RASopathy Mutations Demonstrate the Critical Function of the Cysteine-rich Domain in Maintaining BRAF in an Autoinhibited State to Prevent Aberrant Signaling

The RAF kinases are essential effectors of the RAS-GTPases, and germline mutations in BRAF or CRAF are disease drivers in the RASopathy developmental syndromes, with mutations in the cysteine-rich domain (CRD) often observed in BRAF but not CRAF. Although the CRD is known for roles in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the interplay of these activities and their impact on BRAF function in normal and disease states is unclear. Here, we analyze a panel of BRAF-CRD RASopathy mutations and show that they all increase the biological activity of BRAF by relieving autoinhibition and/or enhancing PS/membrane binding. Strikingly, we find that relief of autoinhibition is the predominant factor determining the severity of the mutations. Comparison of the BRAF and CRAF CRDs further indicates that the BRAF-CRD is a stronger mediator of both autoinhibition and PS binding, and we identify residues contributing to these differences. Moreover, given the increased catalytic activity of BRAF versus CRAF, our studies reveal a more critical role for the CRD and autoinhibition in BRAF regulation and demonstrate that CRD-mediated autoinhibition is required to prevent aberrant BRAF signaling, a finding consistent with the high frequency of mutations that disrupt this function in the RASopathies.