Mechanism of MEK1 phosphorylation by the N-terminal acidic motif-mediated asymmetric BRAF dimer

The RAF/MEK/ERK signaling cascade regulates cell proliferation and differentiation and is frequently dysregulated in cancer. Approximately 90% of RAF-mutant cancers harbour mutations in B-type of Rapidly Accelerated Fibrosarcoma (BRAF). Its proto-oncogenicity has been attributed to a four-residue N-terminal acidic (NtA) motif. While a long-standing model proposes that the NtA promotes an activating asymmetric RAF dimerization, this idea has lacked structural support. Here, we present the first X-ray crystal structure of an NtA-mediated asymmetric BRAF dimer bound to its substrate Mitogen-activated Protein kinase Kinase (MEK1). Further cellular and biochemical analyses reveal that this asymmetric dimer is not an intermediate along the BRAF activation pathway, but rather a catalytically active state of the kinase. We validate this with the structure of BRAF bound to Ser222-phosphorylated MEK1, capturing the complex in a post-catalytic state. This combination of structural and cellular analysis establishes the relevance of NtA-driven asymmetry in BRAF activation and resolves a long-standing disconnect between RAF cancer genetics and structural biology.