title: Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization.
abstract: RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.
authors.affiliation: Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signalling, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Hugo
authors.initials: H
authors.lastname: Lavoie
authors.forename: Neroshan
authors.initials: N
authors.lastname: Thevakumaran
authors.forename: Gwenaëlle
authors.initials: G
authors.lastname: Gavory
authors.forename: John J
authors.initials: JJ
authors.lastname: Li
authors.forename: Abbas
authors.initials: A
authors.lastname: Padeganeh
authors.forename: Sébastien
authors.initials: S
authors.lastname: Guiral
authors.forename: Jean
authors.initials: J
authors.lastname: Duchaine
authors.forename: Daniel Y L
authors.initials: DY
authors.lastname: Mao
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.forename: Marc
authors.initials: M
authors.lastname: Therrien
mesh_terms: D015374:Biosensing Techniques; D003460:Crystallization; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004735:Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D049449:Luminescence; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D018076:DNA, Complementary; D047428:Protein Kinase Inhibitors; D019908:Proto-Oncogene Proteins c-raf