Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.
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.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Carrier Proteins, Conserved Sequence, Cross-Linking Reagents, HEK293 Cells, Humans, Lysine, Mass Spectrometry, Molecular Sequence Data, Multiprotein Complexes, Mutation, Peptides, Phosphoproteins, Protein Binding, Protein Structure, Tertiary, Rats, Substrate Specificity, TOR Serine-Threonine Kinases
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Carrier Proteins, Conserved Sequence, Cross-Linking Reagents, HEK293 Cells, Humans, Lysine, Mass Spectrometry, Molecular Sequence Data, Multiprotein Complexes, Mutation, Peptides, Phosphoproteins, Protein Binding, Protein Structure, Tertiary, Rats, Substrate Specificity, TOR Serine-Threonine Kinases
J. Biol. Chem.
Date: Feb. 21, 2014
PubMed ID: 24403073
View in: Pubmed Google Scholar
Download Curated Data For This Publication
166708
Switch View:
- Interactions 5