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Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

Gangwani L, Mikrut M, Galcheva-Gargova Z, Davis RJ - J. Cell Biol. (1998)

Bottom Line: The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation.Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth.Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

ABSTRACT
The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

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Coimmunoprecipitation analysis demonstrates that  eEF-1α and ZPR1 interact in vivo. (A) COS-7 cells were mock-transfected (CONTROL) or transfected with a vector expressing  epitope-tagged ZPR1. Flag-ZPR1 was immunoprecipitated with  the M2 monoclonal antibody. eEF-1α in the immunoprecipitates  (IP) was detected by protein immunoblot analysis with a monoclonal antibody to eEF-1α. (B) Coimmunoprecipitation of ZPR1  with eEF-1α from cells treated with serum or EGF. A431 epidermoid carcinoma cells were incubated in serum-free medium for  24 h. The cells were untreated or treated with 5% calf serum, 100  nM EGF, or 100 nM PMA for 15 min. Extracts were prepared  from the cells and eEF-1α was isolated by immunoprecipitation.  Specificity of the immunoprecipitation was examined using mock  immunoprecipitates prepared using protein A–Sepharose beads  without antibody to eEF-1α (CONTROL). ZPR1 in the immunoprecipitates was detected by protein immunoblot analysis using a  rabbit polyclonal antibody. (C) EGF causes the rapid formation  of ZPR1/eEF-1α complexes that are detected by coimmunoprecipitation analysis. A431 cells were serum starved for 24 h. The  cells were incubated with 100 nM EGF for various times, extracts  were prepared, and then eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined  using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). The presence of ZPR1 in the immunoprecipitates was examined by protein immunoblot analysis with a rabbit polyclonal antibody.
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Figure 4: Coimmunoprecipitation analysis demonstrates that eEF-1α and ZPR1 interact in vivo. (A) COS-7 cells were mock-transfected (CONTROL) or transfected with a vector expressing epitope-tagged ZPR1. Flag-ZPR1 was immunoprecipitated with the M2 monoclonal antibody. eEF-1α in the immunoprecipitates (IP) was detected by protein immunoblot analysis with a monoclonal antibody to eEF-1α. (B) Coimmunoprecipitation of ZPR1 with eEF-1α from cells treated with serum or EGF. A431 epidermoid carcinoma cells were incubated in serum-free medium for 24 h. The cells were untreated or treated with 5% calf serum, 100 nM EGF, or 100 nM PMA for 15 min. Extracts were prepared from the cells and eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). ZPR1 in the immunoprecipitates was detected by protein immunoblot analysis using a rabbit polyclonal antibody. (C) EGF causes the rapid formation of ZPR1/eEF-1α complexes that are detected by coimmunoprecipitation analysis. A431 cells were serum starved for 24 h. The cells were incubated with 100 nM EGF for various times, extracts were prepared, and then eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). The presence of ZPR1 in the immunoprecipitates was examined by protein immunoblot analysis with a rabbit polyclonal antibody.

Mentions: To test whether eEF-1α and ZPR1 may interact in vivo, we performed coimmunoprecipitation analysis. Epitope-tagged mZPR1 was immunoprecipitated from serum-treated COS-7 cells and the presence of eEF-1α in the immunoprecipitates was examined by Western blot analysis. Fig. 4 A shows that eEF-1α was coimmunoprecipitated with ZPR1. Control experiments using cells that did not express epitope-tagged mZPR1 demonstrated no coimmunoprecipitation of eEF-1α. These data indicate that ZPR1 and eEF-1α may interact in vivo.


Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

Gangwani L, Mikrut M, Galcheva-Gargova Z, Davis RJ - J. Cell Biol. (1998)

Coimmunoprecipitation analysis demonstrates that  eEF-1α and ZPR1 interact in vivo. (A) COS-7 cells were mock-transfected (CONTROL) or transfected with a vector expressing  epitope-tagged ZPR1. Flag-ZPR1 was immunoprecipitated with  the M2 monoclonal antibody. eEF-1α in the immunoprecipitates  (IP) was detected by protein immunoblot analysis with a monoclonal antibody to eEF-1α. (B) Coimmunoprecipitation of ZPR1  with eEF-1α from cells treated with serum or EGF. A431 epidermoid carcinoma cells were incubated in serum-free medium for  24 h. The cells were untreated or treated with 5% calf serum, 100  nM EGF, or 100 nM PMA for 15 min. Extracts were prepared  from the cells and eEF-1α was isolated by immunoprecipitation.  Specificity of the immunoprecipitation was examined using mock  immunoprecipitates prepared using protein A–Sepharose beads  without antibody to eEF-1α (CONTROL). ZPR1 in the immunoprecipitates was detected by protein immunoblot analysis using a  rabbit polyclonal antibody. (C) EGF causes the rapid formation  of ZPR1/eEF-1α complexes that are detected by coimmunoprecipitation analysis. A431 cells were serum starved for 24 h. The  cells were incubated with 100 nM EGF for various times, extracts  were prepared, and then eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined  using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). The presence of ZPR1 in the immunoprecipitates was examined by protein immunoblot analysis with a rabbit polyclonal antibody.
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Figure 4: Coimmunoprecipitation analysis demonstrates that eEF-1α and ZPR1 interact in vivo. (A) COS-7 cells were mock-transfected (CONTROL) or transfected with a vector expressing epitope-tagged ZPR1. Flag-ZPR1 was immunoprecipitated with the M2 monoclonal antibody. eEF-1α in the immunoprecipitates (IP) was detected by protein immunoblot analysis with a monoclonal antibody to eEF-1α. (B) Coimmunoprecipitation of ZPR1 with eEF-1α from cells treated with serum or EGF. A431 epidermoid carcinoma cells were incubated in serum-free medium for 24 h. The cells were untreated or treated with 5% calf serum, 100 nM EGF, or 100 nM PMA for 15 min. Extracts were prepared from the cells and eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). ZPR1 in the immunoprecipitates was detected by protein immunoblot analysis using a rabbit polyclonal antibody. (C) EGF causes the rapid formation of ZPR1/eEF-1α complexes that are detected by coimmunoprecipitation analysis. A431 cells were serum starved for 24 h. The cells were incubated with 100 nM EGF for various times, extracts were prepared, and then eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). The presence of ZPR1 in the immunoprecipitates was examined by protein immunoblot analysis with a rabbit polyclonal antibody.
Mentions: To test whether eEF-1α and ZPR1 may interact in vivo, we performed coimmunoprecipitation analysis. Epitope-tagged mZPR1 was immunoprecipitated from serum-treated COS-7 cells and the presence of eEF-1α in the immunoprecipitates was examined by Western blot analysis. Fig. 4 A shows that eEF-1α was coimmunoprecipitated with ZPR1. Control experiments using cells that did not express epitope-tagged mZPR1 demonstrated no coimmunoprecipitation of eEF-1α. These data indicate that ZPR1 and eEF-1α may interact in vivo.

Bottom Line: The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation.Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth.Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

ABSTRACT
The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

Show MeSH
Related in: MedlinePlus