PMID- 10364159
OWN - NLM
STAT- MEDLINE
DCOM- 19990727
LR  - 20201209
IS  - 0890-9369 (Print)
IS  - 0890-9369 (Linking)
VI  - 13
IP  - 11
DP  - 1999 Jun 1
TI  - Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.
PG  - 1422-37
AB  - 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.
FAU - Gingras, A C
AU  - Gingras AC
AD  - Department of Biochemistry and McGill Cancer Center, McGill University, Montreal,
      Quebec, H3G 1Y6, Canada.
FAU - Gygi, S P
AU  - Gygi SP
FAU - Raught, B
AU  - Raught B
FAU - Polakiewicz, R D
AU  - Polakiewicz RD
FAU - Abraham, R T
AU  - Abraham RT
FAU - Hoekstra, M F
AU  - Hoekstra MF
FAU - Aebersold, R
AU  - Aebersold R
FAU - Sonenberg, N
AU  - Sonenberg N
LA  - eng
GR  - T32 HG000035/HG/NHGRI NIH HHS/United States
GR  - T32HG00035-3/HG/NHGRI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Genes Dev
JT  - Genes & development
JID - 8711660
RN  - 0 (Adaptor Proteins, Signal Transducing)
RN  - 0 (Antigen-Antibody Complex)
RN  - 0 (Carrier Proteins)
RN  - 0 (Cell Cycle Proteins)
RN  - 0 (Chromones)
RN  - 0 (Culture Media, Serum-Free)
RN  - 0 (EIF4EBP1 protein, human)
RN  - 0 (Eukaryotic Initiation Factor-4E)
RN  - 0 (Morpholines)
RN  - 0 (Peptide Initiation Factors)
RN  - 0 (Phosphoproteins)
RN  - 0 (Protein Isoforms)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 2ZD004190S (Threonine)
RN  - 31M2U1DVID (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one)
RN  - EC 2.7.- (Protein Kinases)
RN  - EC 2.7.1.- (Phosphotransferases (Alcohol Group Acceptor))
RN  - EC 2.7.1.1 (MTOR protein, human)
RN  - EC 2.7.1.1 (TOR Serine-Threonine Kinases)
RN  - EC 5.2.1.8 (Immunophilins)
RN  - W36ZG6FT64 (Sirolimus)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing
MH  - Amino Acid Sequence
MH  - Antigen-Antibody Complex
MH  - Binding Sites
MH  - *Carrier Proteins
MH  - Cell Cycle Proteins
MH  - Cell Line
MH  - Chromones/pharmacology
MH  - Culture Media, Serum-Free
MH  - Eukaryotic Initiation Factor-4E
MH  - Humans
MH  - Immunophilins/metabolism
MH  - Molecular Sequence Data
MH  - Morpholines/pharmacology
MH  - Peptide Initiation Factors/*metabolism
MH  - Phosphoproteins/*metabolism
MH  - Phosphorylation
MH  - Phosphotransferases (Alcohol Group Acceptor)/metabolism
MH  - Protein Isoforms
MH  - *Protein Kinases
MH  - Recombinant Fusion Proteins/metabolism
MH  - Sirolimus/pharmacology
MH  - TOR Serine-Threonine Kinases
MH  - Threonine/metabolism
PMC - PMC316780
EDAT- 1999/06/11 00:00
MHDA- 1999/06/11 00:01
CRDT- 1999/06/11 00:00
PHST- 1999/06/11 00:00 [pubmed]
PHST- 1999/06/11 00:01 [medline]
PHST- 1999/06/11 00:00 [entrez]
AID - 10.1101/gad.13.11.1422 [doi]
PST - ppublish
SO  - Genes Dev. 1999 Jun 1;13(11):1422-37. doi: 10.1101/gad.13.11.1422.