Hierarchical phosphorylation of the translation inhibitor 4E-BP1
Open accessAbstract
In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5′ end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.
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Activated mTOR phosphorylates 4E-BP1, leading to inhibition of 4E-BP1's repression of 5'cap-dependent translation and initiation of 5'cap-dependent translation.
“Activated mTOR phosphorylates 4E-BP1, which leads to inhibition of 4E-BP1's repression of 5'capdependent translation resulting in the initiation of 5'cap-dependent translation (23,24).”
From Enhanced mTORC1 signaling and Protein Synthesis in Parkinson’s Disease Pathogenesis Disease Pathogenesis (2022) · cites this paper as [24]
The Abstract explains that 4E-BP1 inhibits cap-dependent translation and that its hyperphosphorylation prevents binding to eIF4E, relieving that inhibition. It also says mTOR signaling is required for 4E-BP1’s release from eIF4E. But the Discussion states that the kinase responsible for phosphorylating 4E-BP1 in vivo is unknown, so the claim that activated mTOR directly phosphorylates it is stronger than the paper supports. (LLM, conf=0.88)
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- Authors
- Gingras, A.-C., Raught, B., Gygi, S. P., Niedzwiecka, A., Miron, M., Burley, S. K., Polakiewicz, R. D., Wyslouch-Cieszynska, A., Aebersold, R., & Sonenberg, N.
- Affiliations
- McGill University (CA)
- Howard Hughes Medical Institute (US)
- ORCID
- Venue
- Genes & Development
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2001-11-01
- Volume
- 15
- Issue
- 21
- Pages
- 2852-2864
- ISSN
- 0890-9369 (ISSN-L)
- 1549-5477
- PMC
- PMC312813
- OpenAlex
- W2139424276
- Cited by
- 883
- References
- 0
- Citations by year
- 2026: 20 · 2025: 19 · 2024: 27 · 2023: 30 · 2022: 25 · 2021: 35 · 2020: 35 · 2019: 26 · 2018: 46 · 2017: 38 · 2016: 39 · 2015: 42 · 2014: 43 · 2013: 39 · 2012: 51
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genes & Development (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Science Foundation
- Howard Hughes Medical Institute
- Cancer Research Society
- National Institutes of Health (HG00041)
- Canadian Institutes of Health Research
- National Human Genome Research Institute (HG000 41, T32HG000041, K22HG000041)
- National Cancer Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
- Keywords
- 4E-BP1, eIF4E-binding protein, eIF4E, translation initiation, protein phosphorylation, serum deprivation, rapamycin, phosphorylation sites
- MeSH terms
- Amino Acid Sequence, Carrier Proteins, Cell Line, DNA Mutational Analysis, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Enzyme Inhibitors, Humans, Isoelectric Focusing, Molecular Sequence Data, Mutation, Peptide Mapping, Phosphoproteins, Phosphorylation, Ribosomes, RNA, Messenger, Serine, Spectrometry, Fluorescence, Mass Spectrometry, Threonine, Transfection, Protein Biosynthesis, Blotting, Western, Sequence Homology, Amino Acid, Cell Cycle Proteins, Sirolimus, Adaptor Proteins, Signal Transducing
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