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.

中文翻译：

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丝氨酸 365 在 BRAF V600E 对 RAF 抑制的敏感性中的作用

丝氨酸-苏氨酸激酶 BRAF 是 MEK-ERK1/2 通路的上游调节因子，通常在癌症中发生突变。14-3-3 蛋白与 BRAF 中的两个位点结合，即 N 端 S365 和 C 端 S729。14-3-3 结合调节 BRAF 的野生型和非 V600 突变形式的活性和二聚化。在 BRAF V600E 突变体中，C 末端 S729 位点影响截短剪接变体的二聚化。N 末端 S365 在 BRAF V600E 剪接变体中被去除，但其在全长 BRAF V600 突变体中的重要性仍不确定。我们在全长 BRAF V600E 中测试了 S365 在二聚化和 RAF 抑制剂抗性中的作用。将 BRAF S365 位点突变为丙氨酸 (S365A) 可减少 14-3-3 结合并增加 BRAF V600E 同二聚化。BRAF V600E S365A 在 MEK-ERK1/2 信号水平上对 RAF 抑制剂的敏感性降低，细胞生长和细胞活力。这些数据表明，S365 14-3-3 结合位点的改变或去除可能导致 RAF 抑制剂耐药。