Fig 2: Conformational changes induced by NRAS mutants alter BRAF binding affinity.a Representative conformations of NRAS61R, NRAS61K, and NRAS61P extracted from their highly populated replica-exchange molecular dynamics (REMD) structural ensembles. Interactions with the codon 61 sidechain are listed below each structure. b Binding orientation of NRAS61R and NRAS61P with the BRAF-RBDCRD as generated using Hex molecular docking simulations. The average conformation representing highly populated structural ensembles extracted from each NRAS codon 61 mutant trajectory was docked against the BRAF-RBDCRD. In the cartoon representation, the NRAS codon 61 mutant and bound nucleotide are shown in licorice, the BRAF-RBDCRD in gray and polar interactions for each mutant, and its surrounding residues are indicated by blue dashed lines. Comparisons of the interaction energy and the number of contacts between the BRAF-RBDCRD and each NRAS mutant suggest that highly melanomagenic NRAS mutants (NRAS61R, NRAS61K) bind BRAF with higher affinity than NRAS61H, NRAS61L, and NRAS61P. The number of autoinhibitory contacts relieved by NRAS mutant binding is listed in parentheses. BRET protein–protein interaction data from Venus-tagged NRAS mutant and Rluc8-tagged BRAF (c) or CRAF (d) constructs co-transfected into 293T cells at increasing receptor to donor ratios. The data shown are representative of two replicates. Best fit BRET50 values (binding affinity) and standard error, determined by non-linear regression, are shown for each mutant. Bolded values indicate statistically significant values as compared to both NRAS61H and NRAS61P. p-values determined by t-tests with 20 degrees of freedom representing the number of measures per curve. Source data are provided as a Source Data file.

Fig 3: Oncogenic NRAS mutants mediate differential MAPK activation via a RAF-dependent mechanism.Representative immunoblots of AKT and ERK activation in homozygous MEF cell lines treated with shRNAs targeting Nras, Hras and Kras, or Sos1 (a) or Araf, Braf or Craf (b). Dot plot data are presented as mean values +/− SD where each dot represents one biological replicate. For a the following biologically independent replicates per genotype were examined over five independent experiments (eGFP arm: Q = 5, P = 5, R = 5; NRAS arm: Q = 3, P = 3, R = 3; H/KRAS arm: Q = 3, P = 4, R = 3; SOS1 arm: Q = 5, P = 5, R = 5). For b the following biologically independent replicates per genotype were examined over 8 independent experiments (eGFP arm: Q = 8, P = 8, R = 8; ARAF arm: Q = 6, P = 6, R = 6; BRAF arm: Q = 7, P = 7, R = 6; CRAF arm: Q = 7, P = 7, R = 6). Adjusted p-values were generated using a one-way ANOVA with a Tukey’s multiple comparisons test. Statistics denoted in the figure indicate significant differences between shRNA-treated NRAS mutant MEFs and their respective eGFP control. ** p < 0.01, † p < 0.001. A complete list of adjusted p-values can be found in Supplementary Table 4a, b. Source data are provided as a Source Data file.

Fig 4: Melanomagenic NRAS mutants enhance RAF dimerization.a Schematic representation of the RAF NanoBiT assay in which each RAF isoform is tagged with either LgBiT or SmBiT. Dot plots of normalized luminescence intensity in TN61X/X MEFs infected with adenovirus expressing BRAF-LgBiT and BRAF-SmBiT (b), BRAF-LgBiT and Craf-SmBiT (c), CRAF-LgBiT and CRAF-SmBiT (d), ARAF-LgBiT and BRAF-SmBiT (e), ARAF-LgBiT and ARAF-SmBiT (f), or ARAF-LgBiT and CRAF-SmBiT (g). Luminescence intensity was normalized to crystal violet staining for each well. Dot plot data are presented as mean values +/− SD where each dot represents one biological replicate. The following biologically independent replicates per genotype were examined over five independent experiments (BRAF-BRAF: Q/Q = 5, R/R = 5, H/H = 4; P/P = 4; BRAF-CRAF: Q/Q = 5, R/R = 5, H/H = 4; P/P = 5; CRAF-CRAF: Q/Q = 5, R/R = 5, H/H = 4; P/P = 4; BRAF-ARAF: Q/Q = 4, R/R = 4, H/H = 4; P/P = 4; ARAF-ARAF: Q/Q = 4, R/R = 4, H/H = 4; P/P = 4; ARAF-CRAF: Q/Q = 4, R/R = 4, H/H = 4; P/P = 4). One-way ANOVA with a Tukey’s post-test was used to compare data between each genotype. NRAS mutant samples statistically different from NRAS61R/R samples are indicated in the figure. Adjusted p-values for all comparisons can be found in Supplementary Table 4d. * p < 0.05, ** p < 0.01, † p < 0.001, ‡ p < 0.0001. Source data are provided as a Source Data file.

Fig 5: Inhibition of PLK1 reduces colorectal cancer cell growth by targeting the CRAF-MEK-ERK signaling pathway. (A) Overexpression of PLK1 increases clonogenicity. The PLK1 overexpressing clones of CRC cells were subjected to clonogenicity assay (n = 3). (B) Forced expression of PLK1 activates CRAF-MEK-ERK signaling pathway. The proteins from the PLK1-overexpressing clones were analyzed using immunoblotting. (C,D) Knockdown of PLK1 decreases clonogenicity. CRC cells were transfected with two different shRNA sequences targeting PLK1, and the PLK1 stable knockdown clones of CRC cells were subjected to clonogenicity assay (n = 3). (E) Knockdown of PLK1 leads to a decrease in the phosphorylation levels of CRAF, MEK1/2, and ERK1/2 proteins. * statistically significant in comparison to the control group, with p < 0.05.