Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAF^V600E and wild‐type BRAF (BRAF^WT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAF^V600E resides in large complexes of higher molecular mass and activity, while BRAF^WT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐Ras^G12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAF^WT into large, active complexes, whereas pharmacological inhibition of BRAF^V600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.