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α4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding protein

Authors

  • William J. McDonald,

    1. Faculty of Medicine, Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
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  • Shirley M. Sangster,

    1. Faculty of Medicine, Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
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  • Lori D. Moffat,

    1. Faculty of Medicine, Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
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  • Michelle J. Henderson,

    1. Children's Cancer Institute Australia for Medical Research, Sydney, New South Wales, Australia
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  • Catherine K.L. Too

    Corresponding author
    1. Faculty of Medicine, Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
    2. Faculty of Medicine, Department of Obstetrics & Gynaecology, Dalhousie University, Halifax, Nova Scotia, Canada
    • Faculty of Medicine, Department of Biochemistry & Molecular Biology, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5.
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Abstract

Mammalian α4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. α4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using α4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of α4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with α4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between α4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. α4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD–α4 immunocomplex. Furthermore, deletion mutants of α4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of α4, independent of the α4-PP2Ac binding site. Therefore, in addition to PP2Ac, α4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123–1129, 2010. Published 2010 Wiley-Liss, Inc.

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