Kinase activity and vemurafenib sensitivity of an ARAF variant discovered in a LCH patient with wild-type BRAF alleles. (A) Expression vectors encoding wild-type ARAF (ARAF WT), the 6 nucleotide deletion mutation encoding Q347_A348del, the single nucleotide variant encoding the single amino acid substitution F351L, and both ARAF mutations (as discovered in the patient sample) were transfected into HEK293 cells along with activated Src and H-Ras to maximize ARAF activation.¹³  All proteins were tagged with the FLAG epitope. Anti-FLAG immune precipitates were tested for their ability to phosphorylate an artificial MEK substrate in vitro and for activated MEK to phosphorylate artificial ERK. Immunoblots show the presence of phospho-MEK (pMEK), phospho-ERK (pERK), and total ARAF protein (ARAF) and demonstrate the ability of the mutated versions of ARAF to phosphorylate MEK. (B) Substitution of methionine for lysine at position 336 in the ARAF mutant (ARAF Q347_A348del/F351L/K336M), which is known to destroy ARAF kinase activity, prevents the mutant from phosphorylating MEK. (C) Comparison of mutant ARAF kinase activity to BRAF V600E kinase activity shows that ARAF kinase activity is only a little lower than that of BRAF V600E. (Note that lower amounts of ARAF were loaded on this gel compared with BRAF.) (D) Increasing amounts of the kinase reaction mixture (1, 2, 5, and 10 µL) were analyzed by immunoblot for phospho-MEK (pMEK) and total ARAF or BRAF as detected by FLAG immunoblotting. Adding increasing amounts of vemurafenib (Vem) (final concentrations of 0, 16, 63, 250, and 1000 µM) inhibited both the mutant ARAF and BRAF V600E kinase activities.