In vitro reconstitutions suggest a general model for paradoxical activation of ARAF, BRAF, and CRAF by diverse RAF inhibitor types that does not rely on negative allostery.

RAF kinases are central regulators of the RAS/MAP kinase pathway and important targets in cancer therapy. Paradoxically, RAF inhibitors can activate wild-type RAF signaling. Negative allostery is a central feature of the prevailing model for this phenomenon, wherein inhibitors induce RAF dimers in which inhibitor binding to one protomer promotes an active but inhibitor-resistant conformation in the other protomer. Here we systematically examined paradoxical activation of ARAF, BRAF, and CRAF using biochemical assays with isolated RAF/MEK kinase domain complexes. We found that type I and type II inhibitors induce paradoxical activation of all three isoforms, and that phosphomimetic mutation of the N-terminal acidic motif of ARAF and CRAF dramatically sensitized these isoforms to activation by type II inhibitors. The inhibition phase of paradoxical activation curves for type II inhibitors was suggestive of positive cooperativity, a finding in conflict with the prevailing model which implies negative cooperativity. In contrast to the kinase domain RAF/MEK preparations, full-length autoinhibited RAF/MEK/14-3-3 complexes were refractory to activation. Mass photometry confirmed that paradoxical activators promote BRAF dimerization. These findings support a revised model that does not rely on negative allostery. Inhibitors act on the RAS-engaged "open monomer" state to induce dimerization and activation. The open monomer and active dimer are structurally distinct species with differing affinities for inhibitor and ATP, creating a concentration window in which paradoxical activation occurs.