RASopathy Mutations Provide Functional Insight into the BRAF Cysteine-rich Domain and Demonstrate the Importance of Autoinhibition in BRAF Regulation

Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well-characterized. In this study, we have conducted an in-depth analysis of a panel of RASopathy-associated B-Raf CRD mutations for their effects on CRD function using a combination of approaches, including surface plasmon resonance analysis to measure PS binding, a bioluminescence resonance energy transfer (BRET) assay to monitor the Ras/Raf interaction in live cells, and a novel BRET assay developed by our group to quantify Raf autoinhibitory interaction under live cell conditions.

In addition, we have examined the impact of these mutations on the biological function of BRAF in cell culture systems and zebrafish embryos, and we have compared the properties of the BRAF-CRD with those of the CRAF-CRD. Through this analysis, we find that the BRAF-CRD is a stronger mediator of both PS binding and autoinhibition than is the CRAF-CRD and that the RASopathy-associated BRAF-CRD mutations can be classified into three functional groups: Class A, causing relief in autoinhibition; Class B, relieving autoinhibition and enhancing PS/plasma membrane binding; and Class C, enhancing PS/membrane binding alone.