RAF Inhibitors Activate the MAPK Pathway by Relieving Inhibitory Autophosphorylation

•BRAFV600E inhibitors activate recombinant purified BRAFWT and CRAF in vitro•RAFWT but not BRAF oncogenes are autoinhibited through P-loop autophosphorylation•P-loop autophosphorylation is required for inhibitors to activate RAFWT•Preventing P-loop autophosphorylation activates RAF monomers

SummaryATP competitive inhibitors of the BRAFV600E oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAFWT). In this study, we investigate the biochemical mechanism of wild-type RAF (RAFWT) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAFV600E and RAFWT enzymes. We show that activation of RAFWT is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF’s phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAFWT cells but also offers a biochemical mechanism for BRAF oncogenesis.

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