Fig. 1
The Q347A348 deletion activates ARAF by enhancing homodimerization. a ARAF(ΔQA) and ARAF(ΔQA/ F351L) have equal activity. The activity of ARAF mutants in 293T transfectants was measured by anti-phospho-ERK1/2 immunoblot. b The activity of ARAF(ΔQA) does not depend on upstream stimuli. ARAF(ΔQA) was coexpressed with N17Ras in 293T cells, and its activity was measured as in a. c ARAF(ΔQA) has a strong transforming ability independent of endogenous RAF molecules. Foci formation assay of immortalized fibroblasts expressing ARAF(ΔQA) was carried out as described before [36, 37]. d, e ARAF(ΔQA) has an elevated propensity to form homodimers. d The dimer affinity of ARAF(ΔQA) was measured by using complementary split luciferase assay [35]. The dimerization of wild-type ARAF induced by 10 um Vemurafenib served as a positive control (n = 5, ***p < 0.001, ****p < 0.0001). e The dimerization of ARAF(ΔQA) was evaluated by co-immunoprecipitation assay. ARAF mutants with FLAG- or HA-tag were coexpressed in 293T cells, and immunoprecipitated by anti-FLAG beads and detected by anti-HA immunoblot. To exclude the effect of ERK1/2-mediated feedback on ARAF dimerization, all 293T transfectants in d and e were pretreated with 20 um Tramentinib for 1 h before measurements. f ARAF(ΔQA) is activated by dimerization-driven transactivation. Mutations that disrupt the dimer interface (R362H) or block NtA-phosphorylation (AGFF) abolish the activity of ARAF(ΔQA). The activity of ARAF mutants in 293T transfectants was measured as in a. g, h Homologous deletions activate BRAF in a dimer-dependent manner. g The sequence alignment of human ARAF, BRAF, and CRAF reveals conserved residues in the β3-αC loop. H, the activity of BRAF mutants in 293T transfectants was measured as in a. All images are representative of at least three independent experiments
