title: Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.
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.
authors.affiliation: From the Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Coffman
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Jie
authors.initials: J
authors.lastname: Lu
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Tetlow
authors.forename: Emelia
authors.initials: E
authors.lastname: Pelliccio
authors.forename: Shan
authors.initials: S
authors.lastname: Lu
authors.forename: Da-Chuan
authors.initials: DC
authors.lastname: Guo
authors.forename: Chun
authors.initials: C
authors.lastname: Tang
authors.forename: Meng-Qiu
authors.initials: MQ
authors.lastname: Dong
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Tamanoi
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D000076222:Mechanistic Target of Rapamycin Complex 1; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D000076223:Regulatory-Associated Protein of mTOR; D013379:Substrate Specificity / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C499526:Eif4ebp1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D010455:Peptides; D010750:Phosphoproteins; C463659:RPTOR protein, human; D000076223:Regulatory-Associated Protein of mTOR; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D008239:Lysine
keywords: 4E-BP1; Cross-linking Mass Spectrometry; Protein Cross-linking; Protein Kinases; Raptor; Signaling; TOS Motif; mTOR; mTOR Complex (mTORC)