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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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ZPR1 binds to eEF-1α in vitro. (A) A431 cells  were labeled with [35S]methionine. Soluble extracts  were prepared and incubated  with immobilized ZPR1  (GST–ZPR1) proteins from  three species: S. cerevisiae, S.  pombe, and mouse. Bound  proteins were examined by  SDS-PAGE and autoradiography. Arrows, binding of the  180-kD EGF receptor to  mouse ZPR1 and the prominent 50-kD protein that binds  to mouse and yeast ZPR1  proteins. (B) Deletion analysis of mZPR1 sequences required for interaction with  eEF-1α. Immobilized GST  and GST–mZPR1 fusion proteins were incubated with cell  lysates. Bound eEF-1α was  detected by protein immunoblot analysis using a monoclonal antibody to eEF-1α. The binding of eEF-1α to full-length (lane 8) and  truncated (lanes 1–7) mZPR1 proteins is presented. The mZPR1 amino acid residues in the truncated constructs are: 1–99 (1); 94–268  (2); 263–306 (3); 302–469 (4); 1–268 (5); 263–469 (6); 1–306 (7); and 1–469 (8). (C) Recombinant eEF-1α binds to mZPR1 in vitro. Immobilized GST and GST–eEF-1α were incubated with cell lysates. Bound mZPR1 was detected by protein immunoblot analysis using a  rabbit polyclonal antibody to mZPR1. The multiple bands are characteristic of the electrophoretic mobility of mZPR1 (Galcheva-Gargova et al., 1996). (D) Binding of recombinant eEF-1α and cZPR1. Immobilized GST and GST–eEF-1α were incubated with purified  CBP–cZPR1. Bound cZPR1 was detected by protein immunoblot analysis.
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Figure 3: ZPR1 binds to eEF-1α in vitro. (A) A431 cells were labeled with [35S]methionine. Soluble extracts were prepared and incubated with immobilized ZPR1 (GST–ZPR1) proteins from three species: S. cerevisiae, S. pombe, and mouse. Bound proteins were examined by SDS-PAGE and autoradiography. Arrows, binding of the 180-kD EGF receptor to mouse ZPR1 and the prominent 50-kD protein that binds to mouse and yeast ZPR1 proteins. (B) Deletion analysis of mZPR1 sequences required for interaction with eEF-1α. Immobilized GST and GST–mZPR1 fusion proteins were incubated with cell lysates. Bound eEF-1α was detected by protein immunoblot analysis using a monoclonal antibody to eEF-1α. The binding of eEF-1α to full-length (lane 8) and truncated (lanes 1–7) mZPR1 proteins is presented. The mZPR1 amino acid residues in the truncated constructs are: 1–99 (1); 94–268 (2); 263–306 (3); 302–469 (4); 1–268 (5); 263–469 (6); 1–306 (7); and 1–469 (8). (C) Recombinant eEF-1α binds to mZPR1 in vitro. Immobilized GST and GST–eEF-1α were incubated with cell lysates. Bound mZPR1 was detected by protein immunoblot analysis using a rabbit polyclonal antibody to mZPR1. The multiple bands are characteristic of the electrophoretic mobility of mZPR1 (Galcheva-Gargova et al., 1996). (D) Binding of recombinant eEF-1α and cZPR1. Immobilized GST and GST–eEF-1α were incubated with purified CBP–cZPR1. Bound cZPR1 was detected by protein immunoblot analysis.

Mentions: To study the interaction of ZPR1 with other proteins, we performed in vitro binding assays using [35S]methionine human A431 epidermoid carcinoma cell extracts and immobilized GST–ZPR1 fusion proteins. As expected, the mouse ZPR1 protein bound to the 180-kD EGF receptor (Fig. 3 A). In addition, several other [35S]methionine proteins were observed to bind ZPR1. In particular, a 50-kD protein was prominently detected in binding assays using mouse, S. cerevisiae and S. pombe ZPR1 proteins (Fig. 3 A). Similar results were obtained in binding assays using [35S]methionine extracts prepared from COS-7, CHO, and S. cerevisiae (strain L40) cells (data not shown). These data indicate that the yeast and mammalian ZPR1 proteins may have similar biochemical properties, including interaction with a 50-kD protein.


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

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

ZPR1 binds to eEF-1α in vitro. (A) A431 cells  were labeled with [35S]methionine. Soluble extracts  were prepared and incubated  with immobilized ZPR1  (GST–ZPR1) proteins from  three species: S. cerevisiae, S.  pombe, and mouse. Bound  proteins were examined by  SDS-PAGE and autoradiography. Arrows, binding of the  180-kD EGF receptor to  mouse ZPR1 and the prominent 50-kD protein that binds  to mouse and yeast ZPR1  proteins. (B) Deletion analysis of mZPR1 sequences required for interaction with  eEF-1α. Immobilized GST  and GST–mZPR1 fusion proteins were incubated with cell  lysates. Bound eEF-1α was  detected by protein immunoblot analysis using a monoclonal antibody to eEF-1α. The binding of eEF-1α to full-length (lane 8) and  truncated (lanes 1–7) mZPR1 proteins is presented. The mZPR1 amino acid residues in the truncated constructs are: 1–99 (1); 94–268  (2); 263–306 (3); 302–469 (4); 1–268 (5); 263–469 (6); 1–306 (7); and 1–469 (8). (C) Recombinant eEF-1α binds to mZPR1 in vitro. Immobilized GST and GST–eEF-1α were incubated with cell lysates. Bound mZPR1 was detected by protein immunoblot analysis using a  rabbit polyclonal antibody to mZPR1. The multiple bands are characteristic of the electrophoretic mobility of mZPR1 (Galcheva-Gargova et al., 1996). (D) Binding of recombinant eEF-1α and cZPR1. Immobilized GST and GST–eEF-1α were incubated with purified  CBP–cZPR1. Bound cZPR1 was detected by protein immunoblot analysis.
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Figure 3: ZPR1 binds to eEF-1α in vitro. (A) A431 cells were labeled with [35S]methionine. Soluble extracts were prepared and incubated with immobilized ZPR1 (GST–ZPR1) proteins from three species: S. cerevisiae, S. pombe, and mouse. Bound proteins were examined by SDS-PAGE and autoradiography. Arrows, binding of the 180-kD EGF receptor to mouse ZPR1 and the prominent 50-kD protein that binds to mouse and yeast ZPR1 proteins. (B) Deletion analysis of mZPR1 sequences required for interaction with eEF-1α. Immobilized GST and GST–mZPR1 fusion proteins were incubated with cell lysates. Bound eEF-1α was detected by protein immunoblot analysis using a monoclonal antibody to eEF-1α. The binding of eEF-1α to full-length (lane 8) and truncated (lanes 1–7) mZPR1 proteins is presented. The mZPR1 amino acid residues in the truncated constructs are: 1–99 (1); 94–268 (2); 263–306 (3); 302–469 (4); 1–268 (5); 263–469 (6); 1–306 (7); and 1–469 (8). (C) Recombinant eEF-1α binds to mZPR1 in vitro. Immobilized GST and GST–eEF-1α were incubated with cell lysates. Bound mZPR1 was detected by protein immunoblot analysis using a rabbit polyclonal antibody to mZPR1. The multiple bands are characteristic of the electrophoretic mobility of mZPR1 (Galcheva-Gargova et al., 1996). (D) Binding of recombinant eEF-1α and cZPR1. Immobilized GST and GST–eEF-1α were incubated with purified CBP–cZPR1. Bound cZPR1 was detected by protein immunoblot analysis.
Mentions: To study the interaction of ZPR1 with other proteins, we performed in vitro binding assays using [35S]methionine human A431 epidermoid carcinoma cell extracts and immobilized GST–ZPR1 fusion proteins. As expected, the mouse ZPR1 protein bound to the 180-kD EGF receptor (Fig. 3 A). In addition, several other [35S]methionine proteins were observed to bind ZPR1. In particular, a 50-kD protein was prominently detected in binding assays using mouse, S. cerevisiae and S. pombe ZPR1 proteins (Fig. 3 A). Similar results were obtained in binding assays using [35S]methionine extracts prepared from COS-7, CHO, and S. cerevisiae (strain L40) cells (data not shown). These data indicate that the yeast and mammalian ZPR1 proteins may have similar biochemical properties, including interaction with a 50-kD protein.

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