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

J Biol Chem. 2019 Aug 2;294(31):11840-11852. doi: 10.1074/jbc.RA119.008512. Epub 2019 Jun 14.

Abstract

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. Phosphorylation 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 investigated 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 (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ, phosphorylated at Thr-70, Ser-83, and Ser-101, bound to eIF4E during mitosis. Two-dimensional gel electrophoretic 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. Phosphodefective 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.

Keywords: PHAS-I; cell cycle; cyclin-dependent kinase 1 (CDK1); eukaryotic translation initiation; eukaryotic translation initiation factor 4E (eIF4E); eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1); eukaryotic translation initiation factor 4G (eIF4G); mammalian target of rapamycin (mTOR); mitosis; protein phosphorylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / deficiency
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Cell Cycle Proteins / deficiency
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cysteine / analogs & derivatives
  • Cysteine / pharmacology
  • Eukaryotic Initiation Factor-4E / metabolism*
  • Eukaryotic Initiation Factor-4G / metabolism
  • Gene Editing
  • HeLa Cells
  • Humans
  • Interphase
  • Mitosis* / drug effects
  • Phosphorylation
  • Protein Binding
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • EIF4EBP1 protein, human
  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factor-4G
  • Protein Isoforms
  • 3-tritylthio-L-alanine
  • Cysteine