Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis
Kimberly Coffman1, Bing Yang, Jie Lu
1From the Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095.
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.
Insights
Researchers identified the specific interaction sites between Raptor and 4E-BP1, crucial for regulating protein synthesis via mTORC1 signaling. This finding clarifies how mTORC1 controls cell growth and proliferation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of protein synthesis, cell growth, and proliferation.
- mTORC1 phosphorylates 4E-binding protein 1 (4E-BP1), releasing eukaryotic initiation factor 4E (eIF4E) and promoting protein synthesis.
- Raptor, a component of mTORC1, is thought to recruit 4E-BP1 to facilitate its phosphorylation.
Purpose of the Study:
- To elucidate the specific interaction domains between Raptor and 4E-BP1 within the mTORC1 complex.
- To understand how these interactions influence 4E-BP1 phosphorylation and its role in protein synthesis regulation.
Main Methods:
- Chemical cross-linking using bis[sulfosuccinimidyl] suberate.
- Mass spectrometry analysis to identify cross-linked peptides between Raptor and 4E-BP1.
- In vitro and in vivo assays to assess the functional impact of identified interaction regions and mutations.
Main Results:
- Chemical cross-linking identified specific interaction sites between Raptor and 4E-BP1.
- The N-terminal conserved domain of Raptor (residues 89-180) interacts with the Raptor cross-linking region (RCR, residues 56-72) of 4E-BP1.
- Peptides encompassing the 4E-BP1 RCR inhibited Raptor-4E-BP1 cross-linking and interaction, and mutations in this region reduced 4E-BP1's substrate efficiency.
Conclusions:
- The N-terminal domain of Raptor directly interacts with the central RCR of 4E-BP1.
- This interaction is critical for the recruitment of 4E-BP1 by mTORC1 and its subsequent phosphorylation.
- These findings provide a detailed molecular understanding of mTORC1-mediated regulation of protein synthesis.
