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Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

Oncogene volume 27, pages 811–822 (2008)Cite this article

Abstract

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecular-weight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecular-weight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m7GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.

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References

  • Aberle H, Bauer A, Stappert J, Kispert A, Kemler R . (1997). Beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J 16: 3797–3804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Aguilar RC, Wendland B . (2003). Ubiquitin: not just for proteasomes anymore. Curr Opin Cell Biol 15: 184–190.

    Article  CAS  PubMed  Google Scholar 

  • Carroll M, Dyer J, Sossin WS . (2006). Serotonin increases phosphorylation of synaptic 4EBP through TOR, but eukaryotic initiation factor 4E levels do not limit somatic cap-dependent translation in aplysia neurons. Mol Cell Biol 26: 8586–8598.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carter PS, Jarquin-Pardo M, De Benedetti A . (1999). Differential expression of Myc1 and Myc2 isoforms in cells transformed by eIF4E: evidence for internal ribosome repositioning in the human c-myc 5′UTR. Oncogene 18: 4326–4335.

    Article  CAS  PubMed  Google Scholar 

  • Castellvi J, Garcia A, Rojo F, Ruiz-Marcellan C, Gil A, Baselga J et al. (2006). Phosphorylated 4E binding protein 1: a hallmark of cell signaling that correlates with survival in ovarian cancer. Cancer 107: 1801–1811.

    Article  CAS  PubMed  Google Scholar 

  • Chen ZJ . (2005). Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol 7: 758–765.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clemens MJ . (2001). Translational regulation in cell stress and apoptosis. Roles of the eIF4E binding proteins. J Cell Mol Med 5: 221–239.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clemens MJ . (2004). Targets and mechanisms for the regulation of translation in malignant transformation. Oncogene 23: 3180–3188.

    Article  CAS  PubMed  Google Scholar 

  • Constantinou C, Clemens MJ . (2005). Regulation of the phosphorylation and integrity of protein synthesis initiation factor eIF4GI and the translational repressor 4E-BP1 by p53. Oncogene 24: 4839–4850.

    Article  CAS  PubMed  Google Scholar 

  • Constantinou C, Clemens MJ . (2007). Regulation of translation factors eIF4GI and 4E-BP1 during recovery of protein synthesis from inhibition by p53. Cell Death Differ 14: 576–585.

    Article  CAS  PubMed  Google Scholar 

  • Cormier P, Pyronnet S, Morales J, Mulner-Lorillon O, Sonenberg N, Bellé R . (2001). eIF4E association with 4E-BP decreases rapidly following fertilization in sea urchin. Dev Biol 232: 275–283.

    Article  CAS  PubMed  Google Scholar 

  • De Benedetti A, Graff JR . (2004). eIF-4E expression and its role in malignancies and metastases. Oncogene 23: 3189–3199.

    Article  CAS  PubMed  Google Scholar 

  • Fang S, Weissman AM . (2004). A field guide to ubiquitylation. Cell Mol Life Sci 61: 1546–1561.

    Article  CAS  PubMed  Google Scholar 

  • Fawcett J, Hamel FG, Bennett RG, Vajo Z, Duckworth WC . (2001a). Insulin and analogue effects on protein degradation in different cell types. Dissociation between binding and activity. J Biol Chem 276: 11552–11558.

    Article  CAS  PubMed  Google Scholar 

  • Fawcett J, Hamel FG, Duckworth WC . (2001b). Characterization of the inhibition of protein degradation by insulin in L6 cells. Arch Biochem Biophys 385: 357–363.

    Article  CAS  PubMed  Google Scholar 

  • Fuchs SY, Fried VA, Ronai Z . (1998). Stress-activated kinases regulate protein stability. Oncogene 17: 1483–1490.

    Article  CAS  PubMed  Google Scholar 

  • Gingras AC, Raught B, Sonenberg N . (2004). mTOR signaling to translation. Curr Top Microbiol Immunol 279: 169–197.

    CAS  PubMed  Google Scholar 

  • Gingras AC, Svitkin Y, Belsham GJ, Pause A, Sonenberg N . (1996). Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus. Proc Natl Acad Sci USA 93: 5578–5583.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Graff JR, Zimmer SG . (2003). Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs. Clin Exp Metastasis 20: 265–273.

    Article  CAS  PubMed  Google Scholar 

  • Graham B, Gibson SB . (2005). The two faces of NF kappa B in cell survival responses. Cell Cycle 4: 1342–1345.

    Article  CAS  PubMed  Google Scholar 

  • Graves LM, Bornfeldt KE, Argast GM, Krebs EG, Kong X, Lin TA et al. (1995). cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc Natl Acad Sci USA 92: 7222–7226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harhaj EW, Sun SC . (1997). The serine/threonine phosphatase inhibitor calyculin A induces rapid degradation of I kappa B beta. Requirement of both the N- and C-terminal sequences. J Biol Chem 272: 5409–5412.

    Article  CAS  PubMed  Google Scholar 

  • Herbert TP, Tee AR, Proud CG . (2002). The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. J Biol Chem 277: 11591–11596.

    Article  CAS  PubMed  Google Scholar 

  • Hicke L . (2001). Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2: 195–201.

    Article  CAS  PubMed  Google Scholar 

  • Hoover DS, Wingett DG, Zhang J, Reeves R, Magnuson NS . (1997). Pim-1 protein expression is regulated by its 5′-untranslated region and translation initiation factor eIF-4E. Cell Growth Differ 8: 1371–1380.

    CAS  PubMed  Google Scholar 

  • Ishihara H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato Y et al. (1989). Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 159: 871–877.

    Article  CAS  PubMed  Google Scholar 

  • Jeffrey IW, Bushell M, Tilleray VJ, Morley S, Clemens MJ . (2002). Inhibition of protein synthesis in apoptosis: Differential requirements by the tumor necrosis factor α family and a DNA-damaging agent for caspases and the double-stranded RNA-dependent protein kinase. Cancer Res 62: 2272–2280.

    CAS  PubMed  Google Scholar 

  • Johnson P, Chung S, Benchimol S . (1993). Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin. Mol Cell Biol 13: 1456–1463.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Juo P, Kuo CJ, Yuan JY, Blenis J . (1998). Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr Biol 8: 1001–1008.

    Article  CAS  PubMed  Google Scholar 

  • Karin M, Ben-Neriah Y . (2000). Phosphorylation meets ubiquitination: The control of NF-kappa B activity. Annu Rev Immunol 18: 621–663.

    Article  CAS  PubMed  Google Scholar 

  • Kim TK, Maniatis T . (1996). Regulation of interferon-gamma-activated STAT1 by the ubiquitin-proteasome pathway. Science 273: 1717–1719.

    Article  CAS  PubMed  Google Scholar 

  • Krappmann D, Scheidereit C . (2005). A pervasive role of ubiquitin conjugation in activation and termination of I kappa B kinase pathways. EMBO R 6: 321–326.

    Article  CAS  Google Scholar 

  • Kumar KGS, Krolewski JJ, Fuchs SY . (2004). Phosphorylation and specific ubiquitin acceptor sites are required for ubiquitination and degradation of the IFNAR1 subunit of type I interferon receptor. J Biol Chem 279: 46614–46620.

    Article  CAS  PubMed  Google Scholar 

  • Kumar V, Sabatini D, Pandey P, Gingras AC, Majumder PK, Kumar M et al. (2000). Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase. J Biol Chem 275: 10779–10787.

    Article  CAS  PubMed  Google Scholar 

  • Le Bouffant R, Cormier P, Mulner-Lorillon O, Belle R . (2006). Hypoxia and DNA-damaging agent bleomycin both increase the cellular level of the protein 4E-BP. J Cell Biochem 99: 126–132.

    Article  CAS  PubMed  Google Scholar 

  • Li BG, Fang CH, Hasselgren P . (2000). Degradation of methoxysuccinyl-phe-leu-phe-7-amido-4-trifluoromethyl coumarin (FLF) in cultured myotubes and HepG2 cells is proteasome- and calpain/calcium-dependent. Int J Biochem Cell Biol 32: 677–686.

    Article  CAS  PubMed  Google Scholar 

  • Li S, Sonenberg N, Gingras AC, Peterson M, Avdulov S, Polunovsky VA et al. (2002). Translational control of cell fate: availability of phosphorylation sites on translational repressor 4E-BP1 governs its proapoptotic potency. Mol Cell Biol 22: 2853–2861.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin DI, Barbash O, Kumar KG, Weber JD, Harper JW, Klein-Szanto AJ et al. (2006a). Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex. Mol Cell 24: 355–366.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin KI, Baraban JM, Ratan RR . (1998). Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration. Cell Death Differ 5: 577–583.

    Article  CAS  PubMed  Google Scholar 

  • Lin SS, Bassik MC, Suh H, Nishino M, Arroyo JD, Hahn WC et al. (2006b). PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum. J Biol Chem 281: 23003–23012.

    Article  CAS  PubMed  Google Scholar 

  • Lin T-A, Kong X, Haystead TAJ, Pause A, Belsham G, Sonenberg N et al. (1994). PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science 266: 653–656.

    Article  CAS  PubMed  Google Scholar 

  • Lin T-A, Kong X, Saltiel AR, Blackshear PJ, Lawrence Jr JC . (1995). Control of PHAS-I by insulin in 3T3-L1 adipocytes. Synthesis, degradation, and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-independent pathway. J Biol Chem 270: 18531–18538.

    Article  CAS  PubMed  Google Scholar 

  • Mamane Y, Petroulakis E, LeBacquer O, Sonenberg N . (2006). mTOR, translation initiation and cancer. Oncogene 25: 6416–6422.

    Article  CAS  PubMed  Google Scholar 

  • Mamane Y, Petroulakis E, Rong LW, Yoshida K, Ler LW, Sonenberg N . (2004). eIF4E – from translation to transformation. Oncogene 23: 3172–3179.

    Article  CAS  PubMed  Google Scholar 

  • Mendez R, Myers Jr MG, White MF, Rhoads RE . (1996). Stimulation of protein synthesis, eukaryotic translation initiation factor 4Ephosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase. Mol Cell Biol 16: 2857–2864.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murata T, Shimotohno K . (2006). Ubiquitination and proteasome-dependent degradation of human eukaryotic translation initiation factor 4E. J Biol Chem 281: 20788–20800.

    Article  CAS  PubMed  Google Scholar 

  • Musti AM, Treier M, Bohmann D . (1997). Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science 275: 400–402.

    Article  CAS  PubMed  Google Scholar 

  • Nimmanapalli R, Bali P, O'Bryan E, Fuino L, Guo F, Wu J et al. (2003). Arsenic trioxide inhibits translation of mRNA of bcr-abl, resulting in attenuation of Bcr-Abl levels and apoptosis of human leukemia cells. Cancer Res 63: 7950–7958.

    CAS  PubMed  Google Scholar 

  • Nishizawa M, Furuno N, Okazaki K, Tanaka H, Ogawa Y, Sagata N . (1993). Degradation of Mos by the N-terminal proline (Pro2)-dependent ubiquitin pathway on fertilization of Xenopus eggs: possible significance of natural selection for Pro2 in Mos. EMBO J 12: 4021–4027.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Okazaki K, Sagata N . (1995). The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells. EMBO J 14: 5048–5059.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Orford K, Crockett C, Jensen JP, Weissman AM, Byers SW . (1997). Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin. J Biol Chem 272: 24735–24738.

    Article  CAS  PubMed  Google Scholar 

  • Othumpangat S, Kashon M, Joseph P . (2005a). Eukaryotic translation initiation factor 4E is a cellular target for toxicity and death due to exposure to cadmium chloride. J Biol Chem 280: 25162–25169.

    Article  CAS  PubMed  Google Scholar 

  • Othumpangat S, Kashon M, Joseph P . (2005b). Sodium arsenite-induced inhibition of eukaryotic translation initiation factor 4E (eIF4E) results in cytotoxicity and cell death. Mol Cell Biochem 279: 123–131.

    Article  CAS  PubMed  Google Scholar 

  • Paglin S, Lee NY, Nakar C, Fitzgerald M, Plotkin J, Deuel B et al. (2005). Rapamycin-sensitive pathway regulates mitochondrial membrane potential, autophagy, and survival in irradiated MCF-7 cells. Cancer Res 65: 11061–11070.

    Article  CAS  PubMed  Google Scholar 

  • Peterson RT, Desai BN, Hardwick JS, Schreiber SL . (1999). Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin-associated protein. Proc Natl Acad Sci USA 96: 4438–4442.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Proud CG . (2002). Regulation of mammalian translation factors by nutrients. Eur J Biochem 269: 5338–5349.

    Article  CAS  PubMed  Google Scholar 

  • Proud CG . (2005). The eukaryotic initiation factor 4E-binding proteins and apoptosis. Cell Death Differ 12: 541–546.

    Article  CAS  PubMed  Google Scholar 

  • Ren Q, Kari C, Quadros MR, Burd R, McCue P, Dicker AP et al. (2006). Malignant transformation of immortalized HaCaT keratinocytes through deregulated nuclear factor kappa B signaling. Cancer Res 66: 5209–5215.

    Article  CAS  PubMed  Google Scholar 

  • Rosenwald IB, Kaspar R, Rousseau D, Gehrke L, Leboulch P, Chen JJ et al. (1995). Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels. J Biol Chem 270: 21176–21180.

    Article  CAS  PubMed  Google Scholar 

  • Rousseau D, Kaspar R, Rosenwald I, Gehrke L, Sonenberg N . (1996). Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E. Proc Natl Acad Sci USA 93: 1065–1070.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Salaün P, Pyronnet S, Morales J, Mulner-Lorillon O, Bellé R, Sonenberg N et al. (2003). eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo. Dev Biol 255: 428–439.

    Article  PubMed  Google Scholar 

  • Scheidereit C . (2006). I kappa B kinase complexes: gateways to NF-kappa B activation and transcription. Oncogene 25: 6685–6705.

    Article  CAS  PubMed  Google Scholar 

  • Schneider R, Braunstein S, Xi Q, Formenti S . (2005). Ionizing radiation controls protein synthesis through a novel Akt-independent pathway involving regulation of mTOR and 4E-BP1 stability. Int J Radiat Oncol Biol Phys 63: S146.

    Article  Google Scholar 

  • Song Q, Lavin MF . (1993). Calyculin A, a potent inhibitor of phosphatases-1 and -2A, prevents apoptosis. Biochem Biophys Res Commun 190: 47–55.

    Article  CAS  PubMed  Google Scholar 

  • Strudwick S, Borden KLB . (2002). The emerging roles of translation factor eIF4E in the nucleus. Differentiation 70: 10–22.

    Article  CAS  PubMed  Google Scholar 

  • Tee AR, Blenis J . (2005). mTOR, translational control and human disease. Semin Cell Dev Biol 16: 29–37.

    Article  CAS  PubMed  Google Scholar 

  • Tee AR, Proud CG . (2000). DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling. Oncogene 19: 3021–3031.

    Article  CAS  PubMed  Google Scholar 

  • Tilleray V, Constantinou C, Clemens MJ . (2006). Regulation of protein synthesis by inducible wild-type p53 in human lung carcinoma cells. FEBS Lett 580: 1766–1770.

    Article  CAS  PubMed  Google Scholar 

  • Topisirovic I, Guzman ML, McConnell MJ, Licht JD, Culjkovic B, Neering SJ et al. (2003). Aberrant eukaryotic translation initiation factor 4E-dependent mRNA transport impedes hematopoietic differentiation and contributes to leukemogenesis. Mol Cell Biol 23: 8992–9002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Torres C, Francis MK, Lorenzini A, Tresini M, Cristofalo VJ . (2003). Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts. Exp Cell Res 290: 195–206.

    Article  CAS  PubMed  Google Scholar 

  • von der Haar T, Gross JD, Wagner G, McCarthy JEG . (2004). The mRNA cap-binding protein eIF4E in post-transcriptional gene expression. Nat Struct Biol 11: 503–511.

    Article  CAS  Google Scholar 

  • Walsh D, Mohr I . (2004). Phosphorylation of eIF4E by Mnk-1 enhances HSV-1 translation and replication in quiescent cells. Genes Dev 18: 660–672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walsh D, Perez C, Notary J, Mohr I . (2005). Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells. J Virol 79: 8057–8064.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wan X, Mendoza A, Khanna C, Helman LJ . (2005). Rapamycin inhibits ezrin-mediated metastatic behavior in a murine model of osteosarcoma. Cancer Res 65: 2406–2411.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Simon Morley (University of Sussex) for providing Jurkat cells and antibody to eIF4E. This work was supported by the Association for International Cancer Research and the Cancer Prevention Research Trust.

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  1. C Constantinou

    Present address: 2Current address: Yasoo Health Ltd, 1 Poseidon Street, PO Box 25193, 1307 Nicosia, Cyprus.,

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  1. Division of Basic Medical Sciences, Translational Control Group, Centre for Molecular and Metabolic Signalling, St George's, University of London, London, UK

    A Elia, C Constantinou & M J Clemens

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Elia, A., Constantinou, C. & Clemens, M. Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1. Oncogene 27, 811–822 (2008). https://doi.org/10.1038/sj.onc.1210678

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