The MAPK pathway was previously shown to stabilize FOS by phosphorylation and stimulate its gene transactivation activities36,37. We therefore investigated the role of BRAF V600E in the phosphorylation of FOS. As shown in Fig. 6a, BRAF knockdown decreased the phosphorylation of FOS in cells harboring BRAF mutation. Similarly, the BRAF V600E-specific inhibitor PLX4032 significantly decreased the phosphorylation of FOS (Fig. 6b). To test the role of BRAF V600E/MAPK pathway-induced FOS phosphorylation in GABPB and mutant TERT activation, we overexpressed the wild-type FOS (FOS-wt) and a mutant FOS (FOS-mut) containing alanine replacements on all the phosphorylation sites (Thr-232, Thr-325, Thr-331, and Ser-374) of the target of ERK activation38. Overexpression of FOS-wt increased the phosphorylated species of FOS and enhanced the expression of GABPB and TERT (Fig. 6c); it also increased GABPB 5′-UTR and mutant TERT promoter activities (Fig. 6d, e). In contrast, overexpression of FOS-mut had no effect on GABPB and TERT activities (Fig. 6c–e). Binding of FOS to GABPB was considerably decreased after BRAF knockdown as demonstrated in ChIP assay (Fig. 6f). Conversely, BRAF V600E mutation knock-in increased the phosphorylation of FOS (Fig. 6g), enhanced its binding to the 5′-UTR of GABPB (Fig. 6h), increased the activities of the GABPB promoter (Fig. 6i), and upregulated the expression of the GABPB gene (Fig. 6g). Similar results were obtained in WRO cells induced to stably express BRAF V600E (Fig. 6j).Fig. 6BRAF V600E promoted FOS binding to GABPB by upregulating FOS phosphorylation. a Western blotting analyses for phosphorylated-FOS (p-FOS), total FOS (t-FOS), BRAF, and beta-actin in K1 and A375 cells with or without stable BRAF knockdown. b Western blotting analyses of p-FOS, t-FOS, p-ERK, and beta-actin in K1 and A375 cells treated with 0.5 μm PLX4032 for 0, 1, and 24 h. c, d, e Effects of FOS phosphorylation on GABPB and TERT activation. c KAT18 cells were serum-starved for 24 h and transiently transfected with FOS wild-type (FOS-wt) or mutant (FOS-mut) bearing none of the potential ERK-targeted phosphorylation sites, followed by western blotting analysis for TERT, GABPB, p-FOS, t-FOS, and beta-actin. d KAT18 cells were transfected with FOS-wt or FOS-mut along with GABPB 5′-UTR luciferase reporter and Renilla luciferase (pRL-TK) plasmids in the absence of serum for 24 h, and the luciferase activities were then measured. e KAT18 cells were transiently transfected with FOS-wt or FOS-mut, together with TERT promoter luciferase reporters harboring the C228T or C250T mutation, and pRL-TK for 24 h, followed by luciferase assays. f ChIP assay for FOS binding to the 5′-UTR of GABPB in K1 and A375 cells. g Western blotting analyses for GABPB, p-FOS, p-ERK, and t-ERK in the parental and BRAF-V600E knock-in SW48 cells. h ChIP assay for FOS binding to the 5′-UTR of GABPB in SW48 cells. i GABPB 5′-UTR region-luciferase reporter assays in SW48 cells. j Wild-type (WT) BRAF and BRAF V600E were stably introduced to express in WRO cells, followed by western blotting analyses of the expression of GABPB, p-FOS, BRAF, p-ERK, and t-ERK after serum starving overnight. **P < 0.01, ***P < 0.001, by two-tailed Student’s t test. n.s. not significant. All values represent the average ± standard deviation (SD) of triplicate samples and similar results were obtained in two additional independent experiments