BRAF oncogenic mutants evade autoinhibition through a common mechanism.
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| Title: | BRAF oncogenic mutants evade autoinhibition through a common mechanism. |
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| Authors: | Lavoie, Hugo, Jin, Ting, Lajoie, Driss, Decossas, Marion, Gendron, Patrick, Wang, Bing, Filandr, Frantisek, Sahmi, Malha, Hwa Jo, Chang, Weber, Sandra, Arseneault, Geneviève, Tripathy, Sasmita, Beaulieu, Pierre, Schuetz, Doris A., Schriemer, David C., Marinier, Anne, Rice, William J., Maisonneuve, Pierre, Therrien, Marc |
| Source: | Science. 5/29/2025, Vol. 388 Issue 6750, p1-12. 12p. |
| Subjects: | Sarcoma, Genetic mutation, Dimerization, Guanosine triphosphatase, Conformational analysis |
| Abstract: | Uncontrolled activation of the rat sarcoma (RAS)–extracellular signal–regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving monomeric autoinhibition and activation by dimerization, has been intensely scrutinized, but mechanisms enabling oncogenic mutants to evade regulation remain unclear. By using cryo–electron microscopy, we solved the three-dimensional structures of the three oncogenic BRAF mutant classes, including the common V600E variant. These mutations disrupted wild-type BRAF's autoinhibited state, mediated by interactions between the cysteine-rich domain and kinase domain, thereby shifting the kinase domain into a preactivated conformation. This structural change likely results from helix αC displacement. PLX8394, a BRAF inhibitor that stabilizes helix αC in an inactive conformation, restored the autoinhibited conformation of oncogenic BRAF, explaining the properties of this class of compounds. Editor's summary: The protein kinase BRAF functions in mitogenic signaling from the RAS small guanosine triphosphatase to activate mitogen-activated protein kinases. Activated mutants of BRAF are oncogenic. Lavoie et al. solved structures of such mutants using cryo–electron microscopy, which helps to clarify how conformational changes relieve an autoinhibitory domain interaction within the enzyme. A BRAF inhibitor was found to restore the interaction of the autoinhibitory domain with the kinase domain. These results help to explain the regulation of normal and oncogenic BRAF and may assist in the development of more effective inhibitors for cancer therapy. —L. Bryan Ray INTRODUCTION: Cells utilize a complex network of signaling pathways to integrate and respond to external cues. The rat sarcoma (RAS)–extracellular signal–regulated kinase (ERK) signaling pathway regulates cell proliferation and differentiation. Upon activation by receptors on the cell surface, the small guanosine triphosphatase RAS recruits and activates kinases from the rapidly accelerated fibrosarcoma (RAF) family. These kinases initiate a phosphorylation cascade, ultimately modulating numerous target proteins that drive specific cellular responses. One prominent member of the RAF family, BRAF, is frequently activated by mutations in human cancers. RATIONALE: Under quiescent conditions, wild-type (WT) BRAF is maintained in a monomeric autoinhibited state, stabilized by interactions between its cysteine-rich domain (CRD) and kinase domain (KD; CRD-in conformation). Upon growth factor stimulation, BRAF undergoes activation through KD dimerization. The most prevalent class of BRAF mutations (Class 1) appear to induce constitutive activation independently of RAS activity and bypass the requirement for KD dimerization. However, the structural basis for these properties has remained speculative. We used cryo–electron microscopy (cryo-EM) to determine the three-dimensional (3D) structures of oncogenic BRAF variants and compared them to those of WT BRAF. This revealed the alterations caused by the mutations, offering insights into the molecular mechanisms underlying BRAF oncogenic activation. RESULTS: The 3D structure of monomeric BRAF V600E, encoded by the most prevalent Class 1 oncogenic mutation in the BRAF gene, revealed features characteristic of an active dimer despite the monomeric state. The inhibitory interaction between the CRD and the KD was disrupted (CRD-out conformation), and the KD adopted an active-like orientation, resembling the conformation of a single BRAF molecule in an active dimer. The inward positioning of helix αC within the KD was consistent with the active state. This inward movement of helix αC, driven by the V600E mutation, was shown to be the main mechanism underlying the structural reorganization of BRAF. By using small-molecule inhibitors that stabilize either the inward (active) or outward (inactive) position of helix αC, we demonstrated that this conformational shift is central to the activation mechanism induced by the V600E mutation. We also solved the 3D structures of representative class 2 and 3 oncogenic BRAF variants. These variants exhibited the same global reorganization of the KD observed in BRAF V600E, including the disruption of autoinhibitory interactions. Our findings are consistent with a common mechanism by which oncogenic BRAF mutations induce kinase activation through the release of autoinhibition and the adoption of an active-like KD conformation driven by helix αC inward movement. CONCLUSION: This study establishes a structural and mechanistic framework that unifies the mode of action of diverse oncogenic BRAF mutations, providing crucial insights into their common activation mechanisms and potential opportunities for targeted therapeutic intervention. The active-like conformation adopted by these oncogenic variants resembles a hybrid state between the autoinhibited monomer and active dimer configurations observed in WT BRAF. This similarity suggests that the oncogenic variants may mimic a transition state that WT BRAF undergoes during its shift from an inactive monomer to an active dimer. Cryo-EM structural analysis of oncogenic BRAF variants (red shading) compared with WT BRAF (blue shading), leading to their unrestrained activity.: WT BRAF adopts a stable, autoinhibited monomeric state in which its CRD (C) forms inhibitory contacts with the KD (K), stabilized by a 14-3-3 protein dimer (CRD-in). Upon binding to activated RAS at the plasma membrane, WT BRAF forms a catalytically active dimer, enabling downstream signaling to mitogen-activated protein kinase kinase (MEK) and ERK. Oncogenic BRAF mutations induce an inward shift of the KD's helix αC, disrupting CRD-KD interactions and repositioning the KD into a preactive conformation (CRD-out). Class 1 mutants are constitutively active as stable monomers, independent of dimerization. Class 2 mutants also form CRD-out monomers but require dimerization for full activation. Class 3 mutants, which are catalytically impaired, adopt a CRD-out monomeric state that enhances RAS binding and facilitates dimerization and transactivation of another catalytically competent RAF protein, such as CRAF. [ABSTRACT FROM AUTHOR] |
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| Database: | Psychology and Behavioral Sciences Collection |
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| Abstract: | Uncontrolled activation of the rat sarcoma (RAS)–extracellular signal–regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving monomeric autoinhibition and activation by dimerization, has been intensely scrutinized, but mechanisms enabling oncogenic mutants to evade regulation remain unclear. By using cryo–electron microscopy, we solved the three-dimensional structures of the three oncogenic BRAF mutant classes, including the common V600E variant. These mutations disrupted wild-type BRAF's autoinhibited state, mediated by interactions between the cysteine-rich domain and kinase domain, thereby shifting the kinase domain into a preactivated conformation. This structural change likely results from helix αC displacement. PLX8394, a BRAF inhibitor that stabilizes helix αC in an inactive conformation, restored the autoinhibited conformation of oncogenic BRAF, explaining the properties of this class of compounds. Editor's summary: The protein kinase BRAF functions in mitogenic signaling from the RAS small guanosine triphosphatase to activate mitogen-activated protein kinases. Activated mutants of BRAF are oncogenic. Lavoie et al. solved structures of such mutants using cryo–electron microscopy, which helps to clarify how conformational changes relieve an autoinhibitory domain interaction within the enzyme. A BRAF inhibitor was found to restore the interaction of the autoinhibitory domain with the kinase domain. These results help to explain the regulation of normal and oncogenic BRAF and may assist in the development of more effective inhibitors for cancer therapy. —L. Bryan Ray INTRODUCTION: Cells utilize a complex network of signaling pathways to integrate and respond to external cues. The rat sarcoma (RAS)–extracellular signal–regulated kinase (ERK) signaling pathway regulates cell proliferation and differentiation. Upon activation by receptors on the cell surface, the small guanosine triphosphatase RAS recruits and activates kinases from the rapidly accelerated fibrosarcoma (RAF) family. These kinases initiate a phosphorylation cascade, ultimately modulating numerous target proteins that drive specific cellular responses. One prominent member of the RAF family, BRAF, is frequently activated by mutations in human cancers. RATIONALE: Under quiescent conditions, wild-type (WT) BRAF is maintained in a monomeric autoinhibited state, stabilized by interactions between its cysteine-rich domain (CRD) and kinase domain (KD; CRD-in conformation). Upon growth factor stimulation, BRAF undergoes activation through KD dimerization. The most prevalent class of BRAF mutations (Class 1) appear to induce constitutive activation independently of RAS activity and bypass the requirement for KD dimerization. However, the structural basis for these properties has remained speculative. We used cryo–electron microscopy (cryo-EM) to determine the three-dimensional (3D) structures of oncogenic BRAF variants and compared them to those of WT BRAF. This revealed the alterations caused by the mutations, offering insights into the molecular mechanisms underlying BRAF oncogenic activation. RESULTS: The 3D structure of monomeric BRAF V600E, encoded by the most prevalent Class 1 oncogenic mutation in the BRAF gene, revealed features characteristic of an active dimer despite the monomeric state. The inhibitory interaction between the CRD and the KD was disrupted (CRD-out conformation), and the KD adopted an active-like orientation, resembling the conformation of a single BRAF molecule in an active dimer. The inward positioning of helix αC within the KD was consistent with the active state. This inward movement of helix αC, driven by the V600E mutation, was shown to be the main mechanism underlying the structural reorganization of BRAF. By using small-molecule inhibitors that stabilize either the inward (active) or outward (inactive) position of helix αC, we demonstrated that this conformational shift is central to the activation mechanism induced by the V600E mutation. We also solved the 3D structures of representative class 2 and 3 oncogenic BRAF variants. These variants exhibited the same global reorganization of the KD observed in BRAF V600E, including the disruption of autoinhibitory interactions. Our findings are consistent with a common mechanism by which oncogenic BRAF mutations induce kinase activation through the release of autoinhibition and the adoption of an active-like KD conformation driven by helix αC inward movement. CONCLUSION: This study establishes a structural and mechanistic framework that unifies the mode of action of diverse oncogenic BRAF mutations, providing crucial insights into their common activation mechanisms and potential opportunities for targeted therapeutic intervention. The active-like conformation adopted by these oncogenic variants resembles a hybrid state between the autoinhibited monomer and active dimer configurations observed in WT BRAF. This similarity suggests that the oncogenic variants may mimic a transition state that WT BRAF undergoes during its shift from an inactive monomer to an active dimer. Cryo-EM structural analysis of oncogenic BRAF variants (red shading) compared with WT BRAF (blue shading), leading to their unrestrained activity.: WT BRAF adopts a stable, autoinhibited monomeric state in which its CRD (C) forms inhibitory contacts with the KD (K), stabilized by a 14-3-3 protein dimer (CRD-in). Upon binding to activated RAS at the plasma membrane, WT BRAF forms a catalytically active dimer, enabling downstream signaling to mitogen-activated protein kinase kinase (MEK) and ERK. Oncogenic BRAF mutations induce an inward shift of the KD's helix αC, disrupting CRD-KD interactions and repositioning the KD into a preactive conformation (CRD-out). Class 1 mutants are constitutively active as stable monomers, independent of dimerization. Class 2 mutants also form CRD-out monomers but require dimerization for full activation. Class 3 mutants, which are catalytically impaired, adopt a CRD-out monomeric state that enhances RAS binding and facilitates dimerization and transactivation of another catalytically competent RAF protein, such as CRAF. [ABSTRACT FROM AUTHOR] |
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| ISSN: | 00368075 |
| DOI: | 10.1126/science.adp2742 |
