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

Nature Structural & Molecular Biology volume 13, pages 55–62 (2006)Cite this article

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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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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Authors and Affiliations

  1. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10021, New York, USA

    Stéphane Larochelle, Jasmin Batliner, Matthew J Gamble, Nora M Barboza & Robert P Fisher

  2. Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, 8093, Switzerland

    Jasmin Batliner

  3. Department of Cellular and Molecular Pharmacology, University of California, 94143, San Francisco, California, USA

    Brian C Kraybill, Justin D Blethrow & Kevan M Shokat

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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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