Phosphorylation of the Eukaryotic Translation Initiation Factor 4E-Transporter (4E-T) by c-Jun N-Terminal Kinase Promotes Stress-Dependent P-Body Assembly
Phosphorylation of the Eukaryotic Translation Initiation Factor 4E-Transporter (4E-T) by c-Jun N-Terminal Kinase Promotes Stress-Dependent P-Body Assembly
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DOI：
10.1128/mcb.00544-12
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发表时间：
2012-11-01
影响因子：
5.3
通讯作者：
Roux, Philippe P.
中科院分区：
文献类型：
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作者：
Cargnello, Marie;Tcherkezian, Joseph;Roux, Philippe P.
加工体（PB或P体）是参与mRNA储存和降解的细胞质颗粒，参与基因表达的调节。PB浓缩了非翻译的mRNA和参与mRNA衰变和翻译抑制的几种因子，包括真核翻译起始因子4 E-转运蛋白（4 E-T）。4 E-T是PB组装所必需的，但对调节其功能的分子机制知之甚少。在这里，我们证明了氧化应激促进多位点4 E-T磷酸化。我们发现c-Jun N-末端激酶（JNK）在氧化应激反应中靶向PB，并促进4 E-T的磷酸化。定量质谱分析表明，JNK磷酸化4 E-T的六个脯氨酸导向的网站，所需的4 E-T复合物的形成压力。我们已经开发了一种基于图像的计算方法来量化细胞中PB的大小，数量和密度，并且我们发现，虽然4 E-T是稳态PB组装所需的，但其磷酸化促进了氧化应激后更大PB的形成。使用多聚体mRNA分析，我们评估了整体和特异性mRNA翻译，但没有发现4 E-T磷酸化影响翻译控制。总的来说，这些数据支持一个模型，PB组装是由两步机制，涉及4 E-T依赖性组装阶段在非应激细胞和4 E-T磷酸化依赖性聚集阶段，在应激刺激的调节。
Processing bodies (PBs, or P bodies) are cytoplasmic granules involved in mRNA storage and degradation that participate in the regulation of gene expression. PBs concentrate nontranslated mRNAs and several factors involved in mRNA decay and translational repression, including the eukaryotic translation initiation factor 4E-transporter (4E-T). 4E-T is required for PB assembly, but little is known about the molecular mechanisms that regulate its function. Here, we demonstrate that oxidative stress promotes multisite 4E-T phosphorylation. We show that the c-Jun N-terminal kinase (JNK) is targeted to PBs in response to oxidative stress and promotes the phosphorylation of 4E-T. Quantitative mass spectrometry analysis reveals that JNK phosphorylates 4E-T on six proline-directed sites that are required for the formation of the 4E-T complex upon stress. We have developed an image-based computational method to quantify the size, number, and density of PBs in cells, and we find that while 4E-T is required for steady-state PB assembly, its phosphorylation facilitates the formation of larger PBs upon oxidative stress. Using polysomal mRNA profiling, we assessed global and specific mRNA translation but did not find that 4E-T phosphorylation impacts translational control. Collectively, these data support a model whereby PB assembly is regulated by a two-step mechanism involving a 4E-T-dependent assembly stage in unstressed cells and a 4E-T phosphorylation-dependent aggregation stage in response to stress stimuli.