Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism
Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism
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DOI：
10.1101/gad.13.11.1422
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发表时间：
1999-06-01
影响因子：
10.5
通讯作者：
Sonenberg, N
中科院分区：
文献类型：
--
作者：
Gingras, AC;Gygi, SP;Sonenberg, N
多亚基真核生物翻译起始因子（eIF）CF将40 S核糖体亚基募集到mRNA的5'端。eIF 4F亚基eIF 4 E直接与mRNA 5'帽结构相互作用。eIF 4F复合物的组装受到阻遏多肽家族eIF 4 E结合蛋白（4 E-BP）的抑制。4 E-BP与eIF 4 E的结合受磷酸化调节：低磷酸化4 E-BP亚型与eIF 4 E强烈相互作用，而高磷酸化亚型则不相互作用。4 E-BP 1在静止细胞中是低磷酸化的，但在暴露于多种细胞外刺激后在多个位点上是高磷酸化的。PI 3-激酶/Akt通路和激酶FRAP/mTOR信号传导至4 E-BP 1。已经报道FRAP/mTOR在体外直接磷酸化4 E-BP 1。然而，目前尚不清楚FRAP/mTOR是否负责所有4 E-BP 1位点的磷酸化，也不知道哪些位点必须被磷酸化以从eIF 4 E释放4 E-BP 1。为了解决这些问题，在体外激酶测定中利用重组FRAP/mTOR蛋白和FRAP/mTOR免疫沉淀物来磷酸化4 E-BP 1。体外标记蛋白的磷酸肽图谱产生了两个4 E-BP 1磷酸肽，它们与体内产生的磷酸肽共同迁移。质谱分析表明，这些肽含有磷酸化的Thr-37和Thr-46。当4 E-BP 1与eIF 4 E结合时，Thr-37和Thr-46在体外被FRAP/mTOR有效磷酸化。然而，这些位点的磷酸化与eIF 4 E结合的丧失无关。在所有磷酸化的体内4 E-BPI同种型中检测到磷酸化Thr-37和Thr-SB，包括与eIF 4 E相互作用的那些。最后，突变分析表明，Thr-37/Thr-46的磷酸化是随后几个羧基末端血清敏感位点磷酸化所必需的。综上所述，我们的结果表明，4 E-BP 1磷酸化FRAP/mTOR的Thr-37和Thr-46是一个引发事件，随后的羧基末端血清敏感位点的磷酸化。
The multisubunit eukaryotic translation initiation factor (eIF) CF recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-SB are detected in all phosphorylated in vivo 4E-BPI isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.