Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization

Abstract

The RAS–RAF–MEK–ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAFKD) dimers that are critical for activity. Here we present a 2.9-Å-resolution crystal structure of human BRAFKD in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAFKD. We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAFKD–14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAFKD assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: ACP breaks BRAFKD dimers.
Fig. 2: 14-3-3 stabilizes BRAF dimers in presence of ATP and acts as a molecular switch.
Fig. 3: 14-3-3 stabilizes BRAF dimer and increases BRAF catalytic activity.
Fig. 4: Oncogenic mutations in BRAF perturb ACP-induced monomer conformation.
Fig. 5: ACP-bound BRAFKD—implications for drug design.
Fig. 6: Model for dimerization-based regulation of RAF kinase activity.

Data Availability

Coordinates for the X-ray crystal structures have been deposited in the PDB with accession codes 6U2G and 6U2H.

References

  1. 1.

    Lavoie, H. & Therrien, M. Regulation of RAF protein kinases in ERK signalling. Nat. Rev. Mol. Cell Biol. 16, 281–298 (2015).

    CAS  PubMed  Google Scholar 

  2. 2.

    Haling, J. R. et al. Structure of the BRAF–MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling. Cancer Cell 26, 402–413 (2014).

    CAS  PubMed  Google Scholar 

  3. 3.

    Thorson, J. A. et al. 14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity. Mol. Cell. Biol. 18, 5229–5238 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Tzivion, G., Luo, Z. J. & Avruch, J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 394, 88–92 (1998).

    CAS  PubMed  Google Scholar 

  5. 5.

    Rajakulendran, T., Sahmi, M., Lefrancois, M., Sicheri, F. & Therrien, M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature 461, 542–545 (2009).

    CAS  PubMed  Google Scholar 

  6. 6.

    Kubiniok, P., Lavoie, H., Therrien, M. & Thibault, P. Time-resolved phosphoproteome analysis of paradoxical RAF activation reveals novel targets of ERK. Mol. Cell Proteom. 16, 663–679 (2017).

    CAS  Google Scholar 

  7. 7.

    Kondo, Y. et al. Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases. Science 366, 109–115 (2019).

    CAS  PubMed  Google Scholar 

  8. 8.

    Park, E. et al. Architecture of autoinhibited and active BRAF–MEK1–14-3-3 complexes. Nature 575, 545–550 (2019).

    CAS  PubMed  Google Scholar 

  9. 9.

    Tate, J. G. et al. COSMIC: the catalogue of somatic mutations in cancer. Nucleic Acids Res. 47, D941–D947 (2019).

    CAS  PubMed  Google Scholar 

  10. 10.

    Poulikakos, P. I. et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480, 387–390 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Vido, M. J., Le, K., Hartsough, E. J. & Aplin, A. E. BRAF splice variant resistance to RAF inhibitor requires enhanced MEK association. Cell Rep. 25, 1501–1510 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010).

    CAS  PubMed  Google Scholar 

  13. 13.

    Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Carnahan, J. et al. Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth. Mol. Cancer Ther. 9, 2399–2410 (2010).

    CAS  PubMed  Google Scholar 

  15. 15.

    Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Freeman, A. K., Ritt, D. A. & Morrison, D. K. The importance of Raf dimerization in cell signaling. Small GTPases 4, 180–185 (2013).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Durrant, D. E. & Morrison, D. K. Targeting the Raf kinases in human cancer: the Raf dimer dilemma. Br. J. Cancer 118, 3–8 (2018).

    CAS  PubMed  Google Scholar 

  18. 18.

    Lavoie, H. et al. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat. Chem. Biol. 9, 428–436 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Bridges, D. & Moorhead, G. B. 14-3-3 proteins: a number of functions for a numbered protein. Sci. STKE 2005, re10 (2005).

    PubMed  Google Scholar 

  20. 20.

    Muslin, A. J., Tanner, J. W., Allen, P. M. & Shaw, A. S. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 (1996).

    CAS  PubMed  Google Scholar 

  21. 21.

    Knighton, D. R. et al. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 414–420 (1991).

    CAS  PubMed  Google Scholar 

  22. 22.

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

    CAS  PubMed  Google Scholar 

  23. 23.

    Xie, T. et al. Structural insights into RIP3-mediated necroptotic signaling. Cell Rep. 5, 70–78 (2013).

    CAS  PubMed  Google Scholar 

  24. 24.

    Huang, C. S. et al. Crystal structure of Ripk4 reveals dimerization-dependent kinase activity. Structure 26, 767–777 (2018).

    CAS  PubMed  Google Scholar 

  25. 25.

    Wan, P. T. et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).

    CAS  PubMed  Google Scholar 

  26. 26.

    Molzan, M. et al. Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers. ACS Chem. Biol. 8, 1869–1875 (2013).

    CAS  PubMed  Google Scholar 

  27. 27.

    Morrison, D. K., Heidecker, G., Rapp, U. R. & Copeland, T. D. Identification of the major phosphorylation sites of the Raf-1 kinase. J. Biol. Chem. 268, 17309–17316 (1993).

    CAS  PubMed  Google Scholar 

  28. 28.

    Shaw, A. S., Kornev, A. P., Hu, J., Ahuja, L. G. & Taylor, S. S. Kinases and pseudokinases: lessons from RAF. Mol. Cell. Biol. 34, 1538–1546 (2014).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Thevakumaran, N. et al. Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation. Nat. Struct. Mol. Biol. 22, 37–43 (2015).

    CAS  PubMed  Google Scholar 

  30. 30.

    Schreiber, G., Haran, G. & Zhou, H. X. Fundamental aspects of protein–protein association kinetics. Chem. Rev. 109, 839–860 (2009).

    CAS  PubMed  Google Scholar 

  31. 31.

    Morrison, J. F. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta 185, 269–286 (1969).

    CAS  PubMed  Google Scholar 

  32. 32.

    Yao, Z. et al. Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS. Nature 548, 234–238 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Yao, Z. et al. RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling. Nat. Med. 25, 284–291 (2019).

    CAS  PubMed  Google Scholar 

  34. 34.

    Michaud, N. R. et al. KSR stimulates Raf-1 activity in a kinase-independent manner. Proc. Natl Acad. Sci. USA 94, 12792–12796 (1997).

    CAS  PubMed  Google Scholar 

  35. 35.

    Lavoie, H. et al. MEK drives BRAF activation through allosteric control of KSR proteins. Nature 554, 549–553 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Verlande, A. et al. Metabolic stress regulates ERK activity by controlling KSR-RAF heterodimerization. EMBO Rep. 19, 320–336 (2018).

    CAS  PubMed  Google Scholar 

  37. 37.

    Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl Acad. Sci. USA 105, 3041–3046 (2008).

    CAS  PubMed  Google Scholar 

  38. 38.

    Mccoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Buss, O., Rudat, J. & Ochsenreither, K. FoldX as protein engineering tool: better than random based approaches? Comput. Struct. Biotechnol. J. 16, 25–33 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Schrodinger, L. L. C. The PyMOL Molecular Graphics System, Version 1.3r1. (2010).

  43. 43.

    Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).

    CAS  PubMed  Google Scholar 

  44. 44.

    Bern, M., Cai, Y. H. & Goldberg, D. Lookup peaks: a hybrid of de novo sequencing and database search for protein identification by tandem mass spectrometry. Anal. Chem. 79, 1393–1400 (2007).

    CAS  PubMed  Google Scholar 

  45. 45.

    Bern, M., Kil, Y. J. & Becker, C. Byonic: advanced peptide and protein identification software. Curr. Protoc. Bioinformatics 40, 13.20.1–13.20.14 (2012).

    Google Scholar 

Download references

Acknowledgements

We thank the BioMolecular Resources (BMR) group at Genentech for their support in generating constructs and biomass for protein production; Stanford Synchrotron Radiation Lightsource (SSRL) and Advanced Light Source (ALS) for access to the synchrotrons; and W. Wang, C. Noland for help with data collection and C. Sneeringer for assistance with enzyme assays. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Beamline 5.0.2 of the Advanced Light Source, a DOE Office of Science User Facility under Contract No. DE-AC02-05CH11231, is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169-01.

Author information

Affiliations

Authors

Contributions

N.P.D.L, T.J.W and J.S. designed, generated and biochemically characterized proteins and their complexes. N.P.D.L, S.G.H. and J.S. solved crystal structures. M.S., A.S.S. and J.G.Q. designed experiments and generated SPR data. F.J.I. and S.I. ran MD simulations. W.P., J.T. and P.L. generated mass spectrometry data. T.J.W., N.P.D.L., S.G.H., S.M., A.S.S. and J.S. analyzed all the data and wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Sarah G. Hymowitz or Jawahar Sudhamsu.

Ethics declarations

Competing interests

All authors were employees and/or shareholders of Genentech/Roche at the time the work was done.

Additional information

Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 ATP analog interacts intimately with and stabilizes monomers of BRAF and CRAF kinase domains.

a, SDS-PAGE gels from SEC traces in Fig. 1a. b, Size exclusion chromatography traces (left) showing CRAFKD-MEK1 complex alone (blue), in the presence of ACP (red) and in the presence of GDC-0879, a RAF dimer promoter (magenta). SDS-PAGE analysis of size exclusion experiments (right). The positions of CRAF and MEK1 are labeled to the right of each gel. Elution volumes at top correspond to all gels. c, BRAF-ACP key interactions diagram. d, BRAF-ACP interaction with key residues highlighted. Blue mesh represents electron density of ACP bound to BRAF, 2Fo-Fc map contoured at 2 σ.

Extended Data Fig. 2 14-3-3 induced RAF dimer (+/- MEK) is stable in presence of ATP analog when C-terminus of RAF is phosphorylated.

a, CRAFKD(FCT) (left) and BRAFKD(CFT) co-purify as a 2:2 complex with 14-3-3 as judged by preparative SEC (top) and SDS-PAGE (bottom). b, Identification of phosphorylation on BRAF-S729 and CRAF-S621 quantified at >90% by LC-MS/MS analysis. c, Proteins co-purifying with RAFKD(FCT) identified as insect 14-3-3ζ and 14-3-3ε by LC-MS/MS analysis. d, SDS-PAGE analysis of size exclusion experiments in Fig. 2a. The corresponding SEC trace for each gel is labeled to the left, while the positions of CRAF, BRAF, MEK and 14-3-3 are labeled to the right of each gel. Elution volumes at top correspond to all gels.

Extended Data Fig. 3 BRAFKD:14-3-3 complex assembled in vitro results in active conformation of BRAFKD, with BRAFKD dimer and 14-3-3 dimer flexibly tethered by phosphopeptide:14-3-3 interactions.

a, Identification of phosphorylation on pBRAFKD(BRAFKD-14m phosphorylated at S729) - S729 phosphorylation was quantified at >99% by LC-MS/MS analysis. b, SDS-PAGE analysis of size exclusion experiments in Fig. 2b. c, Alignment of the BRAF dimer (green) in an active conformation from the previously reported BRAFKD:MEK1 hetero-tetramer structure (PDB ID = 4MNE) aligned with the BRAF dimer (blue) from the BRAFKD:14-3-3 hetero-tetramer in Fig. 2c. d, Equilibrium molecular dynamics simulation showing crystal lattice induced BRAF-14-3-3 interface is transient, with increased solvent accessibility between BRAF and 14-3-3 in 3/5 simulations over time (top left, green, blue and black vs red and purple) and a decreased number of intermolecular contacts in 3/5 simulations over time (bottom left). Right panels show initial (cyan) and dissociated (red) conformations and beginning and ending non-pS729 region contacts (bottom). e, BRAF:BRAF, 14-3-3:14-3-3 and pS729 contacts do not change over time, indicating a real interaction.

Extended Data Fig. 4 Model of BRAF-KD:MEK:14-3-3 hexamer and unambiguous electron density of pS729 in BRAF bound to 14-3-3.

a, Model of hetero-hexamer formed by MEK1 (red, salmon), BRAF (blue, marine) and 14-3-3 (orange, yellow). Compared to the BRAF/14-3-3 crystal structure (Fig. 2c), the BRAF dimer must undock from the α9 helix of one 14-3-3 protomer to bind MEK1 (as observed by SEC in Fig. 2a). Undocking is easily feasible (inset) while maintaining pS729 binding (peptide shown in pink) to 14-3-3. b, Detailed view of one BRAF-pSer729 (blue) – 14-3-3 (orange) interaction. Electron density map is 2Fo – Fc contoured to 2.0σ.

Extended Data Fig. 5 Active site titration of various BRAF variants to estimate active enzyme concentration using Morrison kinetics.

(Left) Active site titration of GDC-0879 against indicated enzymes. Activities for each enzyme were normalized to a no inhibitor control and fitted to Morrison Ki. For BRAFKD, low inhibitor concentration points below the observed paradoxical activation maximum activity were removed to allow for fitting of Morrison Ki. Each point represents three time points assayed in duplicate (n=6), normalized to reaction time. Error bars represent SD of six replicates. (Right) Zoomed version of active site titration of BRAFKD including all inhibitor points, showing paradoxical activation.

Extended Data Fig. 6 Comparison of BRAF:14-3-3 complex from this study (x-ray) with recently published BRAF:14-3-3 complexes (CryoEM).

a, Crystal structure of BRAF dimer (blue) bound to 14-3-3 (yellow, orange) reported in this work, aligned by 14-3-3 dimer onto the BRAF:14-3-3 structure (left) reported in PDB: 6UAN, and (right) the BRAF:MEK1:14-3-3 structure reported by in PDB:6Q0J (BRAF-14-3-3 in grey, MEK1 in pink). b, Overlay of structures aligned by BRAF dimer, with structure pairs and coloring as described in a.

Extended Data Fig. 7 Conserved residues in all RAF isoforms stabilize ACP induced inactive state, which is disrupted by activating mutations like V600E in BRAF.

a, Alignment of ARAF, BRAF and CRAF across species shows conservation of key residues involved in the hydrophobic packing interactions displayed in Fig. 3c. b, BRAFKD-V600E:MEK1 complex alone (top), in the presence of ACP (middle) and in the presence of GDC-0879 (bottom) from SEC experiments in main Fig. 3c were analyzed by SDS-PAGE. The positions of MEK1 and BRAFV600E are labeled to the right of each gel.

Extended Data Fig. 8 BRAF inhibitors that are “Paradox busters” disrupt BRAF dimers by causing steric clash at the dimer interface.

BRAFKD dimer (yellow) overlaid with two PLX-4720 bound BRAFKD monomers (PDB code 4WO5) (blue), aligned by their C-lobes. While most of the N-lobe and C-lobe regions overlay well, PLX4720 induces a large shift of the aC helix, causing a steric clash at the dimer interface, destabilizing the dimer interface. This is likely the mechanism of the current series of “paradox buster” molecules. With PLX4720, this shift can be accommodated in a BRAF dimer, however this requires slight conformational changes at the dimer interface and in the binding orientation of one of two bound PLX4720 molecules (PDB code 3C4C).

Supplementary information

41594_2019_365_MOESM2_ESM.mpg

Conformational changes at the BRAF dimer interface upon ACP binding

41594_2019_365_MOESM3_ESM.mpg

Conformational changes in BRAF upon ACP binding (only one molecule shown)

41594_2019_365_MOESM4_ESM.mov

Molecular dynamics simulation showing that the BRAF N-lobe–14-3-3 interaction is unstable and that the BRAF dimer and 14-3-3 dimer interaction via phospho-peptide–14-3-3 interaction is stable

41594_2019_365_MOESM5_ESM.mov

Alignment of all known BRAFKD crystal structures suggesting that the BRAF-ATP analogue-bound structure is unique amongst all known BRAF kinase domain structures

Reporting Summary

Supplementary Video 1

Conformational changes at the BRAF dimer interface upon ACP binding

Supplementary Video 2

Conformational changes in BRAF upon ACP binding (only one molecule shown)

Supplementary Video 3

Molecular dynamics simulation showing that the BRAF N-lobe–14-3-3 interaction is unstable and that the BRAF dimer and 14-3-3 dimer interaction via phospho-peptide–14-3-3 interaction is stable

Supplementary Video 4

Alignment of all known BRAFKD crystal structures suggesting that the BRAF-ATP analogue-bound structure is unique amongst all known BRAF kinase domain structures

Source data

Source Data Fig. 3

Data used to generate curves in Fig. 3 for SPR data (BRAF–14-3-3 binding) and kinase activity data for various forms of BRAF tested

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liau, N.P.D., Wendorff, T.J., Quinn, J.G. et al. Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization. Nat Struct Mol Biol 27, 134–141 (2020). https://doi.org/10.1038/s41594-019-0365-0

Download citation

Further reading