Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling

Abstract

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-Raf^V600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-Raf^V600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-Raf^V600E-induced melanomas.

Figure 1.. B-Raf-MEK Complex.

(a) Sequence alignments of B-Raf kinase domain C-lobe. Mutations in the solubilized construct are shown in red. Mutations introduced in this study are shown with various symbols. Conserved residues with respect to wild-type B-Raf with the V600E mutation are marked by an asterisk below the alignment. (b) Structure of the B-Raf-MEK complex. The two proteins are shown as cartoons with the B-Raf N-lobe in pink, B-Raf C-lobe in purple, MEK N-lobe in blue, and MEK C-lobe in cyan. The residue that was mutated to produce active B-Raf with 15 surface mutations is shown in sticks and highlighted with a red star. The α-helix under study is indicated and the αC helix is labeled. Figure generated using PDB ID: 4MNE . (c) Zoomed in view of the B-Raf α-helix. The α-helix is shown as a purple cartoon with the solubilizing mutation that interacts with MEK shown as sticks. The electrostatic surface potential of the MEK C-lobe is shown (blue is positively charged and red is negatively charged) and the position of D315 is shown with a cyan star. Figure generated using PDB ID: 4MNE .

Figure 2.. Purification of GST-B-Raf^V600E,15mut from E. coli.

(a) FPLC trace of size-exclusion chromatography run of GST-B-Raf ^V600E,15mut. The elution volumes of protein standards (158 and 44 kDa) are indicated by arrows. (b) SDS-PAGE analysis of fractions from the corresponding size-exclusion chromatography run. Fraction numbers are indicated and those kept for analysis are boxed.

Figure 3.. Binding and activity of E. coli expressed B-Raf ^V600E,15mut compared to Sf9-expressed B-Raf^V600E.

(a) SDS-PAGE gel of pull-down assay of GST-tagged full length MEK^WT with B-Raf ^V600E,16mut or B-Raf^V600E,15mut expressed in E. coli, B-Raf^WT expressed in Sf9 cells and B-Raf^V600E, expressed in Sf9 cells. The B-Raf band is indicated with a red star. (b) BLI curves of GST-B-Raf ^V600E,16mut, GST-B-Raf ^V600E,15mut, His-B-Raf^WT, and His-B-Raf^V600E binding to full length MEK. In each case of GST-tagged B-Raf mutants, His-tagged MEK was immobilized onto an anti-His biosensor, and B-Raf constructs were introduced at two or more concentrations, ranging from low micromolar to high micromolar, depending on the mutant tested. In each case of His-tagged B-Raf mutants, B-Raf was immobilized onto an anti-His biosensor, and GST-MEK was introduced at two or more concentrations, ranging from low micromolar to high micromolar, depending on the mutant tested. Global fits are shown as black lines. (c) A representative Western blot for in vitro phosphorylation of MEK, performed in duplicate, using His-tagged B-Raf^V600E and B-Raf^WT expressed in Sf9 cells compared to B-Raf ^V600E,16mut and B-Raf ^V600E,15mut expressed in E. coli with 1mM ATP.

Figure 4.. Improving B-Raf binding to MEK.

(a) The introduced B-Raf Y667 mutation was modeled using PyMOL by keeping the same coordinates of the backbone and Cβ atoms and is shown as purple sticks together with its possible interactions with MEK residues (cyan). Hydrogen bond distances are shown with dashed lines and distances are provided. Figure generated using PDB ID: 4MNE. (b) Representative BLI curves of the GST-B-Raf ^{V600E,15mut,F667Y} mutant binding to immobilized full length His-tagged MEK. B-Raf was introduced at the indicated concentrations. Global fits are shown as black lines.

Figure 5.. Interactions of B-Raf α-helix with the MEK C-lobe.

(a) The B-Raf α-helix under study is shown as a purple cartoon with residues mutated in this study shown as sticks. The MEK C-lobe is shown as a cyan surface and electrostatics surface (b). (c) The potential interaction of the B-Raf residue N661 with the indicated MEK residue shown as sticks. (d) The potential interaction of the B-Raf residue R671 with the indicated MEK residue shown as sticks. Hydrogen bonds and salt-bridges are shown with dashed lines and distances are provided. Figure generated using PDB ID: 4MNE .

Figure 6.. In vitro Phosphorylation of MEK and ERK by B-Raf ^V600E,15mut α-helix mutants.

MEK and ERK were incubated with ATP with or without the indicated constructs of B-Raf for 30 minutes. Reaction mixtures were subjected to Western blot analysis at least twice with the indicated antibodies and a representative blot is shown. Input lanes are shown in blue. B-Raf and ERK were both GST-tagged. The intensities of the p-MEK and p-ERK bands were quantified by ImageJ and normalized to the corresponding band from the mixture with B-Raf ^V600E,15mut.

Figure 7.. Phosphorylation of MEK and ERK by full length B-Raf.

Indicated full length B-Raf and MEK were transfected into HEK293T cells and lysed after 24 hours of expression. Cell lysates were subjected to Western blot analysis with the indicated antibodies. Control lanes are shown in blue. Experiment was performed at least three times and representative blots are shown.