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

The translational regulator protein 4E‐BP1, that binds to eukaryotic initiation factor‐4E (eIF4E) to prevent the formation of the active translation complex, dissociates from eIF4E by phosphorylation through the mammalian target of rapamycin (mTOR) in the cells stimulated by amino acids. 4E‐BP1 has been shown to associate with the scaffold protein raptor through its TOS and RAIP motifs to be recognized by mTOR. We revealed that the TOS motif mutant was phosphorylated by mTOR only at the priming sites of Thr37/46 but the RAIP motif mutant was phosphorylated not only at the priming sites but also at the subsequent site of Thr70 in vitro and in response to amino acid treatment in HEK293 cells. Analysis using the phosphorylation site mutants indicated that phosphorylation of the priming and subsequent sites of 4E‐BP1 was required for dissociation from raptor as well as for the release of eIF4E. The expression of the 4E‐BP1 mutants replacing the TOS motif and phosphorylation sites, that are poor substrates for mTOR and have no or little dissociation ability from raptor and eIF4E, respectively, significantly reduced the size of K562 cells. These results indicate that the the TOS motif has an essential function whereas the RAIP motif has an accessory role in the association with raptor and mTOR‐mediated phosphorylation of 4E‐BP1 to dissociate it from raptor and release eIF4E in response to amino acid stimulation leading to the control of cell size.

[1]  M. Murakami,et al.  Distinct Signaling Events Downstream of mTOR Cooperate To Mediate the Effects of Amino Acids and Insulin on Initiation Factor 4E-Binding Proteins , 2005, Molecular and Cellular Biology.

[2]  M. Murakami,et al.  mTOR Is Essential for Growth and Proliferation in Early Mouse Embryos and Embryonic Stem Cells , 2004, Molecular and Cellular Biology.

[3]  J. Avruch,et al.  Dissociation of raptor from mTOR is a mechanism of rapamycin‐induced inhibition of mTOR function , 2004, Genes to cells : devoted to molecular & cellular mechanisms.

[4]  C. Proud,et al.  Target of Rapamycin (TOR)-signaling and RAIP Motifs Play Distinct Roles in the Mammalian TOR-dependent Phosphorylation of Initiation Factor 4E-binding Protein 1* , 2003, Journal of Biological Chemistry.

[5]  J. Lawrence,et al.  Two Motifs in the Translational Repressor PHAS-I Required for Efficient Phosphorylation by Mammalian Target of Rapamycin and for Recognition by Raptor* , 2003, Journal of Biological Chemistry.

[6]  J. Blenis,et al.  TOS Motif-Mediated Raptor Binding Regulates 4E-BP1 Multisite Phosphorylation and Function , 2003, Current Biology.

[7]  J. Avruch,et al.  The Mammalian Target of Rapamycin (mTOR) Partner, Raptor, Binds the mTOR Substrates p70 S6 Kinase and 4E-BP1 through Their TOR Signaling (TOS) Motif* , 2003, The Journal of Biological Chemistry.

[8]  Michael N. Hall,et al.  TOR signalling in bugs, brain and brawn , 2003, Nature Reviews Molecular Cell Biology.

[9]  C. Proud,et al.  Regulation of mammalian translation factors by nutrients. , 2002, European journal of biochemistry.

[10]  J. Avruch,et al.  Raptor, a Binding Partner of Target of Rapamycin (TOR), Mediates TOR Action , 2002, Cell.

[11]  D. Sabatini,et al.  mTOR Interacts with Raptor to Form a Nutrient-Sensitive Complex that Signals to the Cell Growth Machinery , 2002, Cell.

[12]  J. Blenis,et al.  Identification of a Conserved Motif Required for mTOR Signaling , 2002, Current Biology.

[13]  C. Proud,et al.  Caspase Cleavage of Initiation Factor 4E-Binding Protein 1 Yields a Dominant Inhibitor of Cap-Dependent Translation and Reveals a Novel Regulatory Motif , 2002, Molecular and Cellular Biology.

[14]  S K Burley,et al.  Hierarchical phosphorylation of the translation inhibitor 4E-BP1. , 2001, Genes & development.

[15]  A. Gingras,et al.  Regulation of translation initiation by FRAP/mTOR. , 2001, Genes & development.

[16]  Tobias Schmelzle,et al.  TOR, a Central Controller of Cell Growth , 2000, Cell.

[17]  Kenta Hara,et al.  Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro * , 1999, The Journal of Biological Chemistry.

[18]  E. Hafen,et al.  Drosophila S6 kinase: a regulator of cell size. , 1999, Science.

[19]  G. Brunn,et al.  Mutational analysis of sites in the translational regulator, PHAS‐I, that are selectively phosphorylated by mTOR , 1999, FEBS letters.

[20]  J. Avruch,et al.  Regulation of Translational Effectors by Amino Acid and Mammalian Target of Rapamycin Signaling Pathways , 1999, The Journal of Biological Chemistry.

[21]  K. Kaneko,et al.  Characterization of the phosphoproteins and protein kinase activity in mTOR immunoprecipitates. , 1998, Biochemical and biophysical research communications.

[22]  J. Avruch,et al.  Amino Acid Sufficiency and mTOR Regulate p70 S6 Kinase and eIF-4E BP1 through a Common Effector Mechanism* , 1998, The Journal of Biological Chemistry.

[23]  M. Kasuga,et al.  Regulation of eIF-4E BP1 Phosphorylation by mTOR* , 1997, The Journal of Biological Chemistry.

[24]  M. Sekiguchi.  Genes to cells: edited by Jun-ichi Tomizawa, Blackwell Science Ltd. Institutional: £218.00 (Europe), £242.00 (Rest of World), US$382.00 (USA and Canada). Individual: £65.00 (Europe), £72.00 (Rest of World), US$114.00 (USA and Canada) ISSN 1356 9597 , 1997 .

[25]  T. Haystead,et al.  Identification of Phosphorylation Sites in the Translational Regulator, PHAS-I, That Are Controlled by Insulin and Rapamycin in Rat Adipocytes* , 1997, The Journal of Biological Chemistry.

[26]  Nihon Hassei Seibutsu Gakkai,et al.  Genes to cells , 1996 .

[27]  K. Arndt,et al.  Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. , 1996, Genes & development.

[28]  A. Gingras,et al.  Rapamycin blocks the phosphorylation of 4E‐BP1 and inhibits cap‐dependent initiation of translation. , 1996, The EMBO journal.

[29]  I. Stansfield,et al.  An MBoC Favorite: TOR controls translation initiation and early G1 progression in yeast , 2012, Molecular biology of the cell.

[30]  J. Kunz,et al.  Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression , 1993, Cell.