Extended Data Figure 4: APS-2-79 activity is not dependent on KSR phosphorylation sites in MEK or direct RAF inhibition.

a, APS-2-79 does not affect BRAF(V600E)-induced MAPK activation in cells. BRAF(V600E)–Flag was expressed for 24 h in 293H cells. Cells were then treated for 2 h with DMSO or 5 μM of either APS-2-79, APS-3-77, or dabrafenib before collection and western blot analysis of phosphorylated MEK (MEK1/2(pSer218/pSer222)) and ERK (ERK1/2(pT202/pY204)). b, Removal of putative KSR phosphorylation sites in MEK (MEK(AAAA); S18A, T23A, S24A, S72A; ref. 7) neither hinders KSR-dependent MAPK signalling, nor the activity of APS-2-79. Co-expression of full-length KSR–Flag and wild-type MEK1–GFP or MEK(AAAA)–GFP leads to enhanced MAPK signalling within 293H cells as visualized by immunoblotting for phosphorylated MEK (MEK1/2(pSer218/pSer222)) and ERK (ERK1/2(pT202/pY204)). APS-2-79 impedes KSR-stimulated MAPK signalling within cells through wild-type and MEK(AAAA) equally. Bars and error bars indicate pMEK and pERK intensity and standard deviations, respectively. Signals were normalized relative to lane 5. Error bars indicate the mean ± s.d. (n = 3 biological replicates). ***P < 0.0005 by two-tailed unpaired t-testing. c, The dimer-deficient KSR(R718H) mutant, relative to wild-type KSR, is compromised in MEK-inhibitor-induced feedback. 293H cells were co-transfected with MEK–GFP and KSR–Flag or KSR(R718H)–Flag for 24 h and then treated with increasing concentrations of trametinib (range of 0.13 to 100 nM; threefold dilutions) for an additional 48 h. Cells were collected and analysed by western blot. d, Phospho-AMPK remains unchanged in HCT116 cells upon co-treatment with APS-2-79 and trametinib. HCT116 cells were treated with APS-2-79 and/or trametinib for 48 h. Phospho-AMPK (top), phospho-ERK(pERK), and total MEK (bottom) western blots are shown.