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Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bβ subunit into a replacement for yeast eEF1Bα

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Abstract

Elongation factor subunit eEF1Bβ (formerly EF-1β in plants and EF-1δ in animals) was identified and cloned in a screen for proteins from pea that interact with a cyclin-dependent kinase (CDK). CDKs are enzymes that regulate progression through meiotic and mitotic cell cycles in eukaryotes. eEF1Bβ and the related protein eEF1Bα (formerly EF-1β' in plants and EF-1β in animals and fungi) can catalyze GTP/GDP exchange on the G-protein eEF1A (formerly EF-1α in plants, animals and fungi) during the elongation phase of protein synthesis in eukaryotes. Recombinant Cdc2 and its native homologues from pea extracts associated both in vitro and in vivo with eEF1Bβ. A Cdc2-cyclin B complex phosphorylated recombinant plant eEF1Bβs, but not eEF1Bα. These interactions between CDK and eEF1Bβ prompted investigations into the in vivo consequences of this relationship. Expression of cDNAs encoding rice or pea eEF1Bβ subunits failed to complement a Saccharomyces cerevisiae mutant deleted for the eEF1Bα gene, as was previously observed for the human eEF1Bβ. However, replacement of Thr91, the sole consensus CDK phosphorylation site in pea eEF1Bβ, with alanine allowed the pea protein to substitute for eEF1Bα function in vivo. In addition, this rescued strain was severely cold sensitive, and more sensitive to translational inhibitors than wild-type yeast. Taken together, these results suggest a physiological connection between the cyclin-dependent class of kinases and a translational elongation factor in mitotic cells, and provide the first in vivo evidence that an altered form of eEF1Bβ can serve as the guanine nucleotide exchange factor for eEF1A.

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References

  • Ach R, Durfee T, Miller A, Taranto L, Hanley-Bowdoin L, Zambryski P, Gruissem W (1997) RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein. Mol Cell Biol 17:5077–5086

    CAS  PubMed  Google Scholar 

  • Atherton-Fessler S, Parker L, Geahlen L, Piwnica-Worms H (1993) Mechanisms of p34cdc2 regulation. Mol Cell Biol 13:1675–1685

    CAS  PubMed  Google Scholar 

  • Bec G, Kerjan P, Waller J (1994) Reconstitution in vitro of the valyl-tRNA synthetase-elongation factor (EF) 1βγδ complex. J Biol Chem 269:2086–2092

    CAS  PubMed  Google Scholar 

  • Bellé R, Minella O, Cormier P, Morales J, Poulhe R, Mulner-Lorillon O (1995) Phosphorylation of elongation factor-1 (EF-1) by cdc2 kinase. Prog Cell Cyc Res 1:265–270

    Google Scholar 

  • Blanar M, Rutter W (1992) Interaction cloning: Identification of a helix-loop-helix zipper protein that interacts with c-Fos. Science 256:1014–1018

    CAS  PubMed  Google Scholar 

  • Boeke J, Trueheart J, Natsoulis G, Fink G (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154:164–175

    CAS  PubMed  Google Scholar 

  • Braun D, Stone J, Walker J (1997) Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases-implications for transmembrane signaling in plants. Plant J 12:83–95

    CAS  PubMed  Google Scholar 

  • Brizuela L, Draetta G, Beach D (1987) p13Suc1 acts in the fission yeast cell division cycle as a component of the cdc2 protein kinase. EMBO J 6:3507–3514

    PubMed  Google Scholar 

  • Browning K (1996) The plant translational apparatus. Plant Mol Biol 32:107–144

    CAS  PubMed  Google Scholar 

  • Carr-Schmid A, Valente L, Loik V, Williams T, Starita L, Kinzy T (1999) Mutations in elongation factor 1β, a guanine nucleotide exchange factor, enhance translational fidelity. Mol Cell Biol 19:5257–5266

    CAS  PubMed  Google Scholar 

  • Chang Y, Traugh J (1997) Phosphorylation of elongation factor 1 and ribosomal protein S6 by multipotential S6 kinase and insulin stimulation of translation elongation. J Biol Chem 272:28252–28257

    Article  CAS  PubMed  Google Scholar 

  • Creanor J, Mitchison J (1982) Patterns of protein synthesis during the cell cycle of the fission yeast Schizosaccharomyces pombe. J Cell Sci 58:263–285

    CAS  PubMed  Google Scholar 

  • De Nadal E, Fadden R, Ruiz A, Haystead T, Ariño J (2001) A role for the Ppz Ser/Thr phosphatases in the regulation of translation elongation factor 1Bα. J Biol Chem 276:14829–14834

    Article  PubMed  Google Scholar 

  • De Veylder L, Beeckman T, Beemster G, Krols L, Terras F, Landrieu I, Van Der Schueren E, Maes S, Naudts M, Inzé D (2001) Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13:1653–1667

    PubMed  Google Scholar 

  • De Veylder L, Beeckman T, Beemster G, de Almeida Engler J, Ormenese S, Maes S, Naudts M, Van Der Schueren E, Jacqmard A, Engler G, Inzé D (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor. EMBO J 21:1360–1368

    PubMed  Google Scholar 

  • Fankhauser C, Marks J, Reymond A, Simanis V (1993) The S. pombe cdc16 gene is required both for maintenance of p34(cdc2) kinase activity and regulation of septum formation—a link between mitosis and cytokinesis. EMBO J 12:2697–2704

    CAS  PubMed  Google Scholar 

  • Gietz R, Sugino A (1988) New yeast- Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534

    CAS  PubMed  Google Scholar 

  • Guerrucci M, Monnier A, Delalande C, Bellé R (1999) The elongation factor-1δ (EF-1δ) originates from gene duplication of an EF-1β ancestor and fusion with a protein-binding domain. Gene 233:83–87

    Article  CAS  PubMed  Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    CAS  PubMed  Google Scholar 

  • Izumi T, Maller J (1991) Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions. Mol Cell Biol 11:3860–3867

    CAS  PubMed  Google Scholar 

  • Janssen G, Möller W (1988) Elongation factor 1 βγ from Artemia. Purification and properties of its subunits. Eur J Biochem 171:119–129

    CAS  PubMed  Google Scholar 

  • Janssen G, Maessen G, Amons R, Möller W (1988) Phosphorylation of elongation factor 1β by an endogenous kinase affects its catalytic nucleotide exchange activity. J Biol Chem 263:11063–11066

    CAS  PubMed  Google Scholar 

  • Janssen G, van Damme H, Kriek J, Amons R, Möller W (1994) The subunit structure of elongation factor 1 from Artemia: why two α-chains in this complex? J Biol Chem 269:31410–31417

    Google Scholar 

  • Kanki J, Newport J (1991) The cell cycle dependence of protein synthesis during Xenopus laevis development. Dev Biol 146:198–213

    CAS  PubMed  Google Scholar 

  • King R, Deshaies R, Peters J, Kirschner M (1996) How proteolysis drives the cell cycle. Science 274:1652–1658

    CAS  PubMed  Google Scholar 

  • Kinzy T, Woolford J Jr (1995) Increased expression of Saccharomyces cerevisiae translation elongation factor 1α bypasses the lethality of a TEF5 null allele encoding elongation factor 1β. Genetics 141:481–489

    CAS  PubMed  Google Scholar 

  • Kinzy T, Ripmaster T, Woolford J Jr (1994) Multiple genes encode the translational elongation factor 1-γ in Saccharomyces cerevisiae. Nucleic Acids Res 22:2703–2707

    CAS  PubMed  Google Scholar 

  • Koepp D, Harper J, Elledge S (1999) How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97:431–434

    CAS  PubMed  Google Scholar 

  • Kosugi S, Ohashi Y (2002) E2F sites that can interact with E2F proteins cloned from rice are required for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters. Plant J 29:45–59

    Article  CAS  PubMed  Google Scholar 

  • Lacy M, Voss E (1986) A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells. J Immunol Methods 87:169–177

    Article  CAS  PubMed  Google Scholar 

  • Mariconti L, Pellegrini B, Cantoni R, Stevens R, Bergounioux C, Cella R, Albani D (2002) The E2F family of transcription factors from Arabiodpsis thaliana. Novel and conserved components of the retinoblastoma/E2F pathway in plants. J Biol Chem 277:9911–9919

    CAS  PubMed  Google Scholar 

  • Merrick W (1979) Assays for eukaryotic protein synthesis. Methods Enzymol 60:108–123

    CAS  PubMed  Google Scholar 

  • Merrick W, Nyborg J (2000) The Protein biosynthesis elongation cycle. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  • Minella O, Cormier P, Morales J, Poulhe R, Bellé R, Mulner-Lorillon O (1994) CDC2 kinase sets a memory phosphorylation signal on elongation factor EF-1δ during meiotic cell division, which perdures in early development. Cell Mol Biol 40:521–525

    CAS  Google Scholar 

  • Minella O, Mulner-Lorillon O, Bec G, Cormier P, Bellé R (1998) Multiple phosphorylation sites and quaternary organization of guanine-nucleotide exchange complex of elongation factor-1 (EF-1βγδ/ValRS) control the various functions of EF-1α. Biosci Rep 18:119–127

    Article  CAS  PubMed  Google Scholar 

  • Mironov V, De Veylder L, Van Montagu M, Inzé D (1999) Cyclin-dependent kinases and cell division in plants-the nexus. Plant Cell 11:509–521

    CAS  PubMed  Google Scholar 

  • Monnier A, Bellé R, Morales J, Cormier P, Boulben S, Mulner-Lorillon O (2001) Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation. Nucleic Acids Res 29:1453–1457

    CAS  PubMed  Google Scholar 

  • Moreno S, Nurse P (1990) Substrates for p34 cdc2: In vivo veritas? Cell 61:549–551

  • Mulner-Lorillon O, Poulhe R, Cormier P, Capony J, Cavadore J (1989) Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34 cdc2 substrate. FEBS Lett 251:219–224

    Article  CAS  PubMed  Google Scholar 

  • Mulner-Lorillon O, Cormier P, Cavadore J, Morales J, Poulhe R, Bellé R (1992) Phosphorylation of Xenopus elongation factor-1γ by cdc2 protein kinase: Identification of the phosphorylation site. Exp Cell Res 202:549–551

    CAS  PubMed  Google Scholar 

  • Mulner-Lorillon O, Minella O, Cormier P, Jean-Paul C, Cavadore J, Morales J, Poulhe R, Bellé R (1994) Elongation factor EF-1δ, a new target for maturation-promoting factor in Xenopus oocytes. J Biol Chem 269:20201–20207

    CAS  PubMed  Google Scholar 

  • Pedersen L, Anderson G, Knudsen C, Kinzy T, Nyborg J (2001) Crystallization of the yeast elongation factor complex eEF1A-eEF1Bα. Acta Cryst D57:159–161

    CAS  Google Scholar 

  • Peters H, Chang Y, Traugh J (1995) Phosphorylation of elongation factor 1 (EF-1) by protein kinase C stimulates GDP/GTP-exchange activity. Eur J Biochem 234:550–556

    CAS  PubMed  Google Scholar 

  • Pitluk Z, McDonough M, Sangan P, Gonda D (1995) Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis. Mol Cell Biol 15:1210–1219

    CAS  PubMed  Google Scholar 

  • Pomerening J, Sontag E, Ferrell J Jr (2003) Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2. Nat Cell Biol 5:346–351

    Article  CAS  PubMed  Google Scholar 

  • Ramirez-Parra E, Xie Q, Boniotti M, Gutierrez C (1999) The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G(1)/S regulators. Nucleic Acids Res. 27:3527–3533

    Google Scholar 

  • Redpath N, Price N, Severinov K, Proud C (1993) Regulation of elongation factor-2 by multisite phosphorylation. Eur J Biochem 213:689–699

    CAS  PubMed  Google Scholar 

  • Rosenthal E, Brandhorst B, Ruderman J (1982) Translationally mediated changes in patterns of protein synthesis during maturation of starfish oocytes. Dev Biol 91:215–220

    CAS  PubMed  Google Scholar 

  • Ryazanov A, Spirin A (1993) Phosphorylation of elongation factor 2: a mechanism to shut off protein synthesis for reprogramming gene expression. In: Ilan J (ed) Plenum Press, New York, pp 433–455

  • Sanders J, Raggiaschi R, Morales J, Moller W (1993) The human leucine zipper-containing guanine-nucleotide exchange protein elongation factor-1 delta. Biochim Biophys Acta 1174:87–90

    Article  CAS  PubMed  Google Scholar 

  • Sekine M, Ito, M, Uemukai K, Maeda Y, Nakagami H, Shinmyo A (1999) Isolation and characterization of the E2F-like gene in plants. FEBS Lett 460:117–122

    CAS  PubMed  Google Scholar 

  • Sherman F, Fink G, Hicks J (1986) Methods in yeast genetics: A laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sheu G, Traugh J (1997) Recombinant subunits of mammalian elongation factor 1 (EF-1) expressed in E. coli; subunit interactions, elongation activity and phosphorylation by protein kinase CKII. J Biol Chem 272:33290–33297

    Article  CAS  PubMed  Google Scholar 

  • Sheu G, Traugh J (1999) A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII. Mol Cell Biochem 191:181–186

    Article  CAS  PubMed  Google Scholar 

  • Terui Y, Tsutsumi K, Kidou S, Sawazaki T, Kuroiwa Y, Yamaki M, Ejiri S (1998) A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2. Biochim Biophys Acta 1442:369–372

    Article  CAS  PubMed  Google Scholar 

  • Venema R, Peters H, Traugh J (1991) Phosphorylation of valyl-tRNA synthetase and elongation factor 1 in response to phorbol esters is associated with stimulation of both activities. J Biol Chem 266:11993–11998

    CAS  PubMed  Google Scholar 

  • Wang H, Qi Q, Schorr P, Cutler A, Crosby W, Fowke L (1998) ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid. Plant J 15:501–510

    PubMed  Google Scholar 

  • Yamaguchi M, Umedea M, Uchimiya H (1998) A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II. Plant J 16:613–619

    Article  CAS  PubMed  Google Scholar 

  • Yang X, Hubbard J, Carlson M (1992) A protein kinase substrate identified by the two-hybrid system. Science 257:680–682

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Philip Wakeley for his conception of and assistance with the library screening, past and present members of the Jacobs lab for helpful discussion, and Russell Kallis and Gracia Zabala for their contributions to this paper. Special thanks to Jim Ferrell and members of his laboratory in the Stanford University School of Medicine-Department of Molecular Pharmacology for their critical review of the manuscript. This work was supported by grants to T.W.J. from the U.S. Department of Energy, U.S. Department of Agriculture, and the Research Board of the University of Illinois. J.R.P. was supported in part by a Clark Research Support Grant and a UIUC Graduate College Dissertation Research Grant. T.G.K. is supported by NSF MCB-9983565, and L.V. by NIH F31 GM-20445. This work was performed in compliance with the current laws governing genetic experimentation in the United States of America.

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Authors and Affiliations

  1. Department of Molecular Pharmacology, Stanford University School of Medicine, 269 West Campus Drive, CCSR 3160, Stanford, CA 94305-5174, USA

    J. R. Pomerening

  2. Department of Molecular Genetics, Microbiology, and Immunology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

    L. Valente & T. G. Kinzy

  3. The Cancer Institute of New Jersey, 195 Little Albany St., New Brunswick, NJ 08901, USA

    T. G. Kinzy

  4. Department of Plant Biology , University of Illinois at Urbana-Champaign, 1201 West Gregory Drive, 191 Edward R. Madigan Laboratory, Urbana, IL 61801-3838, USA

    T. W. Jacobs

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  1. J. R. Pomerening
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  2. L. Valente
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Correspondence to J. R. Pomerening.

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Communicated by C. P. Hollenberg

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Pomerening, J.R., Valente, L., Kinzy, T.G. et al. Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bβ subunit into a replacement for yeast eEF1Bα. Mol Gen Genomics 269, 776–788 (2003). https://doi.org/10.1007/s00438-003-0888-1

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