Characterization of the Raptor/4E-BP1 Interaction by Chemical Cross-linking Coupled with Mass Spectrometry Analysis
| Autor: | Da-Chuan Guo, Kimberly Coffman, Emelia Pelliccio, Chun Tang, Bing Yang, Fuyuhiko Tamanoi, Ashley L. Tetlow, Meng-Qiu Dong, Shan Lu, Jie Lu |
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| Rok vydání: | 2014 |
| Předmět: |
Molecular Sequence Data
Protein domain Peptide mTORC1 Mechanistic Target of Rapamycin Complex 1 Biology environment and public health Biochemistry Mass Spectrometry Substrate Specificity Protein biosynthesis Animals Humans Amino Acid Sequence Molecular Biology Conserved Sequence PI3K/AKT/mTOR pathway Adaptor Proteins Signal Transducing chemistry.chemical_classification Kinase Lysine TOR Serine-Threonine Kinases fungi EIF4E Intracellular Signaling Peptides and Proteins Regulatory-Associated Protein of mTOR Cell Biology Phosphoproteins Protein Structure Tertiary Rats enzymes and coenzymes (carbohydrates) Cross-Linking Reagents HEK293 Cells chemistry Multiprotein Complexes Mutation Phosphorylation biological phenomena cell phenomena and immunity Carrier Proteins Peptides Signal Transduction Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 289:4723-4734 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m113.482067 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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