Fig. 3: Structural basis of EGFR R252C-dependent ERK1/2 activation.
From: An alternative EGFR activation by patient-derived R252C mutation promotes cancer progression
a 293T cells were co-transfected with EV, HA-Tagged or Flag-Tagged WT or R252C. Flag-EGFR was immunoprecipitated using anti-Flag agarose beads. Semi-quantitative scoring was carried out. The HA to Flag ratio in the IP samples was quantified and normalized to the EGFR WT group. b Conformations of the ligand-free EGFR R252C whole model, which contain the ECD, TM, JM and KD. c The distribution of the center-of-mass between the two kinase domains in the simulated ECD + TM + JM + KD dimers. The values of Center-mass Distance (Å) were colored in green and salmon for ligand-free EGFR R252C and ligand-bound EGFR WT, respectively. d Schematic diagram of the NanoBiT protein-protein interaction assay. EGFR WT or R252C-SmBiT together with EGFR WT or R252C-LgBiT and control Renilla plasmid were used to transfect HEK293T cells. Cells transfected with EGFR WT were serum-starved for 24 h before treated with 10 mg/ml EGF for 30 min. Biosensor activity was determined 36 h after transfection using the NanoBiT assay. Relative luminescence was normalized to the EGFR WT group. e The binding mode of ERK1 and KD dimer in EGFR WT and R252C, and the total binding free energy were shown. In conformation, the KD dimer is colored in cyan and green in EGFR WT and R252C, respectively, and the ERK1 in orange and slate, respectively. While in the binding site model, the dominant residues shown in sticks and colored, respectively. f The dominant residues of the EGFR in the complex of EGFR and ERK1 binding are listed, and all energies are in units of kcal/mol. g Immunoprecipitation assay was performed with HEK293T cells stably expressing Flag-EGFR WT, Flag-EGFR R252C, and indicated mutants. h Cell proliferation assay was performed with U87 cells stably expressing Flag-EGFR WT or mutants. Relative cell proliferation of U87 cells was normalized to day 1 value. i Colony formation assay was performed with U87 cells stably expressing Flag-EGFR WT or mutants. a, d, h, i. Data represent the mean ± s.d. of three biologically independent experiments (unpaired, two-tailed t-test).
