Advertisement

Characterization of the Raptor/4E-BP1 Interaction by Chemical Cross-linking Coupled with Mass Spectrometry Analysis*

  • Kimberly Coffman
    Affiliations
    Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095
    Search for articles by this author
  • Bing Yang
    Affiliations
    National Institute of Biological Sciences, Beijing, Beijing 102206, China
    Search for articles by this author
  • Jie Lu
    Affiliations
    Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095
    Search for articles by this author
  • Ashley L. Tetlow
    Affiliations
    Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095
    Search for articles by this author
  • Emelia Pelliccio
    Affiliations
    Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095
    Search for articles by this author
  • Shan Lu
    Affiliations
    National Institute of Biological Sciences, Beijing, Beijing 102206, China
    Search for articles by this author
  • Da-Chuan Guo
    Affiliations
    Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
    Search for articles by this author
  • Chun Tang
    Affiliations
    Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
    Search for articles by this author
  • Meng-Qiu Dong
    Correspondence
    To whom correspondence may be addressed: National Institute of Biological Sciences, Beijing 102206, China. Tel.: 86-10-8070-6046; Fax: 86-10-8070-6053
    Affiliations
    National Institute of Biological Sciences, Beijing, Beijing 102206, China
    Search for articles by this author
  • Fuyuhiko Tamanoi
    Correspondence
    To whom correspondence may be addressed: Dept. of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, CA 90095-1489. Tel.: 310-206-7318; Fax: 310-206-5231
    Affiliations
    Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant CA41996 (to F. T.). This work was also supported by Ministry of Science and Technology of China Grants 2010CB835203 (to M.-Q. D.) and 2013CB910200 (to C. T.).
    This article contains supplemental Figs. S1 and S2.
Open AccessPublished:January 08, 2014DOI:https://doi.org/10.1074/jbc.M113.482067
      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.

      Introduction

      The mammalian target of rapamycin (mTOR)
      The abbreviations used are: mTOR
      mammalian target of rapamycin
      mTORC1
      mTOR complex 1
      CXMS
      chemical cross-linking coupled with mass spectrometry analysis
      BS3
      bis[sulfosuccinimidyl] suberate
      RNC
      Raptor N-terminal conserved
      RCR
      Raptor cross-linking region
      TOS
      TOR signaling.
      signaling pathway has attracted attention because of its involvement in a variety of cellular processes in response to stimuli such as growth factors, nutrients, and energy conditions (
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling at a glance.
      ,
      • Polak P.
      • Hall M.N.
      mTOR and the control of whole body metabolism.
      ,
      • Wullschleger S.
      • Loewith R.
      • Hall M.N.
      TOR signaling in growth and metabolism.
      ,
      • Tamanoi F.
      • Hall M.N.
      ). Up-regulation of this signaling pathway is frequently found in a variety of human cancers (
      • Karbowniczek M.
      • Spittle C.S.
      • Morrison T.
      • Wu H.
      • Henske E.P.
      mTOR is activated in the majority of malignant melanomas.
      ,
      • Robb V.A.
      • Karbowniczek M.
      • Klein-Szanto A.J.
      • Henske E.P.
      Activation of the mTOR signaling pathway in renal clear cell carcinoma.
      ,
      • Molinolo A.A.
      • Hewitt S.M.
      • Amornphimoltham P.
      • Keelawat S.
      • Rangdaeng S.
      • Meneses García A.
      • Raimondi A.R.
      • Jufe R.
      • Itoiz M.
      • Gao Y.
      • Saranath D.
      • Kaleebi G.S.
      • Yoo G.H.
      • Leak L.
      • Myers E.M.
      • Shintani S.
      • Wong D.
      • Massey H.D.
      • Yeudall W.A.
      • Lonardo F.
      • Ensley J.
      • Gutkind J.S.
      Dissecting the Akt/mammalian target of rapamycin signaling network. Emerging results from the head and neck cancer tissue array initiative.
      ). Single amino acid changes can confer constitutive activation of mTOR, and we have identified activating mutations R2505P and S2215Y by mining the human cancer genome database (
      • Sato T.
      • Nakashima A.
      • Guo L.
      • Coffman K.
      • Tamanoi F.
      Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer.
      ). mTOR is a protein kinase belonging to the PI3K-related kinase family that includes PI3K-, ATM-, ATR-(ATM- and Rad3-related), and DNA-dependent protein kinase (
      • Abraham R.T.
      PI 3-kinase related kinases. “Big” players in stress-induced signaling pathways.
      ). Protein kinases in this group share similar structural features including the presence of the HEAT repeat, FAT, FATC, and the kinase domains. mTOR forms two different multiprotein complexes, mTORC1 and mTORC2 (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.
      ,
      • Sarbassov D.D.
      • Ali S.M.
      • Kim D.H.
      • Guertin D.A.
      • Latek R.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and Raptor-independent pathway that regulates the cytoskeleton.
      ); mTORC1 consists mainly of mTOR, Raptor, and mLST8 (GβL), whereas mTORC2 consists of mTOR, Rictor, mLST8, and Sin1. Substrates of mTORC1 include 4E-BP1 and S6K, whose phosphorylation causes promotion of protein synthesis. In the case of 4E-BP1, its phosphorylation results in relieving inhibitory action of this protein on eIF4E, a cap-binding protein (
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling at a glance.
      ,
      • Polak P.
      • Hall M.N.
      mTOR and the control of whole body metabolism.
      ,
      • Wullschleger S.
      • Loewith R.
      • Hall M.N.
      TOR signaling in growth and metabolism.
      ,
      • Tamanoi F.
      • Hall M.N.
      ,
      • Gingras A.C.
      • Gygi S.P.
      • Raught B.
      • Polakiewicz R.D.
      • Abraham R.T.
      • Hoekstra M.F.
      • Aebersold R.
      • Sonenberg N.
      Regulation of 4E-BP1 phosphorylation. A novel two-step mechanism.
      ). S6K stimulates protein synthesis by phosphorylating ribosomal protein S6. In cancer cells, it is believed that 4E-BP1 has an important role in growth and proliferation (
      • She Q.B.
      • Halilovic E.
      • Ye Q.
      • Zhen W.
      • Shirasawa S.
      • Sasazuki T.
      • Solit D.B.
      • Rosen N.
      4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors.
      ,
      • Hsieh A.C.
      • Costa M.
      • Zollo O.
      • Davis C.
      • Feldman M.E.
      • Testa J.R.
      • Meyuhas O.
      • Shokat K.M.
      • Ruggero D.
      Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP1-eIF4E.
      ).
      Phosphorylation of 4E-BP1 by mTOR is assisted by Raptor, which functions as a scaffold protein. Raptor recruits substrate proteins to mTORC1 so that they can be phosphorylated by mTOR (
      • Kim D.H.
      • Sarbassov D.D.
      • Ali S.M.
      • King J.E.
      • Latek R.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      mTOR interacts with Raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.
      ,
      • Hara K.
      • Maruki Y.
      • Long X.
      • Yoshino K.
      • Oshiro N.
      • Hidayat S.
      • Tokunaga C.
      • Avruch J.
      • Yonezawa K.
      Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
      ). Raptor is a protein of 150 kDa that contains the Raptor N-terminal conserved (RNC) motif, the HEAT repeat, and the WD40 motif (
      • Kim D.H.
      • Sarbassov D.D.
      • Ali S.M.
      • King J.E.
      • Latek R.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      mTOR interacts with Raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.
      ,
      • Hara K.
      • Maruki Y.
      • Long X.
      • Yoshino K.
      • Oshiro N.
      • Hidayat S.
      • Tokunaga C.
      • Avruch J.
      • Yonezawa K.
      Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
      ). Mutations in the RNC motif decrease interaction with 4E-BP1 (
      • Kim D.H.
      • Sarbassov D.D.
      • Ali S.M.
      • King J.E.
      • Latek R.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      mTOR interacts with Raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.
      ,
      • Hara K.
      • Maruki Y.
      • Long X.
      • Yoshino K.
      • Oshiro N.
      • Hidayat S.
      • Tokunaga C.
      • Avruch J.
      • Yonezawa K.
      Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
      ). We wanted to further investigate how mTORC1 interacts with 4E-BP1. Specifically, we sought to gain insight into the region of proximity between Raptor and 4E-BP1.
      The recognition of substrate proteins by Raptor is believed to involve a five-amino acid sequence called the TOR signaling (TOS) motif, because mutations of this motif result in decreased interaction with Raptor (
      • Schalm S.S.
      • Fingar D.C.
      • Sabatini D.M.
      • Blenis J.
      TOS motif-mediated Raptor binding regulates 4E-BP1 multisite phosphorylation and function.
      ,
      • Eguchi S.
      • Tokunaga C.
      • Hidayat S.
      • Oshiro N.
      • Yoshino K.
      • Kikkawa U.
      • Yonezawa K.
      Different roles for the TOS and RAIP motifs of the translational regulator protein 4E-BP1 in the association with Raptor and phosphorylation by mTOR in the regulation of cell size.
      ,
      • Lee V.H.
      • Healy T.
      • Fonseca B.D.
      • Hayashi A.
      • Proud C.G.
      Analysis of the regulatory motifs in eukaryotic initiation factor 4E binding protein 1.
      ). Another motif called “RAIP” is also proposed to be involved (
      • Schalm S.S.
      • Fingar D.C.
      • Sabatini D.M.
      • Blenis J.
      TOS motif-mediated Raptor binding regulates 4E-BP1 multisite phosphorylation and function.
      ,
      • Eguchi S.
      • Tokunaga C.
      • Hidayat S.
      • Oshiro N.
      • Yoshino K.
      • Kikkawa U.
      • Yonezawa K.
      Different roles for the TOS and RAIP motifs of the translational regulator protein 4E-BP1 in the association with Raptor and phosphorylation by mTOR in the regulation of cell size.
      ,
      • Lee V.H.
      • Healy T.
      • Fonseca B.D.
      • Hayashi A.
      • Proud C.G.
      Analysis of the regulatory motifs in eukaryotic initiation factor 4E binding protein 1.
      ). Although these motifs are important, other regions of 4E-BP1 are also implicated in the interaction with Raptor (
      • Lee V.H.
      • Healy T.
      • Fonseca B.D.
      • Hayashi A.
      • Proud C.G.
      Analysis of the regulatory motifs in eukaryotic initiation factor 4E binding protein 1.
      ).
      Chemical cross-linking coupled with mass spectrometry analysis (CXMS) has emerged as a powerful method to characterize protein-protein interactions (
      • Tabb D.L.
      Evaluating protein interactions through cross-linking mass spectrometry.
      ,
      • Yang B.
      • Wu Y.J.
      • Zhu M.
      • Fan S.B.
      • Lin J.
      • Zhang K.
      • Li S.
      • Chi H.
      • Li Y.X.
      • Chen H.F.
      • Luo S.K.
      • Ding Y.H.
      • Wang L.H.
      • Hao Z.
      • Xiu L.Y.
      • Chen S.
      • Ye K.
      • He S.M.
      • Dong M.Q.
      Identification of cross-linked peptides from complex samples.
      ,
      • Sinz A.
      Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.
      ,
      • Leitner A.
      • Walzthoeni T.
      • Kahraman A.
      • Herzog F.
      • Rinner O.
      • Beck M.
      • Aebersold R.
      Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics.
      ,
      • Chen Z.A.
      • Jawhari A.
      • Fischer L.
      • Buchen C.
      • Tahir S.
      • Kamenski T.
      • Rasmussen M.
      • Lariviere L.
      • Bukowski-Wills J.C.
      • Nilges M.
      • Cramer P.
      • Rappsilber J.
      Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.
      ,
      • Rinner O.
      • Seebacher J.
      • Walzthoeni T.
      • Mueller L.N.
      • Beck M.
      • Schmidt A.
      • Mueller M.
      • Aebersold R.
      Identification of cross-linked peptides from large sequence databases.
      ,
      • Singh P.
      • Panchaud A.
      • Goodlett D.R.
      Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique.
      ). A basic principle of the method is to use chemical cross-linkers to link reactive amino acid residues that are closely located. After protease digestion, cross-linked peptide pairs are separated by liquid chromatography and can be identified using mass spectrometry. Because only the residues located in close proximity can be cross-linked, intramolecular cross-links can provide insights into the folding of a protein, and intermolecular cross-links can reveal the site(s) of interaction between two proteins. As such, CXMS has been applied to gain information about subunit interactions within multiprotein complexes, which is particularly advantageous for protein complexes that are difficult to purify and crystallize. One of the challenges concerning CXMS is to develop a software tool for identification of cross-linked peptides from their fragmentation spectra (
      • Yang B.
      • Wu Y.J.
      • Zhu M.
      • Fan S.B.
      • Lin J.
      • Zhang K.
      • Li S.
      • Chi H.
      • Li Y.X.
      • Chen H.F.
      • Luo S.K.
      • Ding Y.H.
      • Wang L.H.
      • Hao Z.
      • Xiu L.Y.
      • Chen S.
      • Ye K.
      • He S.M.
      • Dong M.Q.
      Identification of cross-linked peptides from complex samples.
      ,
      • Rinner O.
      • Seebacher J.
      • Walzthoeni T.
      • Mueller L.N.
      • Beck M.
      • Schmidt A.
      • Mueller M.
      • Aebersold R.
      Identification of cross-linked peptides from large sequence databases.
      ). The pLink software (
      • Yang B.
      • Wu Y.J.
      • Zhu M.
      • Fan S.B.
      • Lin J.
      • Zhang K.
      • Li S.
      • Chi H.
      • Li Y.X.
      • Chen H.F.
      • Luo S.K.
      • Ding Y.H.
      • Wang L.H.
      • Hao Z.
      • Xiu L.Y.
      • Chen S.
      • Ye K.
      • He S.M.
      • Dong M.Q.
      Identification of cross-linked peptides from complex samples.
      ) has been optimized using a large data set and shown to be effective on a variety of samples including protein complexes.
      In this paper, we report analysis of Raptor-4E-BP1 interaction using CXMS. We show that Raptor cross-links with 4E-BP1 and that several cross-linked peptides can be identified. Our results show that the cross-links mainly involve the RNC1 domain of Raptor and the central region of 4E-BP1, suggesting their close proximity. Identification of intramolecular cross-links raises the possibility that Raptor contains two structured domains. Finally, we have been able to block the formation of Raptor-4E-BP1 cross-links by synthetic peptides corresponding to the sequence within the RCR of 4E-BP1. We also show that mutations of 4E-BP1 residues within the RCR decrease the ability of 4E-BP1 to serve as a substrate of mTORC1 in vitro and in vivo.

      DISCUSSION

      In this paper, we characterized the interaction of mTORC1 and its substrate 4E-BP1 using the CXMS technology. First, we found that 4E-BP1 could be cross-linked with Raptor using purified mTORC1 as well as free Raptor. We then characterized the cross-linked band by mass spectrometry and identified cross-linked peptides that contain both Raptor and 4E-BP1 sequences. Multiple cross-linked peptides were identified that were clustered primarily to a single region for each protein. These results represent the first observation of Raptor and 4E-BP1 cross-links, and the study revealed that the RNC of Raptor and the RCR of 4E-BP1 are in close proximity. Computational structural prediction (Fig. 4B) can be made regarding the residues in the two proteins.
      Our cross-link results were further substantiated by first carrying out peptide inhibition studies. Two peptides that encompass the Raptor interaction site of 4E-BP1 inhibited Raptor-4E-BP1 cross-links, whereas the control peptides each having a scrambled sequence did not. Peptide 2 gave strong inhibition at 15 μm concentration. Second, we have mutated residues within the 4E-BP1 region we defined by our cross-linking study and showed that that the mutations dramatically decrease binding of 4E-BP1 to mTORC1 and reduce phosphorylation by mTORC1. In cells, phosphorylation of the mutant 4E-BP1 was significantly decreased compared with that of the wild type protein. In addition, Raptor binding was significantly decreased with the mutant 4E-BP1.
      Our results on 4E-BP1 suggest that a region encompassing residues 56 and 72 is in close proximity with Raptor. This region is highly conserved among 4E-BP family members (
      • Poulin F.
      • Gingras A.C.
      • Olsen H.
      • Chevalier S.
      • Sonenberg N.
      4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family.
      ). The sequence KFLMECRNSPVAKTPP in 4E-BP1 corresponds to the sequence KFLLDRRNSPMAQTPP in 4E-BP2 and KFLLECKNSPIARTPP in 4E-BP3. Two mTORC1-mediated phosphorylation sites (
      • Gingras A.C.
      • Raught B.
      • Gygi S.P.
      • Niedzwiecka A.
      • Miron M.
      • Burley S.K.
      • Polakiewicz R.D.
      • Wyslouch-Cieszynska A.
      • Aebersold R.
      • Sonenberg N.
      Hierarchical phosphorylation of the translation inhibitor 4E-BP1.
      ) are present in this region. A sequence that is known to interact with eIF4E, as defined by the co-crystallization of eIF4E and a 4E-BP1 peptide (
      • Matsuo H.
      • Li H.
      • McGuire A.M.
      • Fletcher C.M.
      • Gingras A.C.
      • Sonenberg N.
      • Wagner G.
      Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein.
      ), is located on the N-terminal side of the region. The TOS and RAIP motifs have been proposed to play important roles for the interaction between Raptor and mTORC1 substrates. However, the location of the TOS motif in 4E-BP1 is distant from the Raptor cross-linking site that we have identified. It should be noted that our cross-linking method provides information on close proximity of residues, and our results do not exclude the possibility that the TOS and RAIP motifs are playing important roles in the interaction with Raptor. In fact, in earlier studies with 4E-BP1, it has been suggested that regions other than the TOS and RAIP are also involved in Raptor interaction (
      • Lee V.H.
      • Healy T.
      • Fonseca B.D.
      • Hayashi A.
      • Proud C.G.
      Analysis of the regulatory motifs in eukaryotic initiation factor 4E binding protein 1.
      ). It may be the case that multiple sites of 4E-BP1 are involved in the interaction with Raptor. Further analysis is needed to fully understand how Raptor interacts with 4E-BP1.
      On the Raptor side, our results point to the importance of the RNC region. We found that the 4E-BP1 cross-links are clustered within the first RNC domain (RNC1) of Raptor close to the N terminus. The significance of the RNC1 region for interaction with substrate proteins was further supported by our recent preliminary experiments that sought to identify cross-links between Raptor and S6K. In this experiment, unphosphorylated S6K was purified from HEK293T cells transfected with FLAG-S6K and treated with mTOR inhibitor pp242. Incubation of this protein with mTORC1 in the presence of a cross-linker BS3 identified cross-linked peptides that were between Lys120 of Raptor and Lys450 of S6K. Therefore, the same RNC1 of Raptor was involved in the interaction with S6K and 4E-BP1. This site on S6K that interacts with Raptor is distant from the TOS motif that is located close to the N terminus.
      Our study also revealed intramolecular cross-links that occur within the Raptor protein. The locations of these intramolecular cross-links suggest that the Raptor protein has two separate structured domains: one in the N-terminal half and the other in the C-terminal half of the protein. The N-terminal structured domain encompasses RNC1 and RNC2 domains. Interestingly, the lysine residues found to be cross-linked with 4E-BP1 are all located in a small region within this structured domain (Fig. 5). On the other hand, our recent preliminary results raise the possibility that the interaction with mTOR takes place mainly at the C-terminal region. We have recently identified Raptor-mTOR cross-linked peptides. Three cross-linked peptides were identified, and all cross-linked lysines in Raptor were located within the C-terminal structured domain (Lys1332 and Lys1008). Two cross-linked peptides had Raptor cross-linked to the mTOR residue 2507, a residue located within the kinase domain of mTOR. These results suggest that 4E-BP1 and mTOR interact with different regions of Raptor: 4E-BP1 at the N-terminal structured region and mTOR at the C-terminal structured region. Based on our identification of Raptor120-Raptor894 cross-links, Raptor is likely to be folded as depicted in Fig. 5B. If this were the case, the N-terminal region and the C-terminal region of Raptor are located in close proximity, and this will result in bringing the mTOR kinase domain and 4E-BP1 together.
      4E-BP1 in its nonphosphorylated form functions to inhibit protein synthesis by binding to eIF4E, a cap-binding protein that facilitates initiation of protein synthesis. eIF4E is also an oncogene as demonstrated by its ability to confer transformed phenotypes and tumor formation in mice (
      • Lazaris-Karatzas A.
      • Montine K.S.
      • Sonenberg N.
      Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5′ cap.
      ), and overexpression of eIF4E is observed in a variety of cancer cells, most notably in acute myelogenous leukemia (
      • Ruggero D.
      • Pandolfi P.P.
      Does the ribosome translate cancer?.
      ). Ectopic expression of nonphosphorylated form of 4E-BP1 partially reverses transformed phenotypes (
      • Rousseau D.
      • Gingras A.C.
      • Pause A.
      • Sonenberg N.
      The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth.
      ). Thus, blocking the interaction between Raptor and 4E-BP1 is expected to increase the concentration of nonphosphorylated 4E-BP1, which in turn would inhibit eIF4E and tumor growth when the tumor is driven by the overexpression of eIF4E. Small molecule inhibitors have been identified to inhibit the action of eIF4E (
      • Moerke N.J.
      • Aktas H.
      • Chen H.
      • Cantel S.
      • Reibarkh M.Y.
      • Fahmy A.
      • Gross J.D.
      • Degterev A.
      • Yuan J.
      • Chorev M.
      • Halperin J.A.
      • Wagner G.
      Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G.
      ,
      • Assouline S.
      • Culjkovic B.
      • Cocolakis E.
      • Rousseau C.
      • Beslu N.
      • Amri A.
      • Caplan S.
      • Leber B.
      • Roy D.C.
      • Miller Jr., W.H.
      • Borden K.L.
      Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML). A proof-of-principle clinical trial with ribavirin.
      ). Further understanding of how Raptor interacts with 4E-BP1 may open up a novel approach to inhibit eIF4E-mediated tumor formation.

      Acknowledgments

      We thank David Lopez for discussion on structured domains of Raptor. We thank the UCLA Vector Core Facility for the preparation of lentivirus carrying FLAG-Raptor.

      References

        • Laplante M.
        • Sabatini D.M.
        mTOR signaling at a glance.
        J. Cell Sci. 2009; 122: 3589-3594
        • Polak P.
        • Hall M.N.
        mTOR and the control of whole body metabolism.
        Curr. Opin. Cell Biol. 2009; 21: 209-218
        • Wullschleger S.
        • Loewith R.
        • Hall M.N.
        TOR signaling in growth and metabolism.
        Cell. 2006; 124: 471-484
        • Tamanoi F.
        • Hall M.N.
        Structure, Function and Regulation of TOR Complexes from Yeasts to Mammals Part B: The Enzymes. 28. Academic Press/Elsevier, 2010
        • Karbowniczek M.
        • Spittle C.S.
        • Morrison T.
        • Wu H.
        • Henske E.P.
        mTOR is activated in the majority of malignant melanomas.
        J. Invest. Dermatol. 2008; 128: 980-987
        • Robb V.A.
        • Karbowniczek M.
        • Klein-Szanto A.J.
        • Henske E.P.
        Activation of the mTOR signaling pathway in renal clear cell carcinoma.
        J. Urol. 2007; 177: 346-352
        • Molinolo A.A.
        • Hewitt S.M.
        • Amornphimoltham P.
        • Keelawat S.
        • Rangdaeng S.
        • Meneses García A.
        • Raimondi A.R.
        • Jufe R.
        • Itoiz M.
        • Gao Y.
        • Saranath D.
        • Kaleebi G.S.
        • Yoo G.H.
        • Leak L.
        • Myers E.M.
        • Shintani S.
        • Wong D.
        • Massey H.D.
        • Yeudall W.A.
        • Lonardo F.
        • Ensley J.
        • Gutkind J.S.
        Dissecting the Akt/mammalian target of rapamycin signaling network. Emerging results from the head and neck cancer tissue array initiative.
        Clin. Cancer Res. 2007; 13: 4964-4973
        • Sato T.
        • Nakashima A.
        • Guo L.
        • Coffman K.
        • Tamanoi F.
        Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer.
        Oncogene. 2010; 29: 2746-2752
        • Abraham R.T.
        PI 3-kinase related kinases. “Big” players in stress-induced signaling pathways.
        DNA Repair. 2004; 3: 883-887
        • Jacinto E.
        • Loewith R.
        • Schmidt A.
        • Lin S.
        • Rüegg M.A.
        • Hall A.
        • Hall M.N.
        Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.
        Nat. Cell Biol. 2004; 6: 1122-1128
        • Sarbassov D.D.
        • Ali S.M.
        • Kim D.H.
        • Guertin D.A.
        • Latek R.R.
        • Erdjument-Bromage H.
        • Tempst P.
        • Sabatini D.M.
        Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and Raptor-independent pathway that regulates the cytoskeleton.
        Curr. Biol. 2004; 14: 1296-1302
        • Gingras A.C.
        • Gygi S.P.
        • Raught B.
        • Polakiewicz R.D.
        • Abraham R.T.
        • Hoekstra M.F.
        • Aebersold R.
        • Sonenberg N.
        Regulation of 4E-BP1 phosphorylation. A novel two-step mechanism.
        Genes Dev. 1999; 13: 1422-1437
        • She Q.B.
        • Halilovic E.
        • Ye Q.
        • Zhen W.
        • Shirasawa S.
        • Sasazuki T.
        • Solit D.B.
        • Rosen N.
        4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors.
        Cancer Cell. 2010; 18: 39-51
        • Hsieh A.C.
        • Costa M.
        • Zollo O.
        • Davis C.
        • Feldman M.E.
        • Testa J.R.
        • Meyuhas O.
        • Shokat K.M.
        • Ruggero D.
        Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP1-eIF4E.
        Cancer Cell. 2010; 17: 249-261
        • Kim D.H.
        • Sarbassov D.D.
        • Ali S.M.
        • King J.E.
        • Latek R.R.
        • Erdjument-Bromage H.
        • Tempst P.
        • Sabatini D.M.
        mTOR interacts with Raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.
        Cell. 2002; 110: 163-175
        • Hara K.
        • Maruki Y.
        • Long X.
        • Yoshino K.
        • Oshiro N.
        • Hidayat S.
        • Tokunaga C.
        • Avruch J.
        • Yonezawa K.
        Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
        Cell. 2002; 110: 177-189
        • Schalm S.S.
        • Fingar D.C.
        • Sabatini D.M.
        • Blenis J.
        TOS motif-mediated Raptor binding regulates 4E-BP1 multisite phosphorylation and function.
        Curr. Biol. 2003; 13: 797-806
        • Eguchi S.
        • Tokunaga C.
        • Hidayat S.
        • Oshiro N.
        • Yoshino K.
        • Kikkawa U.
        • Yonezawa K.
        Different roles for the TOS and RAIP motifs of the translational regulator protein 4E-BP1 in the association with Raptor and phosphorylation by mTOR in the regulation of cell size.
        Genes Cells. 2006; 11: 757-766
        • Lee V.H.
        • Healy T.
        • Fonseca B.D.
        • Hayashi A.
        • Proud C.G.
        Analysis of the regulatory motifs in eukaryotic initiation factor 4E binding protein 1.
        FEBS J. 2008; 275: 2185-2199
        • Tabb D.L.
        Evaluating protein interactions through cross-linking mass spectrometry.
        Nat. Methods. 2012; 9: 879-881
        • Yang B.
        • Wu Y.J.
        • Zhu M.
        • Fan S.B.
        • Lin J.
        • Zhang K.
        • Li S.
        • Chi H.
        • Li Y.X.
        • Chen H.F.
        • Luo S.K.
        • Ding Y.H.
        • Wang L.H.
        • Hao Z.
        • Xiu L.Y.
        • Chen S.
        • Ye K.
        • He S.M.
        • Dong M.Q.
        Identification of cross-linked peptides from complex samples.
        Nat. Methods. 2012; 9: 904-906
        • Sinz A.
        Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.
        J. Mass Spectrom. 2003; 38: 1225-1237
        • Leitner A.
        • Walzthoeni T.
        • Kahraman A.
        • Herzog F.
        • Rinner O.
        • Beck M.
        • Aebersold R.
        Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics.
        Mol. Cell. Proteomics. 2010; 9: 1634-1649
        • Chen Z.A.
        • Jawhari A.
        • Fischer L.
        • Buchen C.
        • Tahir S.
        • Kamenski T.
        • Rasmussen M.
        • Lariviere L.
        • Bukowski-Wills J.C.
        • Nilges M.
        • Cramer P.
        • Rappsilber J.
        Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.
        EMBO J. 2010; 29: 717-726
        • Rinner O.
        • Seebacher J.
        • Walzthoeni T.
        • Mueller L.N.
        • Beck M.
        • Schmidt A.
        • Mueller M.
        • Aebersold R.
        Identification of cross-linked peptides from large sequence databases.
        Nat. Methods. 2008; 5: 315-318
        • Singh P.
        • Panchaud A.
        • Goodlett D.R.
        Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique.
        Anal. Chem. 2010; 82: 2636-2642
        • Sato T.
        • Nakashima A.
        • Guo L.
        • Tamanoi F.
        Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein.
        J. Biol. Chem. 2009; 284: 12783-12791
        • Pandey A.
        • Andersen J.S.
        • Mann M.
        Use of mass spectrometry to study signaling pathways.
        Sci. STKE. 2000; 2000: PL1
        • Roy A.
        • Kucukural A.
        • Zhang Y.
        I-TASSER. A unified platform for automated protein structure and function prediction.
        Nat. Protoc. 2010; 5: 725-738
        • Schwieters C.D.
        • Kuszewski J.J.
        • Tjandra N.
        • Clore G.M.
        The Xplor-NIH NMR molecular structure determination package.
        J. Magn. Reson. 2003; 160: 65-73
        • Bermejo G.A.
        • Clore G.M.
        • Schwieters C.D.
        Smooth statistical torsion angle potential derived from a large conformational database via adaptive kernel density estimation improves the quality of NMR protein structures.
        Protein Sci. 2012; 21: 1824-1836
        • Tang C.
        • Clore G.M.
        A simple and reliable approach to docking protein-protein complexes from very sparse NOE-derived intermolecular distance restraints.
        J. Biomol. NMR. 2006; 36: 37-44
        • Pierce B.
        • Weng Z.
        ZRANK. Reranking protein docking predictions with an optimized energy function.
        Proteins. 2007; 67: 1078-1086
        • DeLano W.L.
        The PyMOL Molecular Graphics System. Schroedinger, LLC, New York2012 (version 1.5.0.1)
        • Moerke N.J.
        • Aktas H.
        • Chen H.
        • Cantel S.
        • Reibarkh M.Y.
        • Fahmy A.
        • Gross J.D.
        • Degterev A.
        • Yuan J.
        • Chorev M.
        • Halperin J.A.
        • Wagner G.
        Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G.
        Cell. 2007; 128: 257-267
        • Yip C.K.
        • Murata K.
        • Walz T.
        • Sabatini D.M.
        • Kang S.A.
        Structure of the human mTOR complex I and its implications for rapamycin inhibition.
        Mol. Cell. 2010; 38: 768-774
        • Tomoo K.
        • Matsushita Y.
        • Fujisaki H.
        • Abiko F.
        • Shen X.
        • Taniguchi T.
        • Miyagawa H.
        • Kitamura K.
        • Miura K.
        • Ishida T.
        Structural basis for mRNA cap-binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, x-ray crystal structural, and molecular dynamics simulation methods.
        Biochim. Biophys. Acta. 2005; 1753: 191-208
        • Rosettani P.
        • Knapp S.
        • Vismara M.G.
        • Rusconi L.
        • Cameron A.D.
        Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms.
        J. Mol. Biol. 2007; 368: 691-705
        • Brown C.J.
        • McNae I.
        • Fischer P.M.
        • Walkinshaw M.D.
        Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives.
        J. Mol. Biol. 2007; 372: 7-15
        • Liu W.
        • Zhao R.
        • McFarland C.
        • Kieft J.
        • Niedzwiecka A.
        • Jankowska-Anyszka M.
        • Stepinski J.
        • Darzynkiewicz E.
        • Jones D.N.
        • Davis R.E.
        Structural insights into parasite eIF4E binding specificity for m7G and m2,2,7G mRNA caps.
        J. Biol. Chem. 2009; 284: 31336-31349
        • Liu W.
        • Jankowska-Anyszka M.
        • Piecyk K.
        • Dickson L.
        • Wallace A.
        • Niedzwiecka A.
        • Stepinski J.
        • Stolarski R.
        • Darzynkiewicz E.
        • Kieft J.
        • Zhao R.
        • Jones D.N.
        • Davis R.E.
        Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation.
        Nucleic Acids Res. 2011; 39: 8820-8832
        • Siddiqui N.
        • Tempel W.
        • Nedyalkova L.
        • Volpon L.
        • Wernimont A.K.
        • Osborne M.J.
        • Park H.W.
        • Borden K.L.
        Structural insights into the allosteric effects of 4EBP1 on the eukaryotic translation initiation factor eIF4E.
        J. Mol. Biol. 2012; 415: 781-792
        • Fletcher C.M.
        • McGuire A.M.
        • Gingras A.C.
        • Li H.
        • Matsuo H.
        • Sonenberg N.
        • Wagner G.
        4E binding proteins inhibit the translation factor eIF4E without folded structure.
        Biochemistry. 1998; 37: 9-15
        • Poulin F.
        • Gingras A.C.
        • Olsen H.
        • Chevalier S.
        • Sonenberg N.
        4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family.
        J. Biol. Chem. 1998; 273: 14002-14007
        • Gingras A.C.
        • Raught B.
        • Gygi S.P.
        • Niedzwiecka A.
        • Miron M.
        • Burley S.K.
        • Polakiewicz R.D.
        • Wyslouch-Cieszynska A.
        • Aebersold R.
        • Sonenberg N.
        Hierarchical phosphorylation of the translation inhibitor 4E-BP1.
        Genes Dev. 2001; 15: 2852-2864
        • Matsuo H.
        • Li H.
        • McGuire A.M.
        • Fletcher C.M.
        • Gingras A.C.
        • Sonenberg N.
        • Wagner G.
        Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein.
        Nat. Struct. Biol. 1997; 4: 717-724
        • Lazaris-Karatzas A.
        • Montine K.S.
        • Sonenberg N.
        Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5′ cap.
        Nature. 1990; 345: 544-547
        • Ruggero D.
        • Pandolfi P.P.
        Does the ribosome translate cancer?.
        Nat. Rev. Cancer. 2003; 3: 179-192
        • Rousseau D.
        • Gingras A.C.
        • Pause A.
        • Sonenberg N.
        The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth.
        Oncogene. 1996; 13: 2415-2420
        • Assouline S.
        • Culjkovic B.
        • Cocolakis E.
        • Rousseau C.
        • Beslu N.
        • Amri A.
        • Caplan S.
        • Leber B.
        • Roy D.C.
        • Miller Jr., W.H.
        • Borden K.L.
        Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML). A proof-of-principle clinical trial with ribavirin.
        Blood. 2009; 114: 257-260