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

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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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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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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  • DOI: https://doi.org/10.1007/s00438-003-0888-1

Keywords

  • Cyclin-dependent kinase (CDK)
  • Interaction
  • Elongation factor
  • Plant
  • Yeast