Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d

Nature volume 395, pages 244–250 (1998)Cite this article

Abstract

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19INK4d protein has been determined at 1.9 Å resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Electron density at the p19INK4d/Cdk6 interface contoured at 1.2σ.
Figure 2: Three-dimensional structure of the p19INK4d/Cdk6 complex.
Figure 3: Sequence comparisons, secondary structure and residues buried in complex formation.
Figure 4: Surface representation of the p19INK4d/Cdk6 interface.
Figure 5: Comparison of ATP-binding sites in different kinases.

Similar content being viewed by others

References

  1. Sherr, C. J. Cancer cell cycles. Science 274, 1672–1677 (1996).

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Morgan, D. O. Principles of CDK regulation. Nature 374, 131–134 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Parker, L. L., Sylvestre, P. J., Brynes, M. J., Liu, F. & Piwnicaworms, H. Identification of a 95 Kda WEE1-like tyrosine kinase in Hela-cells. Proc. Natl Acad. Sci .USA 92, 9638–9642 (1995).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kumagai, A. & Dunphy, W. G. The Cdc25 protein controls tyrosine dephosphorylation of the Cdc2 protein in a cell-free system. Cell 64, 903–914 (1991).

    Article  CAS  PubMed  Google Scholar 

  5. Sherr, C. & Roberts, J. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9, 1149–1153 (1995).

    Article  CAS  PubMed  Google Scholar 

  6. Harper, J. W. in Checkpoint Control and Cancer(ed Kastan, M. B.) Vol. 29, 91–107 (CSHL Press, New York, 1997).

    Google Scholar 

  7. Soos, T. J. et al. Formation of p27-CDK complexes during the human mitotic cell cycle. Cell Growth Differ. 7, 135–146 (1996).

    CAS  PubMed  Google Scholar 

  8. Poon, R. Y. C., Toyoshima, H. & Hunter, T. Redistribution of the CDK inhibitor p27 between different cyclin-CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation. Mol. Biol. Cell 6, 1197–1213 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Serrano, M., Hannon, G. J. & Beach, D. Anew regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366, 704–707 (1993).

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Hannon, G. J. & Beach, D. p15INK4B is a potential effector of TGF-β-induced cell cycle arrest. Nature 371, 257–261 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Hirai, H., Roussel, M. F., Kato, J-Y., Ashmun, R. A. & Sherr, C. J. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mol. Cell. Biol. 15, 2672–2681 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chan, F. K. M., Zhang, J., Cheng, L., Shaprio, D. N. & Winoto, D. A. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16INK4. Mol. Cell. Biol. 15, 2682–2688 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Guan, K. et al. Isolation and characterization of p19INK4d, a p16 related inhibitor specific to CDK6 and CDK4. Mol. Biol. Cell 7, 57–70 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell 81, 323–330 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. De Bondt, H. L. et al. Crystal structure of cyclin-dependent kinase 2. Nature 363, 595–602 (1993).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Jeffrey, P. D. et al. Mechanism of CDK activation revealed by the structure of a cyclin-CDK2 complex. Nature 376, 313–320 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Bork, P. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally. Proteins Struct. Funct. and Genet. 17, 363–374 (1993).

    Article  CAS  Google Scholar 

  19. Luh, F. Y. et al. Structure of the cyclin-dependent kinase inhibitor p19INK4d. Nature 389, 999–1003 (1997).

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Venkataramani, R., Swaminathan, K. & Marmorstein, R. Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations. Nature Struct. Biol. 5, 74–81 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Byeon, I.-J. L. et al. Tumor suppressor p16INK4a: Determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4. Mol. Cell 1, 421–431 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Reynisdottir, I. & Massagué, J. The subcellular locations of p15INK4b and p27Kip1 coordinate their inhibitory interactions with Cdk4 and Cdk2. Genes Dev. 11, 492–503 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Yang, R., Gombart, A. F., Serrano, M. & Koeffler, H. P. Mutational effects on the p16INK4a tumour-suppressor protein. Cancer Res. 55, 2503–2506 (1995).

    CAS  PubMed  Google Scholar 

  24. Gorina, S. & Pavletich, N. P. Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science 274, 1001–1005 (1996).

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Batchelor, A. H., Piper, D. E., de la Brousse, F. C., McKnight, S. L. & Wolberger, C. The structure of GABPα/β: an ETS domain-ankyrin repeat heterodimer bound to DNA. Science 279, 1037–1041 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Zhang, B. & Peng, Z.-Y. Defective folding of mutant p16INK4a proteins encoded by tumor-derived alleles. J. Biol. Chem. 271, 28734–28737 (1996).

    Article  CAS  PubMed  Google Scholar 

  27. Wölfel, T. et al. Ap16INK4A-insensitive CDK4 mutant targeted by cytolytic T-lymphocytes in a human-melanoma. Science 269, 1281–1284 (1995).

    Article  ADS  PubMed  Google Scholar 

  28. Coleman, K. G. et al. Identification of Cdk4 sequences involved in Cyclin D1 and p16 binding. J. Biol. Chem. 272, 18869–18874 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Zuo, L. et al. Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma. Nature Genet. 12, 97–99 (1996).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Tsao, H., Benoit, E., Sober, A. J., Thiele, C. & Haluska, F. G. Novel mutation in the p16/CDKN2A binding region of cyclin-dependent kinase-4 gene. Cancer Res. 58, 109–113 (1998).

    CAS  PubMed  Google Scholar 

  31. Soufir, N. et al. Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. Hum. Mol. Genet. 7, 209–216 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. Zhang, F., Strand, A., Robbins, D., Cobb, M. H. & Goldsmith, E. J. Atomic structure of the MAP kinase ERK2 at 2.3 Å resolution. Nature 367, 704–710 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  33. Taylor, S. S. & Radzio-Andzelm, E. Three protein kinase structures define a common motif. Structure 2, 345–355 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Zheng, J. H. et al. Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry 32, 2154–2161 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Parry, D., Bates, S., Mann, D. J. & Peters, G. Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/4MTS1 tumour suppressor gene product. EMBO J. 14, 503–511 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. DellaRagione, F. et al. Biochemical characterisation of p16INK4- and p18-containing complexes in human cell lines. J. Biol. Chem. 271, 15942–15949 (1996).

    Article  CAS  Google Scholar 

  37. Russo, A. A., Tong, L., Lee, J.-O., Jeffrey, P. D. & Pavletich, N. P. Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a. Nature 395, 237–244 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  38. Reynisdottir, I., Polyak, A., Iavarone, A. & Massagué, J. Kip/Cip and INK4D CDK inhibitor cooperate to induce cell cycle arrest in response to TGF-β. Genes Dev. 9, 1831–1845 (1995).

    Article  CAS  PubMed  Google Scholar 

  39. Sandhu, C. et al. TGFβ stabilizes p15INK46 protein, increases p15INK46/CDK4 complexes, and inhibits cycD1/CDK4 association in human mammary epithelial cells. Mol. Cell. Biol. 17, 2458–2467 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Russo, A. A., Jeffrey, P. D., Patten, A. K., Massagué, J. & Pavletich, N. P. Crystal structure of the p27Kip1 cyclin-dependent kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382, 325–331 (1996).

    Article  ADS  CAS  PubMed  Google Scholar 

  41. Vlach, J., Hennecke, S. & Amati, B. Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27Kip1. EMBO J. 16, 5334–5344 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Collaborative computing project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  43. Brünger, A. T. X-PLOR, Version 3.1, A System for X-Ray Crystallography and NMR(Yale Univ. Press, New Haven and London, 1992).

    Google Scholar 

  44. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Read, R. J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986).

    Article  Google Scholar 

  46. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron-density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  PubMed  Google Scholar 

  47. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  48. Merrit, E. A. & Murphy, M. E. Raster3D. A program for photorealistic molecular graphics. Acta Crystallogr. D 50, 869–873 (1994).

    Article  Google Scholar 

  49. Hubbard, S. J. & Thornton, J. M. ‘NACCESS’ computer program(Department of Biochemistry and Molecular Biology, Univ. College London, 1993).

  50. Foulkes, W. D., Flanders, T. Y., Pollock, P. M. & Hayward, N. K. The CDKN2A (p16) gene and human cancer. Mol. Med. 3, 5–20 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. J. Sherr (Howard Hughes Medical Institute) for the plasmid expressing GST–p19INK4d; A. Raine for computer programming; M. Nilges for X-PLOR scripts; I. Tickle for advice; F. Luh for the selectively protonated p19INK4d; J. Eckstein for help with molecular graphics; and E. Duke, W. Burmeister and R. C. Kehoe for help with data collection. This work was supported by grants from the BBSRC and the Wellcome Trust. D.H.B. is supported by a studentship from the MRC. M.S. is supported by a grant from DGICYT, Spain, and by a core grant to the Department of Immunology and Oncology from Pharmacia-Upjohn and the Spanish Research Council. The Cambridge Centre for Molecular Recognition is supported by the BBSRC and the Wellcome Trust.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Cambridge Centre for Molecular Recognition, University of Cambridge, 80 Tennis Court Road, CB2 1GA, Cambridge, UK

    Deborah H. Brotherton, Venugopal Dhanaraj, Elena Volyanik, Emilio Parisini, Brian O. Smith, Tom L. Blundell & Ernest D. Laue

  2. Mitotix Inc., One Kendall Square, Building 600, Cambridge, 02139, Massachusetts, USA

    Scott Wick, Leonardo Brizuela & Xu Xu

  3. Du Pont Pharmaceutical Company, Box 80353, Wilmington, 19880-0353, Delaware, USA

    Peter J. Domaille, Sharon J. Archer & Stephen L. Brenner

  4. Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Campus de Cantoblanco, E-28049, Madrid, Spain

    Manuel Serrano

Authors
  1. Deborah H. Brotherton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Venugopal Dhanaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Scott Wick
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Leonardo Brizuela
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter J. Domaille
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elena Volyanik
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Emilio Parisini
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Brian O. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sharon J. Archer
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Manuel Serrano
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Stephen L. Brenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Tom L. Blundell
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Ernest D. Laue
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ernest D. Laue.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brotherton, D., Dhanaraj, V., Wick, S. et al. Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d. Nature 395, 244–250 (1998). https://doi.org/10.1038/26164

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/26164

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing