BRAF 作为 RAS/RAF/MEK/ERK 通路的看门人，在体内平衡中发挥着至关重要的作用。由于该轴的异常信号传导会导致癌症和其他疾病，因此它受到 PI3K/AKT/mTOR 通路和 ERK 介导的反馈环的串扰的严格调节。例如，ERK 通过多个残基的磷酸化来限制 BRAF 信号传导。其中之一，T401，被广泛认为是生长因子急性通路激活后的 ERK 底物。在这里，我们证明，在小鼠和人类来源的各种细胞系中，在没有急性刺激的情况下，已经观察到内源性 BRAF 的显着 T401 磷酸化 (pT401)。重要的是，BRAF/RAF1 抑制剂纳波非尼、MEK 抑制剂曲美替尼和 ERK 抑制剂 ulixertinib 在这些情况下未能降低 pT401 水平，支持另一种不依赖于 ERK 的 T401 磷酸化途径。相比之下，mTOR 抑制剂 torin1 和双特异性 PI3K/mTOR 抑制剂 dactolisib 以时间和浓度依赖性方式显着抑制所有研究细胞类型中的 pT401 水平。相反，致癌性 RHEB (Q64L) 和 mTOR（S2215Y 和 R2505P）突变体激活遗传 mTOR 通路可显着增加 pT401，这种作用可被 dactolisib 和 torin1 逆转，但不能被曲美替尼逆转。我们还表明，shRNAmir 介导的 mTORC1 复合物亚基 Raptor 的消耗显着增强了低剂量 torin1 对 T401 磷酸化的抑制，而 mTORC2 复合物亚基 Rictor 的敲低效果较差。使用质谱分析，我们提供了进一步的证据，证明 torin1 抑制 T401、S405 和 S409 的磷酸化，但不抑制其他重要调节磷酸化位点（如 S446、S729 和 S750）。总之，我们的数据确定 mTOR 轴及其临床（前）相关抑制剂是 T401 BRAF 磷酸化的新型调节剂。© 2024。作者。

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.© 2024. The Author(s).