Abstract
The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.
Keywords:
NMR spectroscopy; atypical kinase; hierarchical phosphorylation; intrinsically disordered protein; kinetic modelling; mTOR signaling; multi‑site binding; protein dynamics; target of rapamycin; translational control.
Copyright © 2021 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / chemistry*
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism
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Binding Sites
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Cell Cycle Proteins / chemistry*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Chaetomium / chemistry
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Chaetomium / genetics
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Cloning, Molecular
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Crystallography, X-Ray
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Eukaryotic Initiation Factor-4E / chemistry*
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Eukaryotic Initiation Factor-4E / genetics
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Eukaryotic Initiation Factor-4E / metabolism
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Humans
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Kinetics
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Mechanistic Target of Rapamycin Complex 1 / chemistry*
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Mechanistic Target of Rapamycin Complex 1 / genetics
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Mechanistic Target of Rapamycin Complex 1 / metabolism
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Models, Molecular
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Phosphorylation
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Protein Processing, Post-Translational
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Regulatory-Associated Protein of mTOR / chemistry*
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Regulatory-Associated Protein of mTOR / genetics
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Regulatory-Associated Protein of mTOR / metabolism
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Signal Transduction
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Structural Homology, Protein
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Substrate Specificity
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TOR Serine-Threonine Kinases / chemistry*
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TOR Serine-Threonine Kinases / genetics
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TOR Serine-Threonine Kinases / metabolism
Substances
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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EIF4E protein, human
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EIF4EBP1 protein, human
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Eukaryotic Initiation Factor-4E
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RPTOR protein, human
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Recombinant Proteins
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Regulatory-Associated Protein of mTOR
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MTOR protein, human
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Mechanistic Target of Rapamycin Complex 1
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TOR Serine-Threonine Kinases