Structure of the BRAF-MEK Complex Reveals a Kinase Activity Independent Role for BRAF in MAPK Signaling

Abstract

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…for B-Raf from the ERK MAPK cascade suggest its symmetric dimerization leaves its kinase domains free to accept substrates (33)(34)(35). This is consistent with the dimerization mode observed for the isolated kinase domains of the MAP3K ASK1 (36).…”

The complexities and versatility of the RAS-to-ERK signalling system in normal and cancer cells

“…Importantly, the activating RAF does not require kinase activity but an N-terminal phosphorylation that functions allosterically to induce cis-autophosphorylation of the receiving RAF. Structural studies of dimeric [72] and monomeric [73] RAF corroborated these findings and demonstrate that RAF proteins dimerize in a side-to-side conformation. Based on these structural data, molecular dynamic simulations revealed that phosphorylations of the so-called Nterminal acidic (NtA) motif facilitate RAF dimerisation by introducing crucial salt bridges, thus providing a structure-based mechanism for the auto-transactivation and asymmetry of RAF dimers [74,75].…”

“…While this was known for some time, only recently, the crystal structures of BRAF-MEK [72] and KSR2-MEK [76] were solved, thus providing novel insights: Both RAF and KSR form heterotetramers with MEK, where either BRAF or KSR2 back-to-back homodimers associate with two MEK1 molecules. While these structural data provide a better understanding on RAF function, available crystal structures are limited to the kinase domain lacking the N-terminal part of RAF.…”

Tunable-Combinatorial Mechanisms of Acquired Resistance Limit the Efficacy of BRAF/MEK Cotargeting but Result in Melanoma Drug Addiction

“…Based on prior structural data of MEK1-BRAF (Haling et al, 2014), vemurafenib-bound V600E BRAF (Bollag et al, 2010), and MEK1-KSR2 (Brennan et al, 2011) and structural alignments of vemurafenib-bound V600E BRAF with BRAF or KSR2, we hypothesized a regulatory V600E BRAF- MUT MEK complex where V600E BRAF arginine 662 makes critical contacts with MEK residues in one complex interface (Figure 6A–B). We predicted that the R662L substitution in V600E BRAF would disrupt this face-to-face V600E BRAF- MUT MEK interaction and attenuate the DDR phenotype.…”

“…Importantly, the R662L mutation in the context of V600E BRAF strongly abolished this enhanced V600E BRAF- MUT MEK1 complex and reduced the overall p-ERK levels. V600E/R509H BRAF also appeared to display reduced interaction with MUT MEK1 but without a reduction in the p-ERK levels, suggesting that this apparent reduction was due to loss of BRAF dimers (Figure 6A) (Haling et al, 2014) or higher-order oligomers (Nan et al, 2013) brought down by anti-FLAG. Consistently, whereas engineered M249 P lines highly over-expressing V600E BRAF or minimally over-expressing V600E/R509H BRAF together with a MEK1 mutant were able to resist robustly BRAFi+MEKi at 1 μM, those cell lines expressing V600E/R662L BRAF or WT BRAF along with a MEK1 mutant grew poorly over 28 or 32 day treatments with BRAFi+MEKi (Figure 6E).…”