Hierarchical phosphorylation of the translation inhibitor 4E-BP1
Anne-Claude Gingras,Brian Raught,Steven P. Gygi,Steven P. Gygi,Anna Niedzwiecka,Mathieu Miron,Stephen K. Burley,Roberto D. Polakiewicz,Aleksandra Wysłouch-Cieszyńska,Ruedi Aebersold,Ruedi Aebersold,Nahum Sonenberg +11 more
TLDR
It is shown thatosphorylation 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 eIF3E, and a novel combination of two-dimensional isoelectric focusing and Western blotting with phosphospecific antibodies is established.Abstract:
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.read more
Citations
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Upstream and downstream of mTOR
Nissim Hay,Nahum Sonenberg +1 more
TL;DR: Both the upstream components of the signaling pathway(s) that activates mammalian TOR (mTOR) and the downstream targets that affect protein synthesis are described.
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Mechanisms of type-I- and type-II-interferon-mediated signalling.
TL;DR: It is anticipated that an increased understanding of the contributions of these recently identified pathways will advance current thinking about how interferons work.
Journal ArticleDOI
Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
Kenta Hara,Yoshiko Maruki,Xiaomeng Long,Ken-ichi Yoshino,Noriko Oshiro,Sujuti Hidayat,Chiharu Tokunaga,Joseph Avruch,Kazuyoshi Yonezawa +8 more
TL;DR: Raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo and yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR.
Journal ArticleDOI
The tor pathway: a target for cancer therapy
TL;DR: In addition to the role of rapamycin as an immune suppressant, emerging data indicate that genetic and metabolic changes accompanying malignant transformation might cause hypersensitivity to TOR inhibition.
Journal ArticleDOI
PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.
Yasemin Sancak,Carson C. Thoreen,Timothy R. Peterson,Robert A. Lindquist,Seong A. Kang,Eric Spooner,Steven A. Carr,David M. Sabatini,David M. Sabatini +8 more
TL;DR: It is proposed that the relative strengths of the rheb- and PRAS40-mediated inputs to m TORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.
References
More filters
Journal ArticleDOI
Correlation between Protein and mRNA Abundance in Yeast
TL;DR: The results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient to predict protein expression levels from quantitative mRNA data.
Journal ArticleDOI
eIF4 Initiation Factors: Effectors of mRNA Recruitment to Ribosomes and Regulators of Translation
TL;DR: The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiated and regulated and suggests that eif4F is also implicated in malignancy and apoptosis.
Journal ArticleDOI
Regulation of translation initiation by FRAP/mTOR
Book
Translational control of gene expression
TL;DR: Origins and Principles of Translational Control, Genetic Approaches to Translation Initiation in Saccharomyces cerevisiae, and Programmed translational Frameshifting, Hopping, an
Journal ArticleDOI
Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function
Arnim Pause,Graham J. Belsham,Anne-Claude Gingras,Olivier Donzé,Tai-An Lin,John C. Lawrence,Nahum Sonenberg +6 more
TL;DR: The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor elF-4E, which binds to the messenger RNA 5'-cap structure, thereby relieving the translational inhibition.