Mitosis-related phosphorylation of the eukaryotic translation suppressor 4E-BP1 and its interaction with eukaryotic translation initiation factor 4E (eIF4E)

Eukaryotic translation initiation factor 4E (eIF4E)–binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phos-phorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investi-gated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (d) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-?—phosphorylated at Thr-70, Ser-83, and Ser-101—bound to eIF4E during mitosis. Two-dimensional gel electropho-retic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46–priming phosphorylation. Moreover, proximity ligation assays for phospho–4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited, but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phospho-defective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA-immunoprecipitation sequencing. Mitotic 5'-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis. Overall design: 1) eIF4G RIP-seq in HeLa cells. 4 groups of samples (M_CtrL, M_PP242, PM_CtrL, PM_PP242) were included. Each group contains Input and IP. 2) eIF4G RIP-seq in mitosis-enriched 4E-BP1WT and 4E-BP1S83A cells. 2 groups of samples (WT, S83A) were included. Each group contains Input and IP. Three independent biological experiments were performed.

真核翻译起始因子4E（eukaryotic translation initiation factor 4E, eIF4E）结合蛋白1（4E-BP1）通过与eIF4G竞争结合eIF4E，从而抑制真核生物的帽依赖型翻译。4E-BP1的Ser-83位点磷酸化发生于有丝分裂期，该修饰由细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1, CDK1）/细胞周期蛋白B（cyclin B）催化，而非经典的雷帕霉素靶蛋白（mTOR）激酶。本研究探究了间期与有丝分裂期内，eIF4E分别与4E-BP1或eIF4G的相互作用情况。结果显示，在间期与有丝分裂期，4E-BP1与eIF4G结合eIF4E的水平相近。磷酸化程度最高的有丝分裂期4E-BP1亚型（d）无法与eIF4E结合；而另一种独特的4E-BP1磷酸化亚型EB-γ，其磷酸化位点包含Thr-70、Ser-83与Ser-101，可在有丝分裂期结合eIF4E。二维凝胶电泳分析验证了eIF4E结合的4E-BP1亚型的磷酸化修饰特征，并发现了一群缺乏Thr-37/Thr-46预磷酸化修饰的磷酸化4E-BP1分子。此外，针对磷酸化4E-BP1与eIF4E的邻近连接实验（proximity ligation assay）揭示，间期与有丝分裂期的原位相互作用存在差异。有丝分裂期细胞中eIF4E与eIF4G的相互作用未被抑制，反而有所增强，这与有丝分裂期翻译起始仍处于活跃状态的结论一致。对4E-BP1的Ser-83位点进行磷酸化缺陷型替换后，通过单细胞新生蛋白质合成实验与eIF4G RNA免疫沉淀测序（RNA immunoprecipitation sequencing, RIP-seq）检测发现，全局翻译水平与单个mRNA的翻译谱均未发生改变。有丝分裂期的5'端寡聚嘧啶RNA（5'-terminal oligopyrimidine RNA, TOP RNA）翻译仍保持活跃，且与间期翻译不同，其对mTOR抑制作用具有抗性。本研究结果阐明了细胞周期全程中4E-BP1亚型的磷酸化特征及其与eIF4E的相互作用模式，并表明4E-BP1并不会特异性抑制有丝分裂期的翻译起始。
实验整体设计如下：
1）在HeLa细胞中开展eIF4G RIP-seq实验，设置4组样本（M_CtrL、M_PP242、PM_CtrL、PM_PP242），每组均包含Input与免疫沉淀（immunoprecipitation, IP）样本；
2）在富集有丝分裂期的4E-BP1野生型（4E-BP1WT）与4E-BP1S83A细胞中开展eIF4G RIP-seq实验，设置2组样本（WT、S83A），每组均包含Input与IP样本，共完成3次独立生物学重复。