Investigations of BRAF Regulation through Auto‐Inhibition and HRAS Interaction

Abstract

BRAF is a key member in the MAPK signaling pathway essential for normal cell growth, reproduction, and differentiation. For this reason, BRAF is also the cause of numerous cancers when misregulated or mutated. Upstream of BRAF is the small, compact GTPase, RAS—a protein implicated in nearly a third of all cancers, yet extremely difficult to target due to its shape and shallow binding pocket. Upon interaction with RAS through the RAS binding domain (RBD) and membrane recruitment, BRAF transitions from an inactive, auto‐inhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. The auto‐inhibited conformation is thought to be stabilized through interactions between the Cysteine Rich Domain (CRD) and the Kinase Domain (KD) of BRAF. Despite its central role in cellular signal transduction, the order of BRAF’s activation steps have yet to be experimentally validated. We propose that by studying the inter‐ and intra‐molecular interactions of BRAF, we will begin to unveil the fine details of RAF regulation, important for developing new strategies towards therapeutic intervention. We performed biophysical, biochemical, and cell‐based assays to further investigate the role of the regulatory regions in BRAF activation and auto‐inhibition. We quantified in vitro binding between the N‐terminal domains of BRAF and the KD as well as binding between the N‐terminal domains of BRAF and HRAS through Surface Plasmon Resonance (SPR). We show that the interaction between RAS and regulatory regions of BRAF disrupt the interaction between the regulatory regions and catalytic region of BRAF, providing direct evidence that RAS binding to BRAF relieves the auto‐inhibitory interactions among BRAF domains. Furthermore, we examined the effect of Raf‐mimetic peptides in disrupting the RAS‐RAF interaction and relief of auto‐inhibition. The studies outlined here are leading to promising new insights into the activation mechanism of BRAF kinase.