RAF Inhibitors Activate the MAPK Pathway by Relieving Inhibitory Autophosphorylation

Summary ATP competitive inhibitors of the BRAF V600E oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF WT ). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF WT ) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF V600E and RAF WT enzymes. We show that activation of RAF WT 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 BRAF WT cells but also offers a biochemical mechanism for BRAF oncogenesis.