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. 2018 Feb 22;554(7693):549-553.
doi: 10.1038/nature25478. Epub 2018 Feb 12.

MEK Drives BRAF Activation Through Allosteric Control of KSR Proteins

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Free PMC article

MEK Drives BRAF Activation Through Allosteric Control of KSR Proteins

Hugo Lavoie et al. Nature. .
Free PMC article

Abstract

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

Figures

Extended Data Figure 1.
Extended Data Figure 1.. MEK levels modulate the formation of BRAF-KSR1 dimers.
a, MEK1/2 expression selectively modulates BRAF-KSR1 interaction but not the KRASG12VBRAFRBD counter screen interaction by BRET. MEK1 and MEK2 expression was driven by the lentiviral vector pLX301. Error bars correspond to mean values ± s.d. of biological triplicates. b, MEK1 and MEK2 modulate BRAF-KSR1 interaction by BRET. c, MEK1 stimulates BRAFKSR1 interaction in a dose-dependent manner. mCherry-tagged MEK1 was titrated in cells expressing the BRAF-KSR1 BRET biosensors. d, MEK1 promotes full-length BRAF-KSR1 interaction by co-immunoprecipitation. e, Reducing endogenous MEK1 and/or MEK2 levels by RNA interference (shMEK1 and/or shMEK2) decreases BRAF-KSR1 interaction by BRET. f, Efficiency of MEK1 and MEK2 shRNA knockdowns in panel d. g, MEK1 stimulates BRAFKSR1 dimerization independently of its kinase activity. mCherry-tagged MEK1 or MEK1K97R similarly stimulate BRAF-KSR1 interaction by BRET. Cells expressing the BRAF-KSR1 BRET biosensor and increasing amounts of WT MEK1 were treated with the MEK inhibitor AZD8330 (10 μM). h, MEK1 stimulates BRAF-KSR1 interaction independently of the phosphorylation status of Serine 218 and 222. mCherry-tagged MEK1, MEK1S218A-S222A or MEK1S218D-S222D similarly stimulate BRAF-KSR1 interaction by BRET. i, and j, mCherry-tagged MEK1 selectively modulates KSR-containing dimers. The impact of MEK was measured on a series of ten biosensors comprising BRAF (h) or CRAF (i) as donor probes expressed with either of ARAF, BRAF, CRAF, KSR1, or KSR2 as acceptor probes. k, mCherry-tagged kinase-dead MEK1K97R selectively modulates KSR-containing dimers. The impact of MEK1K97R was measured on a series of five biosensors comprising BRAF as the donor probe expressed with either of ARAF, BRAF, CRAF, KSR1 or KSR2 as acceptor probes. l, mCherry-tagged interaction-defective MEK1F311S does not modulate dimerization of five biosensors comprising BRAF as the donor probe expressed with either of ARAF, BRAF, CRAF, KSR1 or KSR2 as acceptor probes. In panels c and g-l, BRET log2-transformed fold-changes were reported as a function of log10-transformed mCherry relative fluorescence units (RFU). Experiments were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 2.
Extended Data Figure 2.. Identification of mutations disrupting KSR-MEK and RAF-MEK interactions.
a, Crystal structure of KSR2 bound to MEK1. The structure shows a heterotetrameric arrangement with two KSR2 protomers assembling as a side-to-side dimer. Each KSR2 protomer in turn interacts in a face-to-face manner with one molecule of MEK1. Zoom in views (dashed boxes) highlight the involvement of the αG helices and the activation segments (AS) of both kinase domains in the face-to-face contact (PDB entry 2Y4I). b, Screening scheme used to identify MEK1 and KSR1 binding defective mutants by error-prone-PCR random mutagenesis using the yeasttwo hybrid system. c, Yeast spotting assay showing that the F311S mutation in MEK1 helix αG abrogates interaction with KSR1, BRAF and CRAF. d, Yeast spotting assay showing that KSR1 and BRAF helix αG mutations (W831R and I666R, respectively) disrupt their interaction with MEK1. Experiments in c-d were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 3.
Extended Data Figure 3.. Mutations in helix αG of MEK1 and KSR1 (F311S and W831R, respectively) do not perturb protein fold but abrogate stoichiometric assembly of the BRAFKSR1- MEK1 complex.
a, Size exclusion chromatography followed by multi-angle light scattering (SEC-MALS) analysis of MEK1 WT and F311S mutant. The horizontal black and blue lines correspond to SEC-MALS calculated masses for the indicated proteins. A summary table of measured and calculated molecular weights for each protein is shown below. The experiment was performed in triplicate. b, Fluorescence polarization (FP) binding analysis of MEK1 WT and F311S mutant to a 25 nM fluorophore-conjugated ATP tracer (BODIPY-ATP). Error bars correspond to mean values ± s.d. of technical triplicates. Indicated Kd values ± s.d. are representative of two independent experiments. c, MEK1 and MEK1 F311S expressed in HEK293T cells show equal ability to bind the ATP analog desthiobiotin-ATP as assessed using the Pierce kinase enrichment kit (Thermo- Fisher Scientific). d, and e, WT KSR1 and the W831R mutant assemble to the same extent with the known interaction partners HSP90, CDC37, and 14–3-3, indicating that the protein fold of KSR1 is not overtly perturbed by the W831R mutation. Interaction partners were confirmed by ass spectrometry and by western immunoblotting. f, WT KSR1 and the W831R variant expressed in HEK293T cells show equal binding to the ATP analog desthiobiotin-ATP as assessed using the Pierce kinase enrichment kit (Thermo-Fisher Scientific). g, BRAF-KSR1-MEK1 complexes were purified to near homogeneity by immunoaffinity purification on anti-Flag-agarose resin following treatment of the transfected cells with GDC-0879 (10 μM). BRAF and KSR1 form near stoichiometric complexes in the presence of MEK1 overexpression (compare lanes 3 and 4). The KSR1 W831R mutant (lane 5) and the MEK1 F311S mutant (lane 7) but not the BRAF I666R mutant (lane 6) perturbed this near stoichiometric interaction. Note that the BRAF-KSR1-MEK1 complex also associates at near stoichiometric levels with endogenous 14–3-3 isoforms. In addition, in the absence of MEK1 over expression, appreciable but sub-stoichiometric levels of endogenous MEK are detected by immunoblotting. Protein identity was determined by mass spectrometry. Asterisks indicate the position of non-specific proteins associating with the anti- Flag-agarose resin. h, The basal association of BRAF-KSR1 dimers (panel a, lane 3 and panel b,lane 1) depends on endogenous MEK. Reducing MEK1 and MEK2 levels by short hairpin RNA (shRNA) interference (combination of shMEK1 and shMEK2) decreases the amount of Pyo-KSR1 co-purifying with Flag-BRAF. NT stands for non-target control shRNA. Experiments were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 4.
Extended Data Figure 4.. MEK1 binding to KSR1 is required for the transactivation of BRAF by side-to-side dimerization.
a, BRAF activation by KSR1 depends on an intact side-to-side dimerization surface in each protomer. Co-transfection of WT BRAF and KSR1 induces a strong phospho-MEK signal while KSR1R665H or BRAFR509H mutants do not. b, Human KSR1 and KSR2 can both transactivate BRAF. C-terminal GFP10 fusions of KSR1 and KSR2 were co-transfected with BRAF and the resulting phospho-MEK signal was monitored by western immunoblotting. c, Disruption of KSR1- MEK1 interaction with the W831R mutation perturbs BRAF transactivation by KSR1. The BRAF I666R mutation also prevents MEK phosphorylation. Similarly, MEK1F311S is not phosphorylated when co-transfected with WT BRAF and KSR1. d, Transactivation of Drosophila RAF (dRAF) by Drosophila KSR (dKSR) requires MEK binding (W896R mutation is homologous to W831R in human KSR1) and dRAF-dKSR dimerization (R732H mutation is homologous to R665H in human KSR1). e, The transactivation potential of FRB-dKSRKD towards FKBP-dRAFKD was assessed by monitoring the levels of phosphorylated MEK in the presence or absence of rapamycin in S2 cells (1 μM). Blocking MEK binding to dKSR (W896R mutation) and dRAF-dKSR dimerization (R732H mutation) abrogated RAF transactivation and MEK phosphorylation as judged by western immunoblotting. f, MEK phosphorylation induced by co-transfection of BRAF and KSR1 depends on the catalytic activity of BRAF and not on the integrity of the active site region of KSR1. g, KSR1 A637F (AF) and A637F/YLQE602–604>DDEE602–604 (AFDDEE) gain-offunction mutants are dependent on binding to endogenous MEK1/2 in order to dimerize with endogenous BRAF and to activate the cellular pool of RAF as judged by phospho-MEK1/2 signal. The W831R substitution in KSR1 was employed to abrogate KSR1AF and KSR1AFDDEE mutant binding to MEK. Experiments were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 5.
Extended Data Figure 5.. Identification of MEK1 mutants rescuing the interaction with KSR1W831R.
a, Screening scheme used to identify MEK1 mutants rescuing the interaction with KSR1W831R. Screening was conducted in two rounds. In round #1, MEK1 was randomly mutagenized by errorprone- PCR, which identified 16 mutants corresponding exclusively to M219V and N221Y substitutions. In round #2, saturation mutagenesis was used to test all possible residue combinations in the region encoding amino acids 219–221. This identified 70 mutants that either complemented the Y2H interaction on SD –TLH or on both SD –TLH and SD –TLA. Sequence diversity of the recovered mutants is summarized by sequence logos shown on the right (see Supplementary Table 2 for details regarding mutant sequences). After further phenotypic screening, the fittest mutant on SD-TLA corresponded to MEK1M219W-A220L. b, Position of MEK1 mutations that rescue the interaction with KSR1W831R. Mutations systematically mapped to the MEK1 activation segment between Ser218 and Ser222, which are target phosphorylation sites for RAF proteins. Multiple sequence alignments of MAP2K1–7 activation segments illustrate that residues 219–221 vary within the MAP2K family, suggesting that substitutions of this sequence do not drastically affect enzyme function. The M219V substitution recovered in our screen is also found at the homologous position in MAP2K3 and MAP2K6. c, MEK1 activation segment mutations MEK1M219V, MEK1N221Y, MEK1M219V/N221Y, and MEK1M219W/A220L stimulate MEK1 binding to endogenous KSR1, BRAF, and CRAF. Co-immunoprecipitation of Flag-tagged MEK1 variants was performed in HEK293T cells. d, MEK1M219W-A220L but not WT MEK1 stimulates KSR1W831R-BRAF dimerization in BRET assays. mCherry-tagged MEK1 WT or MEK1M219WA220L were titrated in cells expressing the BRAF-KSR1W831R BRET biosensors. BRET log2 foldchanges were reported as a function of log10 transformed mCherry relative fluorescence units (RFU). e, Strategy to distinguish activator and substrate MEK in BRAF transactivation assays. Eight repeats of a G4S flexible linker (symbolized by an octagon) was added onto WT MEK1 to separate it by size from co-transfected MEK variants lacking the linker. Experimental conditions corresponding to lanes 7 and 8 of Fig. 2d, respectively, are depicted at the top and bottom of the diagram. Experiment in e was repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 6.
Extended Data Figure 6.. BRAF and KSR1 N-terminal regulatory regions (NTRs) selectively interact.
a, MEK expression promotes BRAF-KSR1 interaction but not CRAF-KSR1 interaction. b, BRET titration experiments were used to monitor the interaction between BRAFNTR, CRAFNTR and KSR1NTR. The indicated donor and acceptor pairs were tested for interaction in titration experiments. c, Yeast-two hybrid assays confirm interaction between BRAFNTR and KSR1NTR, and KSR1NTR self-association. d, Domain architecture of full-length mammalian RAF and KSR isoforms and schematic of truncations used to map the molecular determinants of BRAFNTRKSR1NTR interaction. Within the NTRs are highlighted the Ras-binding domain (RBD; orange), Cysteine-rich domain (CRD; yellow), Serine/Threonine-rich (S/T-rich; brown) region, Prolinerich (P-rich; gray) region, BRAF specific (BRS; blue) domain and the Coiled coil - Sterile alpha motif (CC-SAM; green) domain. Co-IP results derived from multiple experiments are indicated (Yes/No) with representative examples shown in panels e and f. e, Co-immunoprecipitation of Pyo-KSR1NTR with the indicated fragments of Flag-tagged BRAF. f, Co-immunoprecipitation of the indicated fragments of Pyo-KSR1 with Flag-tagged BRAFNTR. Experiments were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 7.
Extended Data Figure 7.. The contact surfaces between BRS and CC-SAM domains are required for the physical and functional interactions between BRAF and KSR1.
a, Phylogenetic profiling of BRAF and KSR1 domains demonstrates conservation of the BRS and CC-SAM domains throughout metazoan evolution. A concomitant loss of both domains is observed in the nematode lineage (indicated by “Domain loss”). The conservation of each of BRAF and KSR1 domains with its human ortholog was color-coded by BLAST E-value. RPSBLAST was used to assess conservation of the CC-SAM domain. Conservation of the BRS was established by calculating the sum of the COILS score of amino acids located upstream of the RBD in each BRAF ortholog. b, Sequence alignment of the BRAF-specific (BRS) sequence from 14 metazoan species shows a conserved pattern of hydrophobic residues consistent with a coiledcoil region. Secondary structure elements identified in the BRS crystallographic structure are delineated above the sequence alignment. The heptad repeat (AbcDefg) typical in coiled-coil domains is shown below the alignment: positions A and D usually comprise hydrophobic amino acids while positions b, c, e, f and g usually comprise polar or charged residues. BRS mutations used in this study are indicated by arrowheads. c, Sequence alignment of the coiled-coil-SAM (CC-SAM) sequence from 15 metazoan species. Secondary structure elements identified in the apo and BRS-bound CC-SAM structures are delineated above the sequence alignment. CC-SAM mutations used in this study are indicated by arrowheads below the alignment. Species in the phylogenetic tree and the sequence alignments are: Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Strongylocentrotus purpuratus, Saccoglossus kowalevskii, Lingula anatina, Lottia gigantea, Drosophila melanogaster, Anopheles gambiae, Haemonchus contortus, Dictyocaulus viviparus, Necator americanus, Caenorhabditis briggsae, Caenorhabditis brenneri, Caenorhabditis remanei, Caenorhabditis elegans, Loa loa, Brugia malayi, Bursaphelenchus xylophilus, Trichinella papuae, Trichuris trichiura, Trichuris suis, Nematostella vectensis, Amphimedon queenslandica and Trichoplax adherens. d, Y2H mating matrix of BRAF and KSR1 NTR domains and chimeric fusions (the dashed box indicates the interaction between isolated BRS and CC-SAM domains). e, The BRS and CC-SAM domains specifically interact as determined by BRET titration experiments. In contrast, the BRS-BRS BRET pair did not generate a significant BRET signal. f, Swapping of the BRS and CC-SAM domains supports the BRAFKSR1 interaction and transactivation of BRAF by KSR1. Fusing the BRS domain to CRAF NTR stimulates its interaction with KSR1NTR by co-IP (g) and by yeast-two-hybrid (h). Experiments in d-h were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 8.
Extended Data Figure 8.. NMR characterization of the physical interaction between the BRS and CC-SAM domains.
a, Superposition of the 1H, 15N-HSQC spectra of the [15N]-CC-SAM domain of human KSR1 (hCC-SAM) alone (black) and [15N]-hCC-SAM in the presence of the BRS domain of human BRAF (hBRS) (green) at a 1:2 molar ratio. Binding of BRS to CC-SAM induced both chemical shift perturbations and peak broadening. b, Superposition of the 1H, 15N-HSQC spectra of [15N]- hBRS (black) and [15N]-hBRS in the presence of hCC-SAM (blue) at the indicated molar concentrations. In the [15N]-hBRS HSQC spectra (black) numerous peaks are in intermediate (peak broadening) timescale conformational exchange giving rise to only 50 visible peaks out of 84 expected peaks. In the [15N]-hBRS:hCC-SAM HSQC spectra (blue) the binding of CC-SAM to BRS increases conformational exchange to a faster time regime giving rise to 65 visible peaks. c, Chemical shift perturbation (CSP) of the mouse KSR1 [15N]-CC-SAM (mCC-SAM) domain as a function of titrated hBRS. Superposition of the 1H, 15N-HSQC spectra of [15N]-mCC-SAM alone (black) and [15N]-mCC-SAM in the presence of hBRS (green) at the indicated molar concentrations (top left panel). The Kd value was calculated as the mean ± s.d. of 5 independent peak profiles (right panel) for which one representative chemical shift perturbation profile is shown (bottom left panel). d, CSP of the [15N]-hBRS domain as a function of titrated hCC-SAM. Superposition of the 1H, 15N-HSQC spectra of [15N]-hBRS alone (black) and [15N]-hBRS in the presence of hBRS (blue) at the indicated molar concentrations (top left panel). The Kd value was calculated as the mean ± s.d. of 6 independent peak profiles (right panel) for which one representative chemical shift perturbation profile is shown (bottom left panel). e, Superposition of the 1H, 15N-HSQC spectra of the [15N]-CC-SAM domain of murine KSR1 ([15N]-mCC-SAM) alone (black) and in the presence of the BRS domain of human BRAF (green) at a 1:2 molar ratio. Binding of BRS to mCC-SAM induced both chemical shift perturbations and peak broadening. f, Peak intensity change and CSPs versus residue number for the 1H,15N-HSQC spectra of [15N]- mCC-SAM alone versus [15N]-mCC-SAM with hBRS at a 1:2 molar ratio. Cut-off values indicated by the red line were calculated using the corrected standard deviation method. Vertical green and gray bars indicate residues identified as being part of the interacting surface and unassigned residues, respectively. g, Significant CSPs and peak intensity changes are highlighted in green on the surface of the murine KSR1 CC-SAM domain (PDB code 2LPE). Most CSPs and peak intensity changes map to the coiled-coil region of mCC-SAM. Non shifting peaks are indicated in pale gray while unassigned peaks in the HSQC are colored in dark gray.
Extended Data Figure 9.
Extended Data Figure 9.. Crystal structures of the BRS domain and the BRS:CC-SAM complex.
a, X-Ray data collection and refinement statistics table for the crystal structures of the hBRS domain and the hCC-SAM-hBRS complex. b, Ribbon representation of the crystal structure of the BRS domain of human BRAF. Residues comprising the hydrophobic core of the coiled-coil interactions are shown as sticks. c, Structure of the BRAF BRS: KSR1 CC-SAM domain complex. Contents of the asymmetric unit revealed two CC-SAM:BRS complexes interacting in trans. Disordered (GS)4 linker between CC-SAM and BRS domains is represented by dashed lines. The connectivity between the BRS and the CC-SAM domains was deduced as trans because in this configuration, but not the cis configuration, the carboxyl terminus of the CC-SAM domain is sufficiently close to the amino terminus of the BRS domain (15Å) to be physically spanned by the disordered linker comprised of 15 amino acid residues. For comparison, the connection in cis would need to minimally span 80Å. d, Representative final 2Fo-Fc electron density map contoured at 1.0 σ for the BRS:CC-SAM complex at a region of drastic conformational rearrangement in CC-SAM relative to the isolated CC-SAM structure (indicated by the arrow). e, Size exclusion chromatography followed by multi-angle light scattering (SEC-MALS) analysis of the indicated crystallization fusion proteins (top panel). The horizontal black, red and green lines correspond to SEC-MALS calculated masses for the indicated fusion proteins. Summary table (bottom panel) of measured and theoretical calculated molecular weights for each protein complex analysed. Experiments were performed in triplicate.
Extended Data Figure 10.
Extended Data Figure 10.. Mutational and functional characterization of molecular interactions involving the NTRs and kinase domains of BRAF and KSR1.
a, Peak intensity change and chemical shift perturbation (CSP) versus residue number for the 1H, 15N-HSQC spectra of the [15N]-mCC-SAM L45D mutant (black) and the [15N]-mCC-SAM L45D mutant in the presence of hBRS (green) (left panel), the 1H,15N-HSQC spectra of the [15N]-mCCSAM C60D (black) versus [15N]-mCC-SAM C60D in the presence of hBRS (green) (middle panel) and the 1H, 15N-HSQC spectra of [15N]-mCC-SAM (black) and [15N]-mCC-SAM in the presence of the hBRS M53D mutant (green) (right panel) at the indicated molar ratio. Cut-off values indicated by the red line were calculated using the corrected standard deviation method. Residue coordinates of the CC-SAM correspond to murine KSR1; the corresponding human CC-SAM mutations are L47D and C62D. b, The BRS and CC-SAM domains specifically interact as determined by BRET. The CC-SAM L47D and C62D mutations reduce the BRS - CC-SAM BRET binding signal. Similarly, the BRS M53D mutation disrupts the BRS - CC-SAM BRET binding signal. c, Disruption of the K88-E72 salt interaction disrupts BRS - CC-SAM BRET interaction. Restoring the salt-bridge with reverse polarity as in the K88E-E72K mutant pair rescues complex formation. d, MEK1 expression induces the formation of full-length BRAF-KSR1 heterodimers, but does not induce the formation of heterodimers between the isolated NTRs of BRAF and KSR1 as detected by co-IP. e, BRET analysis demonstrating that MEK expression promotes KSR1 kinase domain homodimerization. Helix αG mutations in either KSR1 (W831R) or MEK1 (F311S) abolish MEK-induced homodimerization of the KSR1 kinase domain. BRET log2-transformed fold-changes were reported as a function of log10-transformed mCherry relative fluorescence units (RFU). f, Co-IP analysis of Pyo-KSR1 with Flag-KSR1 reveals that MEK1 promotes full-length KSR1 homodimerization. Experiments in b-f were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Figure 1.
Figure 1.. MEK1 asymmetrically stimulates BRAF-KSR1 dimerization by interacting with KSR1.
a, Whole-kinome screen identifies MEK1/2 as specific inducers of BRAF-KSR1 dimerization. BRET biosensors comprising RlucII-BRAFKD-CAAX and GFP10-KSR1KD-CAAX were used to screen 558 human kinase-related ORFs expressed from lentiviral vectors. b, Kinase-dead MEK1K97R or MEK inhibitors (AZD8330 and GSK1120212) (10 μM) did not alter MEK1-induced BRAF-KSR1 dimerization in co-IPs. c, mCherry-tagged MEK1F311S did not promote BRAF-KSR1 dimerization by BRET. d, Heterodimerization of BRAFI666R but not KSR1W831R was responsive to MEK1 expression by BRET. e, MEK1 promotes BRAF-KSR1 dimerization and BRAF transactivation by binding KSR1 in co-IP. Experiments in b-e were repeated three times. For gel source data, see Supplementary Fig. 1.
Figure 2.
Figure 2.. KSR1-activated BRAF phosphorylates KSR1-free MEK1.
a, Putative roles of KSR in RAF-mediated MEK phosphorylation. Model 1 was adapted from reference #7. AS stands for activation segment. b, Y2H binding analysis of WT MEK1 and activation segment mutants MEK1M219V (MV), MEK1N221Y (NY), and MEK1M219W/A220L (MW-AL) to KSR1W831R or BRAFI666R. c, MEK1M219W/A220L (MW-AL), but not WT MEK1, induces KSR1W831R-BRAF dimerization and transactivation as determined by co-IP and pMEK levels. d, MEK1M219W/A220L bound to KSR1W831R induces BRAF activity towards MEK1 free of KSR1. Experiments in b-d were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Figure 3.
Figure 3.. The NTRs of KSR1 and BRAF selectively interact.
a, Full-length KSR1 selectively transactivates BRAF. b, The KSR1 NTR associates by co-IP with itself or with the BRAF NTR, but not with the CRAF NTR. c, KSR1 interacts with and transactivates CRAF43−648+BRS to a greater extent than WT CRAF. d, Ribbon representation of the BRS:CC-SAM complex. e, BRS M53D mutation and CC-SAM L47D and C62D mutations disrupt BRAF-KSR1 association and impede KSR1-induced MEK phosphorylation by BRAF. f, Mutational analysis of the K88BRS-E72CC-SAM salt bridge. Experiments in a-c, e and f were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.
Figure 4.
Figure 4.. MEK releases the intramolecular interaction between the NTR and kinase domain of KSR1.
a, The NTR of KSR1 associates with its cognate kinase domain (KSR1KD). MEK1 but not KRASG12V expression disrupts this interaction. The ERK inhibitor SCH772984 (10 μM) was used to exclude a potential ERK negative feedback mechanism. b, The NTR of BRAF associates with its cognate kinase domain (BRAFKD). KRASG12V but not MEK1 expression disrupts this interaction. c, Model of BRAF-KSR1-mediated signal transmission to MEK and ERK. Experiments in a and b were repeated at least 3 times. For gel source data, see Supplementary Fig. 1.

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