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PMID: 10364159 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

Genes & development ·Vol. 13 ·No. 11 ·1999-06-01 ·Pages 1422-37

Gingras AC, Gygi SP, Raught B, Polakiewicz RD, Abraham RT, Hoekstra MF, Aebersold R, Sonenberg N

Abstract

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.

MeSH Terms
Adaptor Proteins, Signal Transducing Amino Acid Sequence Antigen-Antibody Complex Binding Sites Carrier Proteins Cell Cycle Proteins Cell Line Chromones/pharmacology Culture Media, Serum-Free Eukaryotic Initiation Factor-4E Humans Immunophilins/metabolism Molecular Sequence Data Morpholines/pharmacology Peptide Initiation Factors/metabolism Phosphoproteins/metabolism Phosphorylation Phosphotransferases (Alcohol Group Acceptor)/metabolism Protein Isoforms Protein Kinases Recombinant Fusion Proteins/metabolism Sirolimus/pharmacology TOR Serine-Threonine Kinases Threonine/metabolism
Chemicals
Adaptor Proteins, Signal Transducing Antigen-Antibody Complex Carrier Proteins Cell Cycle Proteins Chromones Culture Media, Serum-Free EIF4EBP1 protein, human Eukaryotic Initiation Factor-4E Morpholines Peptide Initiation Factors Phosphoproteins Protein Isoforms Recombinant Fusion Proteins Threonine 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one Protein Kinases Phosphotransferases (Alcohol Group Acceptor) MTOR protein, human TOR Serine-Threonine Kinases Immunophilins Sirolimus
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Gingras A C
Department of Biochemistry and McGill Cancer Center, McGill University, Montréal, Québec, H3G 1Y6, Canada.
Gygi S P
Raught B
Polakiewicz R D
Abraham R T
Hoekstra M F
Aebersold R
Sonenberg N
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Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.13.11.1422
Published
1999-06-01
Pages
1422-37
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC316780
Subset
IM
Grants
NHGRI NIH HHS · T32 HG000035 · United States
NHGRI NIH HHS · T32HG00035-3 · United States
External Links
PubMed source DOI: 10.1101/gad.13.11.1422 PMC Full Text View journal details
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