Department of Experimental Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China.
Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China.
Autophagy. 2024 Mar;20(3):505-524. doi: 10.1080/15548627.2023.2259735. Epub 2023 Sep 29.
MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) orchestrates diverse environmental signals to facilitate cell growth and is frequently activated in cancer. Translocation of MTORC1 from the cytosol to the lysosomal surface by the RRAG GTPases is the key step in MTORC1 activation. Here, we demonstrated that transcription factors MEF2A and MEF2D synergistically regulated MTORC1 activation via modulating its cyto-lysosome shutting. Mechanically, MEF2A and MEF2D controlled the transcription of FNIP1 and FNIP2, the components of the FLCN-FNIP1 or FNIP2 complex that acts as a RRAGC-RRAGD GTPase-activating element to promote the recruitment of MTORC1 to lysosome and its activation. Furthermore, we determined that the pro-oncogenic protein kinase SRC/c-Src directly phosphorylated MEF2D at three conserved tyrosine residues. The tyrosine phosphorylation enhanced MEF2D transcriptional activity and was indispensable for MTORC1 activation. Finally, both the protein and tyrosine phosphorylation levels of MEF2D are elevated in human pancreatic cancers, positively correlating with MTORC1 activity. Depletion of both MEF2A and MEF2D or expressing the unphosphorylatable MEF2D mutant suppressed tumor cell growth. Thus, our study revealed a transcriptional regulatory mechanism of MTORC1 that promoted cell anabolism and proliferation and uncovered its critical role in pancreatic cancer progression. ACTB: actin beta; ChIP: chromatin immunoprecipitation; EGF: epidermal growth factor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FLCN: folliculin; FNIP1: folliculin interacting protein 1; FNIP2: folliculin interacting protein 2; GAP: GTPase activator protein; GEF: guanine nucleotide exchange factors; GTPase: guanosine triphosphatase; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF2: myocyte enhancer factor 2; MEF2A: myocyte enhancer factor 2A; MEF2D: myocyte enhancer factor 2D; MEF2D-3YF: Y131F, Y333F, Y337F mutant; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NR4A1: nuclear receptor subfamily 4 group A member 1; RPTOR: regulatory associated protein of MTOR complex 1; RHEB: Ras homolog, mTORC1 binding; RPS6KB1: ribosomal protein S6 kinase B1; RRAG: Ras related GTP binding; RT-qPCR: real time-quantitative PCR; SRC: SRC proto-oncogene, non-receptor tyrosine kinase; TMEM192: transmembrane protein 192; WT: wild-type.
雷帕霉素靶蛋白（mTOR）激酶复合物 1（mTORC1）协调多种环境信号以促进细胞生长，并且在癌症中经常被激活。RRAG GTPases 将 mTORC1 从细胞质易位到溶酶体表面是 mTORC1 激活的关键步骤。在这里，我们证明转录因子 MEF2A 和 MEF2D 通过调节其胞浆-溶酶体关闭协同调节 mTORC1 的激活。在机制上，MEF2A 和 MEF2D 控制 FNIP1 和 FNIP2 的转录，FNIP1 和 FNIP2 是 FLCN-FNIP1 或 FNIP2 复合物的组成部分，作为 RRAGC-RRAGD GTPase 激活元件，促进 mTORC1 向溶酶体的募集及其激活。此外，我们确定致癌蛋白激酶 SRC/c-Src 可直接在三个保守的酪氨酸残基上磷酸化 MEF2D。酪氨酸磷酸化增强了 MEF2D 的转录活性，对于 mTORC1 的激活是不可或缺的。最后，人胰腺癌细胞中 MEF2D 的蛋白和酪氨酸磷酸化水平均升高，与 mTORC1 活性呈正相关。MEF2A 和 MEF2D 的缺失或表达非磷酸化 MEF2D 突变体均抑制肿瘤细胞生长。因此，我们的研究揭示了促进细胞合成代谢和增殖的 mTORC1 的转录调节机制，并揭示了其在胰腺癌进展中的关键作用。ACTB：肌动蛋白β；ChIP：染色质免疫沉淀；EGF：表皮生长因子；EIF4EBP1：真核翻译起始因子 4E 结合蛋白 1；FLCN：多囊肾蛋白；FNIP1：多囊肾蛋白相互作用蛋白 1；FNIP2：多囊肾蛋白相互作用蛋白 2；GAP：GTPase 激活蛋白；GEF：鸟苷酸交换因子；GTPase：鸟苷三磷酸酶；LAMP2：溶酶体相关膜蛋白 2；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3β；MEF2：肌细胞增强因子 2；MEF2A：肌细胞增强因子 2A；MEF2D：肌细胞增强因子 2D；MEF2D-3YF：Y131F、Y333F、Y337F 突变体；MTOR：雷帕霉素靶蛋白激酶；MTORC1：MTOR 复合物 1；NR4A1：核受体亚家族 4 组 A 成员 1；RPTOR：MTOR 复合物 1 的调节相关蛋白；RHEB：Ras 同源物，mTORC1 结合；RPS6KB1：核糖体蛋白 S6 激酶 B1；RRAG：Ras 相关 GTP 结合；RT-qPCR：实时定量 PCR；SRC：原癌基因 SRC，非受体酪氨酸激酶；TMEM192：跨膜蛋白 192；WT：野生型。
