title: Analysis of the regulatory motifs in eukaryotic initiation factor 4E-binding protein 1.
abstract: Mammalian target of rapamycin complex 1 (mTORC1) phosphorylates proteins such as eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and the S6 kinases. These substrates contain short sequences, termed TOR signalling (TOS) motifs, which interact with the mTORC1 component raptor. Phosphorylation of 4E-BP1 requires an additional feature, termed the RAIP motif (Arg-Ala-Ile-Pro). We have analysed the interaction of 4E-BP1 with raptor and the amino acid residues required for functional RAIP and TOS motifs, as assessed by raptor binding and the phosphorylation of 4E-BP1 in human cells. Binding of 4E-BP1 to raptor strongly depends on an intact TOS motif, but the RAIP motif and additional C-terminal features of 4E-BP1 also contribute to this interaction. Mutational analysis of 4E-BP1 reveals that isoleucine is a key feature of the RAIP motif, that proline is also very important and that there is greater tolerance for substitution of the first two residues. Within the TOS motif, the first position (phenylalanine in the known motifs) is most critical, whereas a wider range of residues function in other positions (although an uncharged aliphatic residue is preferred at position three). These data provide important information on the structural requirements for efficient signalling downstream of mTORC1.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Vivian H Y
authors.initials: VH
authors.lastname: Lee
authors.forename: Timothy
authors.initials: T
authors.lastname: Healy
authors.forename: Bruno D
authors.initials: BD
authors.lastname: Fonseca
authors.forename: Amanda
authors.initials: A
authors.lastname: Hayashi
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Proud
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D001120:Arginine; D005982:Glutathione Transferase; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases