ARAF Activates RAS through Competition with the RASGAP NF1

Activating mutations in the RAS family of small guanosine triphosphatase (GTPase) proteins are common in cancer, and, in their active GTP-bound state, RAS proteins bind and activate effector RAF kinases (ARAF, BRAF, and CRAF) by promoting their homodimerization or heterodimerization. RAF kinases then initiate a mitogenic kinase cascade by phosphorylating and activating MEK kinases which promote proliferation. BRAF and CRAF are active kinases that are required for downstream MEK–ERK signaling, but the function of ARAF, which has lower endogenous kinase activity, still remains to be fully understood. Su, Mukherjee, and colleagues observed that ARAF S214 mutants, which are known oncogenic drivers, function as RAS-independent dimers but do not cause feedback inhibition of RAS-GTP–like activating BRAF and CRAF mutants. Investigation into the ARAF-mediated induction of RAS activation revealed that the unique N-terminus of ARAF and ARAF binding to RAS, but not ARAF dimerization or kinase activity, are necessary for inducing RAS activity. Further studies showed that ARAF reduced GTP hydrolysis as well as the amount of the RAS GTPase activating protein (RASGAP) NF1 (a negative regulator of RAS that accelerates RAS-GTP hydrolysis) bound to RAS, while no other RASGAPs were affected. Additionally, in cells expressing only ARAF, the kinetics of RAS activation and MEK/ERK phosphorylation after ligand-induced EGFR phosphorylation were prolonged compared with cells expressing only BRAF or CRAF. Moreover, ARAF expression was found to alter the duration and consequences of ligand-activated RAS signaling, and its overexpression through gene amplification led to acquired resistance to EGFR inhibition in patients with lung cancer. In preclinical models, resistance could be overcome with ARAF knockdown or cotreatment with an inhibitor of SHP2, a RAS guanine nucleotide exchange factor (RASGEF) typically opposed by NF1 RASGAP activity. Overall, this study shows ARAF not only acts as an effector of RAS but also activates it in a kinase-independent manner, and points to a potential therapeutic strategy in ARAF-altered tumors.

Su W, Mukherjee R, Yaeger R, Son J, Xu J, Na N, et al. ARAF protein kinase activates RAS by antagonizing its binding to RASGAP NF1. Mol Cell 2022 May 24 [Epub ahead of print].

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