (1995).

    Article  CAS  Google Scholar 

  19. Garrett, S. et al. Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T-loop. Mol. Cell. Biol. 21, 88–99 (2001).

    Article  CAS  Google Scholar 

  20. Trigon, S. et al. Characterization of the residues phosphorylated in vitro by different C-terminal domain kinases. J. Biol. Chem. 273, 6769–6775 (1998).

    Article  CAS  Google Scholar 

  21. Ramanathan, Y. et al. Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences. J. Biol. Chem. 276, 10913–10920 (2001).

    Article  CAS  Google Scholar 

  22. Ko, L.J. et al. p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner. Mol. Cell. Biol. 17, 7220–7229 (1997).

    Article  CAS  Google Scholar 

  23. Rochette-Egly, C., Adam, S., Rossignol, M., Egly, J.-M. & Chambon, P. Stimulation of RARα activation function AF-1 through binding to the general transcription factor TFIIH and phosphorylation by CDK7. Cell 90, 97–107 (1997).

    Article  CAS  Google Scholar 

  24. Vandel, L. & Kouzarides, T. Residues phosphorylated by TFIIH are required for E2F–1 degradation during S-phase. EMBO J. 18, 4280–4291 (1999).

    Article  CAS  Google Scholar 

  25. Inamoto, S., Segil, N., Pan, Z.-Q., Kimura, M. & Roeder, R.G. The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors. J. Biol. Chem. 272, 29852–29858 (1997).

    Article  CAS  Google Scholar 

  26. Aprelikova, O., Xiong, Y. & Liu, E.T. Both p16 and p21 families of cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent kinases by the CDK-activating kinase. J. Biol. Chem. 270, 18195–18197 (1995).

    Article  CAS  Google Scholar 

  27. Ubersax, J.A. et al. Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859–864 (2003).

    Article  CAS  Google Scholar 

  28. Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001).

    Article  CAS  Google Scholar 

  29. Rossignol, M., Kolb-Cheynel, I. & Egly, J.-M. Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J. 16, 1628–1637 (1997).

    Article  CAS  Google Scholar 

  30. Yankulov, K.Y. & Bentley, D.L. Regulation of CDK7 substrate specificity by MAT1 and TFIIH. EMBO J. 16, 1638–1646 (1997).

    Article  CAS  Google Scholar 

  31. Larochelle, S. et al. T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity. EMBO J. 20, 3749–3759 (2001).

    Article  CAS  Google Scholar 

  32. Shah, K., Liu, Y., Deirmengian, C. & Shokat, K.M. Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc. Natl. Acad. Sci. USA 94, 3565–3570 (1997).

    Article  CAS  Google Scholar 

  33. Kraybill, B.C., Elkin, L.L., Blethrow, J.D., Morgan, D.O. & Shokat, K.M. Inhibitor scaffolds as new allele specific kinase substrates. J. Am. Chem. Soc. 124, 12118–12128 (2002).

    Article  CAS  Google Scholar 

  34. Cornelis, S. et al. Identification and characterization of a novel cell cycle-regulated internal ribosome entry site. Mol. Cell 5, 597–605 (2000).

    Article  CAS  Google Scholar 

  35. Wada, T. et al. DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12, 343–356 (1998).

    Article  CAS  Google Scholar 

  36. Wada, T., Takagi, T., Yamaguchi, Y., Watanabe, D. & Handa, H. Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro. EMBO J. 17, 7395–7403 (1998).

    Article  CAS  Google Scholar 

  37. Ivanov, D., Kwak, Y.T., Guo, J. & Gaynor, R.B. Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 20, 2970–2983 (2000).

    Article  CAS  Google Scholar 

  38. Russo, A.A., Jeffrey, P.D. & Pavletich, N.P. Structural basis of cyclin-dependent kinase activation by phosphorylation. Nat. Struct. Biol. 3, 696–700 (1996).

    Article  CAS  Google Scholar 

  39. Specht, K.M. & Shokat, K.M. The emerging power of chemical genetics. Curr. Opin. Cell Biol. 14, 155–159 (2002).

    Article  CAS  Google Scholar 

  40. Poon, R.Y. & Hunter, T. Dephosphorylation of Cdk2 Thr160 by the cyclin-dependent kinase-interacting phosphatase KAP in the absence of cyclin. Science 270, 90–93 (1995).

    Article  CAS  Google Scholar 

  41. Cheng, A., Ross, K.E., Kaldis, P. & Solomon, M.J. Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases. Genes Dev. 13, 2946–2957 (1999).

    Article  CAS  Google Scholar 

  42. Chen, J., Larochelle, S., Li, X. & Suter, B. Xpd/Ercc2 regulates CAK activity and mitotic progression. Nature 424, 228–232 (2003).

    Article  CAS  Google Scholar 

  43. Habelhah, H. et al. Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue. J. Biol. Chem. 276, 18090–18095 (2001).

    Article  CAS  Google Scholar 

  44. Liu, Y. et al. Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex. Mol. Cell. Biol. 24, 1721–1735 (2004).

    Article  CAS  Google Scholar 

  45. Rickert, P., Corden, J.L. & Lees, E. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene 18, 1093–1102 (1999).

    Article  CAS  Google Scholar 

  46. Pinhero, R., Liaw, P., Bertens, K. & Yankulov, K. Three cyclin-dependent kinases preferentially phosphorylate different parts of the C-terminal domain of the large subunit of RNA polymerase II. Eur. J. Biochem. 271, 1004–1014 (2004).

    Article  CAS  Google Scholar 

  47. Hu, D., Mayeda, A., Trembley, J.H., Lahti, J.M. & Kidd, V.J. CDK11 complexes promote pre-mRNA splicing. J. Biol. Chem. 278, 8623–8629 (2003).

    Article  CAS  Google Scholar 

  48. Li, T., Inoue, A., Lahti, J.M. & Kidd, V.J. Failure to proliferate and mitotic arrest of CDK11(p110/p58)-null mutant mice at the blastocyst stage of embryonic cell development. Mol. Cell. Biol. 24, 3188–3197 (2004).

    Article  CAS  Google Scholar 

  49. Wen, Y. & Shatkin, A.J. Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev. 13, 1774–1779 (1999).

    Article  CAS  Google Scholar 

  50. Pei, Y. & Shuman, S. Interactions between fission yeast mRNA capping enzymes and elongation factor Spt5. J. Biol. Chem. 277, 19639–19648 (2002).

    Article  CAS  Google Scholar 

  51. Orphanides, G. & Reinberg, D. A unified theory of gene expression. Cell 108, 439–451 (2002).

    Article  CAS  Google Scholar 

  52. Fisher, R.P. Reconstitution of mammalian CDK-activating kinase. Methods Enzymol. 283, 256–270 (1997).

    Article  CAS  Google Scholar 

  53. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

    Article  CAS  Google Scholar 

  54. Gamble, M.J., Erdjument-Bromage, H., Tempst, P., Freedman, L.P. & Fisher, R.P. The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates. Mol. Cell. Biol. 25, 797–807 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D.O. Morgan (University of California at San Francisco) for providing vectors for production of recombinant NDPK and for critical review of the manuscript; M.B. Mathews (University of Medicine and Dentistry of New Jersey) and D.H. Price (University of Iowa) for providing human cyclin T1 cDNAs; Y. Ramanathan for construction of human Cdk9 and cyclin T1 expression vectors; J. Singer for the purification of Csk1; G. Livshits for cloning Cdk11; H. Erdjument-Bromage and P. Tempst for MS identification of proteins; and A. Koff for critical review of the manuscript. HeLa cells were grown by the US National Cell Culture Center. This work was supported by a research fellowship award to S.L. from the National Cancer Institute of Canada, supported with funds provided by the Terry Fox run and by US National Institutes of Health grants GM56985 and DK45460 to R.P.F and EB001987 to K.M.S.

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Correspondence to Robert P Fisher.

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Supplementary information

Supplementary Fig. 1

Structures of 1-NMPP1, N6-(benzyl)-ATP and 3-(benzyl)-PPTP (PDF 123 kb)

Supplementary Fig. 2

Effect of ATP on specificity of labeling. (PDF 593 kb)

Supplementary Fig. 3

Immunoprecipitation of labeled Cdks. (PDF 246 kb)

Supplementary Fig. 4

Initial purification steps for Cdk7-AS substrates. (PDF 326 kb)

Supplementary Table 1

Nucleotide specificity of Cdk7-AS. (PDF 27 kb)

Supplementary Methods (PDF 144 kb)

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Larochelle, S., Batliner, J., Gamble, M. et al. Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics. Nat Struct Mol Biol 13, 55–62 (2006). https://doi.org/10.1038/nsmb1028

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