Role of serine 365 in BRAF V600E sensitivity to RAF inhibition

Abstract The serine‐threonine kinase, BRAF, is an upstream regulator of the MEK‐ERK1/2 pathway and is commonly mutated in cancer. 14‐3‐3 proteins bind to two sites in BRAF, N‐terminal S365, and C‐terminal S729. 14‐3‐3 binding modulates the activity and dimerization of both wild‐type and non‐V600 mutant forms of BRAF. In BRAF V600E mutants, the C‐terminal S729 site affects dimerization of truncated splice variants. The N‐terminal, S365, is removed in BRAF V600E splice variants but its importance in full‐length BRAF V600 mutants remains uncertain. We tested the role of S365 in dimerization and RAF inhibitor resistance in full‐length BRAF V600E. Mutating BRAF S365 site to an alanine (S365A) reduced 14‐3‐3 association and increased BRAF V600E homodimerization. BRAF V600E S365A displayed reduced sensitivity to RAF inhibitor at the level of MEK‐ERK1/2 signaling, cell growth, and cell viability. These data suggest that alteration or removal of the S365 14‐3‐3 binding site may contribute to RAF inhibitor resistance.