The C terminus of initiation factor 4E-binding protein 1 contains multiple regulatory features that influence its function and phosphorylation

Mol Cell Biol. 2003 Mar;23(5):1546-57. doi: 10.1128/MCB.23.5.1546-1557.2003.

Abstract

Eukaryotic initiation factor 4E (eIF4E) binds the mRNA cap structure and forms eIF4F complexes that recruit 40S subunits to the mRNA. Formation of eIF4F is blocked by eIF4E-binding proteins such as 4E-BP1, which interacts with eIF4E via a motif in the center of its 118-residue sequence. 4E-BP1 plays key roles in cell proliferation, growth, and survival. Binding of 4E-BP1 to eIF4E is regulated by hierarchical multisite phosphorylation. Here we demonstrate that three different features in the C terminus of 4E-BP1 play distinct roles in regulating its phosphorylation and function. Firstly, we identify a new phosphorylation site in its C terminus (S101). A serine or glutamate at this position is required for efficient phosphorylation at Ser65. A second C-terminal site, S112, directly affects binding of 4E-BP1 to eIF4E without influencing phosphorylation of other sites. Thirdly, a conserved C-terminal motif influences phosphorylation of multiple residues, including rapamycin-insensitive sites. These relatively long-range effects are surprising given the reportedly unstructured nature of 4E-BP1 and may imply that phosphorylation of 4E-BP1 and/or binding to eIF4E induces a more-ordered structure. 4E-BP2 and -3 lack phosphorylatable residues corresponding to both S101 and S112. However, in 4E-BP3, replacement of the alanine at the position corresponding to S112 by serine or glutamate did not confer the ability to be released from eIF4E in response to insulin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Binding Sites
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins
  • Cell Division
  • Cell Line
  • Cell Survival
  • Enzyme Inhibitors / pharmacology
  • Eukaryotic Initiation Factor-4E / metabolism
  • Genetic Vectors
  • Glutamic Acid / chemistry
  • Glutamine / metabolism
  • Humans
  • Immunoblotting
  • Insulin / pharmacology
  • Models, Genetic
  • Molecular Sequence Data
  • Mutation
  • Peptides / chemistry
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Isoforms
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Serine / chemistry
  • Serine / metabolism
  • Time Factors
  • Transfection

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Cell Cycle Proteins
  • EIF4EBP1 protein, human
  • Enzyme Inhibitors
  • Eukaryotic Initiation Factor-4E
  • Insulin
  • Peptides
  • Phosphoproteins
  • Protein Isoforms
  • Glutamine
  • Glutamic Acid
  • Serine