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:

Activation of tyrosine kinases by mutation of the gatekeeper threonine

Abstract

Protein kinases targeted by small-molecule inhibitors develop resistance through mutation of the 'gatekeeper' threonine residue of the active site. Here we show that the gatekeeper mutation in the cellular forms of c-ABL, c-SRC, platelet-derived growth factor receptor-α and -β, and epidermal growth factor receptor activates the kinase and promotes malignant transformation of BaF3 cells. Structural analysis reveals that a network of hydrophobic interactions—the hydrophobic spine—characteristic of the active kinase conformation is stabilized by the gatekeeper substitution. Substitution of glycine for the residues constituting the spine disrupts the hydrophobic connectivity and inactivates the kinase. Furthermore, a small-molecule inhibitor that maximizes complementarity with the dismantled spine (compound 14) inhibits the gatekeeper mutation of BCR-ABL-T315I. These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases and provide structural insights to guide the development of next-generation inhibitors.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Sequence conservation and structural features of the gatekeeper residue threonine in tyrosine kinases and activation of kinase activity by gatekeeper residue mutation.
Figure 2: Kinase activation and BAF3 cellular transformation by gatekeeper mutants of SRC and ABL.
Figure 3: Kinase activation and BAF3 cellular transformation by gatekeeper residue mutation of receptor tyrosine kinases.
Figure 4: The active conformation of ABL is stabilized by a hydrophobic spine linking the gatekeeper threonine to the activation loop.
Figure 5: The hydrophobic spine in active and inactive SRC kinases.
Figure 6: Disruption of hydrophobic-spine assembly by mutagenesis inactivates the ABL-T334I kinase.
Figure 7: The ATP competitive inhibitor compound 14 disrupts the hydrophobic spine and inhibits c-ABL-T334I and BCR-ABL-T315I.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Cohen, P. Protein kinases—the major drug targets of the twenty-first century? Nat. Rev. Drug Discov. 1, 309–315 (2002).

    Article  CAS  Google Scholar 

  2. Dibb, N.J., Dilworth, S.M. & Mol, C.D. Switching on kinases: oncogenic activation of BRAF and the PDGFR family. Nat. Rev. Cancer 4, 718–727 (2004).

    Article  CAS  Google Scholar 

  3. Sawyers, C. Targeted cancer therapy. Nature 432, 294–297 (2004).

    Article  CAS  Google Scholar 

  4. Sawyers, C.L. Opportunities and challenges in the development of kinase inhibitor therapy for cancer. Genes Dev. 17, 2998–3010 (2003).

    Article  CAS  Google Scholar 

  5. Noble, M.E., Endicott, J.A. & Johnson, L.N. Protein kinase inhibitors: insights into drug design from structure. Science 303, 1800–1805 (2004).

    Article  CAS  Google Scholar 

  6. Liu, Y., Shah, K., Yang, F., Witucki, L. & Shokat, K.M. A molecular gate which controls unnatural ATP analogue recognition by the tyrosine kinase v-Src. Bioorg. Med. Chem. 6, 1219–1226 (1998).

    Article  CAS  Google Scholar 

  7. Azam, M. & Daley, G.Q. Anticipating clinical resistance to target-directed agents: the BCR-ABL paradigm. Mol. Diagn. Ther. 10, 67–76 (2006).

    Article  CAS  Google Scholar 

  8. Daub, H., Specht, K. & Ullrich, A. Strategies to overcome resistance to targeted protein kinase inhibitors. Nat. Rev. Drug Discov. 3, 1001–1010 (2004).

    Article  CAS  Google Scholar 

  9. Druker, B.J. et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med. 2, 561–566 (1996).

    Article  CAS  Google Scholar 

  10. Heinrich, M.C., Blanke, C.D., Druker, B.J. & Corless, C.L. Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT-positive malignancies. J. Clin. Oncol. 20, 1692–1703 (2002).

    Article  CAS  Google Scholar 

  11. Buchdunger, E. et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther. 295, 139–145 (2000).

    CAS  Google Scholar 

  12. Cools, J. et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N. Engl. J. Med. 348, 1201–1214 (2003).

    Article  CAS  Google Scholar 

  13. Gorre, M.E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 (2001).

    Article  CAS  Google Scholar 

  14. Shah, N.P. et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2, 117–125 (2002).

    Article  CAS  Google Scholar 

  15. Tamborini, E. et al. A new mutation in the KIT ATP pocket causes acquired resistance to imatinib in a gastrointestinal stromal tumor patient. Gastroenterology 127, 294–299 (2004).

    Article  CAS  Google Scholar 

  16. Cools, J. et al. PKC412 overcomes resistance to imatinib in a murine model of FIP1L1-PDGFRα-induced myeloproliferative disease. Cancer Cell 3, 459–469 (2003).

    Article  CAS  Google Scholar 

  17. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 352, 786–792 (2005).

    Article  CAS  Google Scholar 

  18. Bell, D.W. et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat. Genet. 37, 1315–1316 (2005).

    Article  CAS  Google Scholar 

  19. Levinson, N.M. et al. A Src-like inactive conformation in the Abl tyrosine kinase domain. PLoS Biol. 4, e144 (2006).

    Article  Google Scholar 

  20. Harrison, S.C. Variation on an Src-like theme. Cell 112, 737–740 (2003).

    Article  CAS  Google Scholar 

  21. Azam, M., Latek, R.R. & Daley, G.Q. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843 (2003).

    Article  CAS  Google Scholar 

  22. Nagar, B. et al. Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 112, 859–871 (2003).

    Article  CAS  Google Scholar 

  23. Hantschel, O. et al. A myristoyl/phosphotyrosine switch regulates c-Abl. Cell 112, 845–857 (2003).

    Article  CAS  Google Scholar 

  24. Xu, W., Harrison, S.C. & Eck, M.J. Three-dimensional structure of the tyrosine kinase c-Src. Nature 385, 595–602 (1997).

    Article  CAS  Google Scholar 

  25. Sicheri, F., Moarefi, I. & Kuriyan, J. Crystal structure of the Src family tyrosine kinase Hck. Nature 385, 602–609 (1997).

    Article  CAS  Google Scholar 

  26. Moarefi, I. et al. Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature 385, 650–653 (1997).

    Article  CAS  Google Scholar 

  27. Takeya, T. & Hanafusa, H. Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus. Cell 32, 881–890 (1983).

    Article  CAS  Google Scholar 

  28. Hunter, T. A tail of two src's: Mutatis mutandis. Cell 49, 1–4 (1987).

    Article  CAS  Google Scholar 

  29. Kato, J.Y. et al. Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein. Mol. Cell. Biol. 6, 4155–4160 (1986).

    Article  CAS  Google Scholar 

  30. Azam, M. et al. Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance. Proc. Natl. Acad. Sci. USA 103, 9244–9249 (2006).

    Article  CAS  Google Scholar 

  31. Kornev, A.P., Haste, N.M., Taylor, S.S. & Eyck, L.F. Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc. Natl. Acad. Sci. USA 103, 17783–17788 (2006).

    Article  CAS  Google Scholar 

  32. Daley, G.Q. & Baltimore, D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein. Proc. Natl. Acad. Sci. USA 85, 9312–9316 (1988).

    Article  CAS  Google Scholar 

  33. Koh, E.Y., Chen, T. & Daley, G.Q. Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation. Oncogene 23, 1214–1220 (2004).

    Article  CAS  Google Scholar 

  34. Carroll, M., Tomasson, M.H., Barker, G.F., Golub, T.R. & Gilliland, D.G. The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGFβR kinase-dependent signaling pathways. Proc. Natl. Acad. Sci. USA 93, 14845–14850 (1996).

    Article  CAS  Google Scholar 

  35. Mizuki, M. et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 96, 3907–3914 (2000).

    CAS  PubMed  Google Scholar 

  36. Jiang, J. et al. Epidermal growth factor-independent transformation of Ba/F3 cells with cancer-derived epidermal growth factor receptor mutants induces gefitinib-sensitive cell cycle progression. Cancer Res. 65, 8968–8974 (2005).

    Article  CAS  Google Scholar 

  37. Levine, R.L. et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7, 387–397 (2005).

    Article  CAS  Google Scholar 

  38. Mathey-Prevot, B., Nabel, G., Palacios, R. & Baltimore, D. Abelson virus abrogation of interleukin-3 dependence in a lymphoid cell line. Mol. Cell. Biol. 6, 4133–4135 (1986).

    Article  CAS  Google Scholar 

  39. Huse, M. & Kuriyan, J. The conformational plasticity of protein kinases. Cell 109, 275–282 (2002).

    Article  CAS  Google Scholar 

  40. Yun, C.H. et al. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11, 217–227 (2007).

    Article  CAS  Google Scholar 

  41. Zhang, X., Gureasko, J., Shen, K., Cole, P.A. & Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137–1149 (2006).

    Article  CAS  Google Scholar 

  42. Okram, B. et al. A general strategy for creating “inactive-conformation” Abl inhibitors. Chem. Biol. 13, 779–786 (2006).

    Article  CAS  Google Scholar 

  43. O'Hare, T. et al. In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer Res. 65, 4500–4505 (2005).

    Article  CAS  Google Scholar 

  44. Schindler, T. et al. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289, 1938–1942 (2000).

    Article  CAS  Google Scholar 

  45. Pfeifer, H. et al. Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Blood 110, 727–734 (2007).

    Article  CAS  Google Scholar 

  46. Skaggs, B.J. et al. Phosphorylation of the ATP-binding loop directs oncogenicity of drug-resistant BCR-ABL mutants. Proc. Natl. Acad. Sci. USA 103, 19466–19471 (2006).

    Article  CAS  Google Scholar 

  47. Griswold, I.J. et al. Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib. Mol. Cell. Biol. 26, 6082–6093 (2006).

    Article  CAS  Google Scholar 

  48. Corbin, A.S., Buchdunger, E., Pascal, F. & Druker, B.J. Analysis of the structural basis of specificity of inhibition of the Abl kinase by STI571. J. Biol. Chem. 277, 32214–32219 (2002).

    Article  CAS  Google Scholar 

  49. Yun, C.H. et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl. Acad. Sci. USA 105, 2070–2075 (2008).

    Article  CAS  Google Scholar 

  50. Emrick, M.A. et al. The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity. Proc. Natl. Acad. Sci. USA 103, 18101–18106 (2006).

    Article  CAS  Google Scholar 

  51. Li, S., Ilaria, R.L. Jr, Million, R.P., Daley, G.Q. & Van Etten, R.A. The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity. J. Exp. Med. 189, 1399–1412 (1999).

    Article  CAS  Google Scholar 

  52. Seeliger, M.A. et al. c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty. Structure 15, 299–311 (2007).

    Article  CAS  Google Scholar 

  53. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  54. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005).

    Article  Google Scholar 

  55. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  56. Afonine, P.V., Grosse-Kunstleve, R.W. & Adams, P.D. A robust bulk-solvent correction and anisotropic scaling procedure. Acta Crystallogr. D Biol. Crystallogr. 61, 850–855 (2005).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from the US National Institutes of Health (NIH), the NIH Director's Pioneer Award of the NIH Roadmap for Medical Research, the Leukemia and Lymphoma Society, and by private funds from the Thomas Anthony Pappas Charitable Foundation. G.Q.D. is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. M.A.S. was supported by a Johnson & Johnson fellowship of the Life Science Research Foundation, Baltimore, and by NIH K99GM08009.

Author information

Authors and Affiliations

Authors

Contributions

M.A. designed and performed the experiments and analyzed and interpreted the data. M.A.S. crystallized the gatekeeper mutant of the SRC kinase. N.S.G. provided the compound 14 and helped in the interpretation of the data. J.K. provided the critical input on gatekeeper mutant kinase models and supervised the solution of the SRC kinase structure. G.Q.D. supervised experimental design and data interpretation. M.A. and G.Q.D wrote the manuscript with input from M.A.S., N.S.G. and J.K. All authors approved the final manuscript.

Corresponding author

Correspondence to George Q Daley.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 5839 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Azam, M., Seeliger, M., Gray, N. et al. Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol 15, 1109–1118 (2008). https://doi.org/10.1038/nsmb.1486

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1486

This article is cited by

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