Letter

MEK drives BRAF activation through allosteric control of KSR proteins

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Accepted:
Published online:

Abstract

RAF family kinases have prominent roles in cancer1. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development2,3. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK)4. Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically5. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF–KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR–MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

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Acknowledgements

We thank L. Volpon, M. Osborne, G. Seabrook, and the Institute for Research in Immunology and Cancer High-Throughput Screening and Proteomics facilities for experimental assistance. H.L. was supported by a Canadian Institutes for Health Research (CIHR) Banting fellowship. N.T. was supported by a CIHR scholarship. Research was supported by Impact Grants from the Canadian Cancer Society (702319 to M.T. and 704116 to F.S.) and by CIHR operating grants (MOP119443 and PJT152873 to M.T. and FDN143277 to F.S.). M.T. and F.S. hold Canada Research Chairs in Intracellular Signaling and in Structural Biology of Signaling. Synchrotron work at the Northeastern Collaborative Access Team beamlines was supported by National Institutes of Health grants (P41 GM103403 and S10 RR029205).

Author information

Author notes

    • Hugo Lavoie
    • , Malha Sahmi
    •  & Pierre Maisonneuve

    These authors contributed equally to this work.

Affiliations

  1. Institute for Research in Immunology and Cancer Laboratory of Intracellular Signaling Université de Montréal C.P. 6128, Succursale Centre-Ville Montréal, Québec H3C 3J7, Canada.

    • Hugo Lavoie
    • , Malha Sahmi
    • , Sara A. Marullo
    • , Ting Jin
    •  & Marc Therrien
  2. Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario M5G 1X5, Canada

    • Pierre Maisonneuve
    • , Neroshan Thevakumaran
    •  & Frank Sicheri
  3. Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Neroshan Thevakumaran
    •  & Frank Sicheri
  4. NE-CAT APS, Building 436E, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois 60439, USA

    • Igor Kurinov
  5. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Frank Sicheri
  6. Département de pathologie et biologie cellulaire, Université de Montréal, Québec H3C 3J7, Canada

    • Marc Therrien

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Contributions

H.L., M.S., P.M., S.A.M., F.S., and M.T. designed the experiments. H.L., P.M., F.S., and M.T. wrote the manuscript. H.L. performed BRET assays with T.J. M.S. performed co-IP and transactivation assays. H.L. and S.A.M. conducted Y2H experiments. P.M., N.T., and I.K. performed structural and biophysical experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Frank Sicheri or Marc Therrien.

Reviewer Information Nature thanks D. Barford, S. Hubbard and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Figure 1

    This file contains gel source data for immunoblots presented in the main and extended data figures including molecular weight markers (kDa).

Excel files

  1. 1.

    Supplementary Table 1

    The raw data of BRET-based whole-kinome screen for modulators of BRAF-KSR1 kinase domains interaction.

  2. 2.

    Supplementary Table 2

    MEK1 mutations that enable KSR1W831R-MEK1 interaction.

Videos

  1. 1.

    Conformational modification of the CC-SAM domain induced by binding of the BRS domain

    Conformational modification of the CC-SAM domain induced by binding of the BRS domain.

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