Abstract A31: Germline RASopathy Mutations Provide Functional Insights into the Raf Cysteine-Rich Domain (CRD)

Abstract The three-member RAF kinase family (A-, B- and C-Raf) are direct Ras effectors and key mediators of the proproliferative MAPK pathway. Somatic driver mutations in the B-Raf kinase domain occur frequently in melanoma and cancers of the thyroid and large intestine. These are predominantly the V600E mutation, which allows B-Raf to signal constitutively as a monomer. Germline mutations in B-Raf also occur and are present in ~67% of the childhood developmental RASopathy-cardiofaciocutaneous (CFC) syndrome. Interestingly, many of these CFC mutations cluster around the cysteine-rich domain (CRD) in the B-Raf regulatory region. CRDs are zinc stabilized structures with two flexible loops that are shown to bind phosphatidylserine (PS), DAG and pro-oncogenic phorbol esters in several PKC family members. Although the B-Raf CRD has not been well characterized, prior studies examining the C-Raf CRD show that it binds to PS through its first flexible loop but does not bind DAG or phorbol esters, likely due to its truncated second loop, which is found in all Raf members. In addition to PS binding, C-Raf CRD may also interact directly with Ras, suggesting a second Ras binding site outside of its Ras binding domain (RBD). Interestingly, B-Raf RASopathy mutations occur in both CRD loops and commonly introduce a positively charged residue suited to interacting with negatively charged lipids in the plasma membrane (PM). Of note, Q257R, which is the most common mutation in CFC, maps to the truncated second loop. Our in-cell studies with full-length B-Raf have shown that RASopathy mutations in both loops enhance biologic activity, PM localization and Ras interaction. However, the biologic activity of loop 1 mutations requires an intact RBD, which is not the case for the loop 2 mutant, Q257R. These data suggest that the Q257R mutant either has increased binding to Ras or allows B-Raf to signal independently of Ras. To further investigate these findings, we are examining differences in localization, Ras interaction and lipid binding using the isolated CRDs of C-Raf, B-Raf and the B-Raf RASopathy mutants. As may be predicted, the RASopathy mutations increased membrane localization over WT B-Raf, with Q257R being the most PM localized. Somewhat surprisingly, expression of the wild-type B-Raf CRD in cells showed greatly enhanced PM localization compared to that of wild-type C-Raf, suggesting functional differences between their CRDs, which we are currently investigating. Citation Format: Russell Spencer-Smith, Elizabeth M. Terrell, Constance Agamasu, Daniel A. Ritt, Alyson K. Freeman, Andrew G. Stephen, Deborah K. Morrison. Germline RASopathy mutations provide functional insights into the Raf cysteine-rich domain (CRD) [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A31.