The Intracellular Cleavage Product of the NG2 Proteoglycan Modulates Translation and Cell-Cycle Kinetics via Effects on mTORC1/FMRP Signaling
The Intracellular Cleavage Product of the NG2 Proteoglycan Modulates Translation and Cell-Cycle Kinetics via Effects on mTORC1/FMRP Signaling
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DOI：
10.3389/fncel.2018.00231
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发表时间：
2018-08
影响因子：
5.3
通讯作者：
Tanmoyita Nayak;J. Trotter;Dominik Sakry
中科院分区：
文献类型：
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作者：
Tanmoyita Nayak;J. Trotter;Dominik Sakry
NG2 蛋白多糖由少突胶质细胞前体细胞 (OPC) 表达，并在黑色素瘤和胶质母细胞瘤等肿瘤中大量表达。 NG2 的功能包括对增殖、迁移和神经调节的影响。与其他 1 型膜蛋白类似，NG2 经历蛋白水解，通过 α 和 γ 分泌酶的连续作用产生大的胞外域、C 末端片段 (CTF) 和细胞内结构域 (ICD)，并通过神经元活性增强。迄今为止，NG2 的功能作用已在全长蛋白、释放的胞外域和 CTF 中得到证实，但在 ICD 中尚未得到证实。在这项研究中，我们描述了 NG2 ICD 在 OPC 和人胚肾 (HEK) 细胞中的作用。过表达的 ICD 主要位于细胞质中，包括 OPC 的远端突起。 ICD 的一部分的核定位取决于核定位信号。免疫沉淀和质谱分析以及功能分析表明 NG2 ICD 调节 mRNA 翻译和细胞周期动力学。在 OPC 和 HEK 细胞中，ICD 过表达导致 mTORC1 依赖性翻译上调，以及细胞群向 S 期的转变。 NG2 ICD 增加 mTOR 的活性（磷酸化）形式并调节下游信号级联，包括增加 p70S6K1 的磷酸化和增加 eEF2 的表达。引人注目的是，FMRP（一种受 mTOR/p70S6K1/eEF2 调节的 RNA 结合蛋白）的水平下降了。在神经元中，FMRP 在活动依赖性控制下充当翻译抑制因子，并在脆性 X 综合征 (FXS) 中发生突变。敲除原代 OPC 中的内源 NG2 会减少 Oli-neu 中的翻译和 mTOR/p70S6K1 磷酸化。在这里，我们通过调节成熟的 mTORC1 通路将 NG2 ICD 确定为 OPC 翻译的调节因子。我们发现 FXS 相关的 FMRP 信号传导不仅存在于神经元中，而且在 OPC 中也发挥着作用。这在 OPC 中提供了一个信号级联，该信号级联可以受到神经元网络的影响，因为 NG2 ICD 已被证明是由组成型和活性依赖性裂解产生的。我们的结果还阐明了 NG2 在肿瘤中的可能作用，表现出增强的翻译速率和快速的细胞周期动力学。
The NG2 proteoglycan is expressed by oligodendrocyte precursor cells (OPCs) and is abundantly expressed by tumors such as melanoma and glioblastoma. Functions of NG2 include an influence on proliferation, migration and neuromodulation. Similar to other type-1 membrane proteins, NG2 undergoes proteolysis, generating a large ectodomain, a C-terminal fragment (CTF) and an intracellular domain (ICD) via sequential action of α- and γ-secretases which is enhanced by neuronal activity. Functional roles of NG2 have so far been shown for the full-length protein, the released ectodomain and CTF, but not for the ICD. In this study, we characterized the role of the NG2 ICD in OPC and Human Embryonic Kidney (HEK) cells. Overexpressed ICD is predominantly localized in the cell cytosol, including the distal processes of OPCs. Nuclear localisation of a fraction of the ICD is dependent on Nuclear Localisation Signals. Immunoprecipitation and Mass Spectrometry followed by functional analysis indicated that the NG2 ICD modulates mRNA translation and cell-cycle kinetics. In OPCs and HEK cells, ICD overexpression results in an mTORC1-dependent upregulation of translation, as well as a shift of the cell population toward S-phase. NG2 ICD increases the active (phosphorylated) form of mTOR and modulates downstream signaling cascades, including increased phosphorylation of p70S6K1 and increased expression of eEF2. Strikingly, levels of FMRP, an RNA-binding protein that is regulated by mTOR/p70S6K1/eEF2 were decreased. In neurons, FMRP acts as a translational repressor under activity-dependent control and is mutated in Fragile X Syndrome (FXS). Knock-down of endogenous NG2 in primary OPC reduced translation and mTOR/p70S6K1 phosphorylation in Oli-neu. Here, we identify the NG2 ICD as a regulator of translation in OPCs via modulation of the well-established mTORC1 pathway. We show that FXS-related FMRP signaling is not exclusive to neurons but plays a role in OPCs. This provides a signal cascade in OPC which can be influenced by the neuronal network, since the NG2 ICD has been shown to be generated by constitutive as well as activity-dependent cleavage. Our results also elucidate a possible role of NG2 in tumors exhibiting enhanced rates of translation and rapid cell cycle kinetics.