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Dual action of epidermal growth factor: extracellular signal-stimulated nuclear-cytoplasmic export and coordinated translation of selected messenger RNA.

Tsai NP, Lin YL, Tsui YC, Wei LN - J. Cell Biol. (2010)

Bottom Line: The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator.Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA.In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455.

ABSTRACT
We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear-cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator. Using the kappa-opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2-containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.

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EGF induces HuR-dependent nuclear export of Grb7. (A) Immunohistochemistry of rat DRG neurons (top) and P19 cells (bottom) transfected with control or HuR siRNA. Colocalization of Grb7 and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (B) Western blot analysis of nuclear and cytoplasmic fractions from P19 cells transfected as indicated in the presence or absence of EGF. (C) Mammalian two-hybrid assay in P19 cells transfected with Gal4-HuR and serial VP16-Grb7 deletion constructs. N-terminal deletions are named according to the number of deleted amino acid residues. (D) FISH detecting endogenous KOR mRNA in DRG neurons (top) and P19 cells (bottom) transfected with WT CFP-Grb7 or Δ9 CFP-Grb7. Colocalization of KOR mRNA and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (A and D) White dotted lines outline the cytoplasm of the individual cells. (A, C, and D) Error bars represent SDs. (E) Western blots of P19 cells transfected as indicated, in the presence or absence of EGF. Bars, 25 µm.
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fig2: EGF induces HuR-dependent nuclear export of Grb7. (A) Immunohistochemistry of rat DRG neurons (top) and P19 cells (bottom) transfected with control or HuR siRNA. Colocalization of Grb7 and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (B) Western blot analysis of nuclear and cytoplasmic fractions from P19 cells transfected as indicated in the presence or absence of EGF. (C) Mammalian two-hybrid assay in P19 cells transfected with Gal4-HuR and serial VP16-Grb7 deletion constructs. N-terminal deletions are named according to the number of deleted amino acid residues. (D) FISH detecting endogenous KOR mRNA in DRG neurons (top) and P19 cells (bottom) transfected with WT CFP-Grb7 or Δ9 CFP-Grb7. Colocalization of KOR mRNA and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (A and D) White dotted lines outline the cytoplasm of the individual cells. (A, C, and D) Error bars represent SDs. (E) Western blots of P19 cells transfected as indicated, in the presence or absence of EGF. Bars, 25 µm.

Mentions: Grb7 does not mobilize RNA directly. However, Grb7 interacts with HuR (Tsai et al., 2008), a ubiquitously expressed RNA-binding protein which can be associated with export complexes (Brennan et al., 2000; Rebane et al., 2004) to regulate the nuclear–cytoplasmic shuttling of mRNA (Doller et al., 2008). We found that HuR-specific siRNA reduced Grb7 export (Fig. 2 A) and blocked EGF-stimulated cytoplasmic accumulation of Grb7 (Fig. 2 B), which was rescued by expressing siRNA off-target HuR (see Materials and methods). The HuR-interacting domain of Grb7 was previously mapped to the N-terminal segment (Tsai et al., 2008). Because the RNA-binding domain of Grb7 is also located in its N terminus (Tsai et al., 2007), a more detailed mapping was performed to determine the minimal HuR-interacting motif (Fig. 2 C). A dominant-negative mutant of Grb7 was made by deleting this HuR-interacting motif, called Δ9. The Δ9 mutant was detected in HuR interaction yet retained the KOR mRNA–binding ability (Fig. S2). The Δ9 and wild-type (WT) Grb7s were each fused to CFP and tested in FISH experiments. It appeared that WT CFP-Grb7 could be exported together with KOR mRNA, whereas Δ9 CFP-Grb7 remained trapped in the nucleus along with KOR mRNA in both DRG neurons and P19 cells (Fig. 2 D). The amount of KOR protein was not elevated by EGF treatment in P19 cells expressing Δ9 Flag-Grb7 but was effectively elevated by EGF in control cells and cells expressing WT Flag-Grb7 (Fig. 2 E). These results confirmed that the HuR–Grb7 complex is involved in exporting KOR mRNA to the cytoplasm.


Dual action of epidermal growth factor: extracellular signal-stimulated nuclear-cytoplasmic export and coordinated translation of selected messenger RNA.

Tsai NP, Lin YL, Tsui YC, Wei LN - J. Cell Biol. (2010)

EGF induces HuR-dependent nuclear export of Grb7. (A) Immunohistochemistry of rat DRG neurons (top) and P19 cells (bottom) transfected with control or HuR siRNA. Colocalization of Grb7 and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (B) Western blot analysis of nuclear and cytoplasmic fractions from P19 cells transfected as indicated in the presence or absence of EGF. (C) Mammalian two-hybrid assay in P19 cells transfected with Gal4-HuR and serial VP16-Grb7 deletion constructs. N-terminal deletions are named according to the number of deleted amino acid residues. (D) FISH detecting endogenous KOR mRNA in DRG neurons (top) and P19 cells (bottom) transfected with WT CFP-Grb7 or Δ9 CFP-Grb7. Colocalization of KOR mRNA and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (A and D) White dotted lines outline the cytoplasm of the individual cells. (A, C, and D) Error bars represent SDs. (E) Western blots of P19 cells transfected as indicated, in the presence or absence of EGF. Bars, 25 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2819679&req=5

fig2: EGF induces HuR-dependent nuclear export of Grb7. (A) Immunohistochemistry of rat DRG neurons (top) and P19 cells (bottom) transfected with control or HuR siRNA. Colocalization of Grb7 and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (B) Western blot analysis of nuclear and cytoplasmic fractions from P19 cells transfected as indicated in the presence or absence of EGF. (C) Mammalian two-hybrid assay in P19 cells transfected with Gal4-HuR and serial VP16-Grb7 deletion constructs. N-terminal deletions are named according to the number of deleted amino acid residues. (D) FISH detecting endogenous KOR mRNA in DRG neurons (top) and P19 cells (bottom) transfected with WT CFP-Grb7 or Δ9 CFP-Grb7. Colocalization of KOR mRNA and DAPI was quantified with the Pearson correlation coefficients and is shown on the right (*, P < 0.05). (A and D) White dotted lines outline the cytoplasm of the individual cells. (A, C, and D) Error bars represent SDs. (E) Western blots of P19 cells transfected as indicated, in the presence or absence of EGF. Bars, 25 µm.
Mentions: Grb7 does not mobilize RNA directly. However, Grb7 interacts with HuR (Tsai et al., 2008), a ubiquitously expressed RNA-binding protein which can be associated with export complexes (Brennan et al., 2000; Rebane et al., 2004) to regulate the nuclear–cytoplasmic shuttling of mRNA (Doller et al., 2008). We found that HuR-specific siRNA reduced Grb7 export (Fig. 2 A) and blocked EGF-stimulated cytoplasmic accumulation of Grb7 (Fig. 2 B), which was rescued by expressing siRNA off-target HuR (see Materials and methods). The HuR-interacting domain of Grb7 was previously mapped to the N-terminal segment (Tsai et al., 2008). Because the RNA-binding domain of Grb7 is also located in its N terminus (Tsai et al., 2007), a more detailed mapping was performed to determine the minimal HuR-interacting motif (Fig. 2 C). A dominant-negative mutant of Grb7 was made by deleting this HuR-interacting motif, called Δ9. The Δ9 mutant was detected in HuR interaction yet retained the KOR mRNA–binding ability (Fig. S2). The Δ9 and wild-type (WT) Grb7s were each fused to CFP and tested in FISH experiments. It appeared that WT CFP-Grb7 could be exported together with KOR mRNA, whereas Δ9 CFP-Grb7 remained trapped in the nucleus along with KOR mRNA in both DRG neurons and P19 cells (Fig. 2 D). The amount of KOR protein was not elevated by EGF treatment in P19 cells expressing Δ9 Flag-Grb7 but was effectively elevated by EGF in control cells and cells expressing WT Flag-Grb7 (Fig. 2 E). These results confirmed that the HuR–Grb7 complex is involved in exporting KOR mRNA to the cytoplasm.

Bottom Line: The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator.Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA.In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455.

ABSTRACT
We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear-cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator. Using the kappa-opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2-containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.

Show MeSH
Related in: MedlinePlus