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.
Highlights
MTORC1 recruits its substrate 4E-BP1 via Raptor/4E-BP1 interaction
Cross-linking of Raptor with 4E-BP1 and Identification of Cross-linked Peptides by Mass Spectrometry—mTORC1 consists of three major proteins: mammalian target of rapamycin (mTOR), Raptor, and mLST8(GL)
Cells were lysed in a buffer containing 0.4% CHAPS, and mTORC1 was affinity-purified by using anti-FLAG beads and eluted by using FLAG peptides as described under “Experimental Procedures.”
Summary
Reagents—Cross-linkers, bis[sulfosuccinimidyl] suberate (BS3) and disuccinimidyl suberate were obtained from Pierce. We subjected the preparation to another round of affinity purification using anti AU1 beads In this case, we transfected HEK293T cells stably expressing FLAG-Raptor with AU1-mTOR DNA [8] before collecting cells. MTORC1 was purified from HEK293T cells expressing FLAG-Raptor using anti-FLAG beads. The FLAG-Raptor lysate was obtained by lysing FLAG-Raptor expressing HEK293T cells in a 15-cm dish with 1 ml of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% CHAPS) and centrifuged for 30 min to remove insoluble fraction. 24 h after transfection, cells were harvested in ice-cold PBS and lysed in lysis buffer (40 mM HEPES, pH 7.4, 150 mM NaCl, 0.4% CHAPS), supplemented with complete protease inhibitors (Roche Applied Science) without EDTA, 1 mM Na3VO4.
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