Gingras A C, Gygi S P, Raught B, Polakiewicz R D, Abraham R T, Hoekstra M F, Aebersold R, Sonenberg N
Department of Biochemistry and McGill Cancer Center, McGill University, Montréal, Québec, H3G 1Y6, Canada.
Genes Dev. 1999 Jun 1;13(11):1422-37. doi: 10.1101/gad.13.11.1422.
The multisubunit eukaryotic translation initiation factor (eIF) 4F 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-46 are detected in all phosphorylated in vivo 4E-BP1 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.
多亚基真核生物翻译起始因子（eIF）4F将40S核糖体亚基募集到mRNA的5'端。eIF4F亚基eIF4E直接与mRNA的5'帽结构相互作用。eIF4F复合物的组装受到一类阻遏多肽即eIF4E结合蛋白（4E-BPs）的抑制。4E-BPs与eIF4E的结合受磷酸化调节：低磷酸化的4E-BP异构体与eIF4E强烈相互作用，而高磷酸化的异构体则不然。4E-BP1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会在多个位点发生高磷酸化。PI3激酶/Akt途径和激酶FRAP/mTOR向4E-BP1发出信号。据报道，FRAP/mTOR在体外可直接使4E-BP1磷酸化。然而，尚不清楚FRAP/mTOR是否负责4E-BP1所有位点的磷酸化，也不清楚哪些位点必须被磷酸化才能使4E-BP1从eIF4E上释放。为了解决这些问题，在体外激酶试验中利用重组FRAP/mTOR蛋白和FRAP/mTOR免疫沉淀产物使4E-BP1磷酸化。对体外标记蛋白的磷酸肽图谱分析产生了两条4E-BP1磷酸肽，它们与体内产生的磷酸肽迁移情况相同。质谱分析表明，这些肽含有磷酸化的苏氨酸-37和苏氨酸-46。当4E-BP1与eIF4E结合时，FRAP/mTOR在体外可有效使苏氨酸-37和苏氨酸-46磷酸化。然而，这些位点的磷酸化与eIF4E结合的丧失无关。在所有体内磷酸化的4E-BP1异构体中都检测到了磷酸化的苏氨酸-37和苏氨酸-46，包括那些与eIF4E相互作用的异构体。最后，突变分析表明，苏氨酸-37/苏氨酸-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，FRAP/mTOR使4E-BP1的苏氨酸-37和苏氨酸-46磷酸化是随后羧基末端血清敏感位点磷酸化的引发事件。
