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

Kondo, Y.; Notbohm, J.; Navas Camacho, I.; Nagy-Davidescu, G.; Mason, T.; Muehle, J.; Standfuss, J.; Perica, T.

doi:10.1101/2025.09.26.678760

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bioRxiv · 10.1101/2025.09.26.678760

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

Kondo, Y.·Notbohm, J.·Navas Camacho, I.·Nagy-Davidescu, G.·Mason, T.·Muehle, J.·Standfuss, J.·Perica, T.

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Abstract

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.

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Kondo, Y., Notbohm, J., Navas Camacho, I., Nagy-Davidescu, G., Mason, T., Muehle, J., Standfuss, J., Perica, T.. 2025-09-26. Mechanism of MEK1 phosphorylation by the N-terminal acidic motif mediated asymmetric BRAF dimer. https://doi.org/10.1101/2025.09.26.678760

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