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Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.

Identifieur interne : 000F09 ( Main/Exploration ); précédent : 000F08; suivant : 000F10

Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.

Auteurs : Kimberly Coffman [États-Unis] ; Bing Yang ; Jie Lu ; Ashley L. Tetlow ; Emelia Pelliccio ; Shan Lu ; Da-Chuan Guo ; Chun Tang ; Meng-Qiu Dong ; Fuyuhiko Tamanoi

Source :

RBID : pubmed:24403073

Descripteurs français

English descriptors

Abstract

mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.

DOI: 10.1074/jbc.M113.482067
PubMed: 24403073
PubMed Central: PMC3931034


Affiliations:


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Le document en format XML

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<term>Adaptor Proteins, Signal Transducing (metabolism)</term>
<term>Amino Acid Sequence (MeSH)</term>
<term>Animals (MeSH)</term>
<term>Carrier Proteins (chemistry)</term>
<term>Carrier Proteins (genetics)</term>
<term>Carrier Proteins (metabolism)</term>
<term>Conserved Sequence (MeSH)</term>
<term>Cross-Linking Reagents (pharmacology)</term>
<term>HEK293 Cells (MeSH)</term>
<term>Humans (MeSH)</term>
<term>Intracellular Signaling Peptides and Proteins (MeSH)</term>
<term>Lysine (metabolism)</term>
<term>Mass Spectrometry (methods)</term>
<term>Mechanistic Target of Rapamycin Complex 1 (MeSH)</term>
<term>Molecular Sequence Data (MeSH)</term>
<term>Multiprotein Complexes (metabolism)</term>
<term>Mutation (genetics)</term>
<term>Peptides (metabolism)</term>
<term>Phosphoproteins (chemistry)</term>
<term>Phosphoproteins (genetics)</term>
<term>Phosphoproteins (metabolism)</term>
<term>Protein Binding (drug effects)</term>
<term>Protein Structure, Tertiary (MeSH)</term>
<term>Rats (MeSH)</term>
<term>Regulatory-Associated Protein of mTOR (MeSH)</term>
<term>Substrate Specificity (drug effects)</term>
<term>TOR Serine-Threonine Kinases (metabolism)</term>
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<term>Animaux (MeSH)</term>
<term>Cellules HEK293 (MeSH)</term>
<term>Complexe-1 cible mécanistique de la rapamycine (MeSH)</term>
<term>Complexes multiprotéiques (métabolisme)</term>
<term>Données de séquences moléculaires (MeSH)</term>
<term>Humains (MeSH)</term>
<term>Liaison aux protéines (effets des médicaments et des substances chimiques)</term>
<term>Lysine (métabolisme)</term>
<term>Mutation (génétique)</term>
<term>Peptides (métabolisme)</term>
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<term>Phosphoprotéines (génétique)</term>
<term>Phosphoprotéines (métabolisme)</term>
<term>Protéine de régulation associée à mTOR (MeSH)</term>
<term>Protéines adaptatrices de la transduction du signal (composition chimique)</term>
<term>Protéines adaptatrices de la transduction du signal (métabolisme)</term>
<term>Protéines de transport (composition chimique)</term>
<term>Protéines de transport (génétique)</term>
<term>Protéines de transport (métabolisme)</term>
<term>Protéines et peptides de signalisation intracellulaire (MeSH)</term>
<term>Rats (MeSH)</term>
<term>Réactifs réticulants (pharmacologie)</term>
<term>Spectrométrie de masse (méthodes)</term>
<term>Spécificité du substrat (effets des médicaments et des substances chimiques)</term>
<term>Structure tertiaire des protéines (MeSH)</term>
<term>Séquence conservée (MeSH)</term>
<term>Séquence d'acides aminés (MeSH)</term>
<term>Sérine-thréonine kinases TOR (métabolisme)</term>
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<term>Adaptor Proteins, Signal Transducing</term>
<term>Carrier Proteins</term>
<term>Phosphoproteins</term>
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<term>Phosphoproteins</term>
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<term>Carrier Proteins</term>
<term>Lysine</term>
<term>Multiprotein Complexes</term>
<term>Peptides</term>
<term>Phosphoproteins</term>
<term>TOR Serine-Threonine Kinases</term>
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<term>Cross-Linking Reagents</term>
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<term>Phosphoprotéines</term>
<term>Protéines adaptatrices de la transduction du signal</term>
<term>Protéines de transport</term>
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<term>Spécificité du substrat</term>
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<term>Phosphoprotéines</term>
<term>Protéines de transport</term>
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<term>Mass Spectrometry</term>
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<term>Complexes multiprotéiques</term>
<term>Lysine</term>
<term>Peptides</term>
<term>Phosphoprotéines</term>
<term>Protéines adaptatrices de la transduction du signal</term>
<term>Protéines de transport</term>
<term>Sérine-thréonine kinases TOR</term>
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<term>Spectrométrie de masse</term>
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<term>Réactifs réticulants</term>
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<term>Amino Acid Sequence</term>
<term>Animals</term>
<term>Conserved Sequence</term>
<term>HEK293 Cells</term>
<term>Humans</term>
<term>Intracellular Signaling Peptides and Proteins</term>
<term>Mechanistic Target of Rapamycin Complex 1</term>
<term>Molecular Sequence Data</term>
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<term>Données de séquences moléculaires</term>
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<term>Protéine de régulation associée à mTOR</term>
<term>Protéines et peptides de signalisation intracellulaire</term>
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<div type="abstract" xml:lang="en">mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo. </div>
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<AbstractText>mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo. </AbstractText>
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