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Importin beta-mediated nuclear import of fibroblast growth factor receptor: role in cell proliferation.

Reilly JF, Maher PA - J. Cell Biol. (2001)

Bottom Line: Here, we show that the nuclear translocation of fibroblast growth factor receptor (FGFR)1 occurs via a mechanism distinct from classical nuclear import but dependent on importin beta, a component of multiple nuclear import pathways.Furthermore, we show that nuclear FGFR1 induces c-Jun and is involved in the regulation of cell proliferation.These data are the first description of a nuclear import pathway for transmembrane growth factor receptors and elucidate a novel signal transduction pathway from the cell surface to the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA. jreilly@neurome.com

ABSTRACT
Although growth factor receptors are generally thought to carry out their role in signal transduction at the cell surface, many of these transmembrane proteins translocate to the nucleus after ligand stimulation. Here, we show that the nuclear translocation of fibroblast growth factor receptor (FGFR)1 occurs via a mechanism distinct from classical nuclear import but dependent on importin beta, a component of multiple nuclear import pathways. Furthermore, we show that nuclear FGFR1 induces c-Jun and is involved in the regulation of cell proliferation. These data are the first description of a nuclear import pathway for transmembrane growth factor receptors and elucidate a novel signal transduction pathway from the cell surface to the nucleus.

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Nuclear import of FGFR1 is mediated by importin β. (a) Coimmunoprecipitation of importin β with FGFR1. Cells were untreated or subjected to ATP depletion by treatment with oligomycin B and 2-deoxyglucose for 2 h, in the absence or presence of FGF-2, and separated into cytosolic (C) and nuclear (N) fractions. FGFR1 was immunoprecipitated from equal amounts of protein, and complexes were separated by SDS-PAGE and immunoblotted for importin β. The whole cell lysate is shown for comparison, the immunoglobulin heavy chain (IgG) is indicated, and molecular weights are indicated in kD. (b–e) Nuclear translocation of FGFR1 in Swiss 3T3 fibroblasts was examined using an in vitro nuclear import assay (Adam et al. 1990), and cells were analyzed by FGFR1 immunostaining and confocal microscopy. Unpermeabilized cells (b) were untreated or incubated for 30 min with FGF-2. Digitonin-permeabilized cells (c–e) were incubated for 30 min with FGF-2 in the absence or presence of exogenous cytosol (c), with mock- or importin β–depleted exogenous cytosol (d) or with exogenous cytosol plus mouse IgG or a neutralizing antibody against importin β (e). Depletion of importin β from exogenous cytosol was confirmed by immunoblotting (d). Bar, 10 μm.
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Figure 4: Nuclear import of FGFR1 is mediated by importin β. (a) Coimmunoprecipitation of importin β with FGFR1. Cells were untreated or subjected to ATP depletion by treatment with oligomycin B and 2-deoxyglucose for 2 h, in the absence or presence of FGF-2, and separated into cytosolic (C) and nuclear (N) fractions. FGFR1 was immunoprecipitated from equal amounts of protein, and complexes were separated by SDS-PAGE and immunoblotted for importin β. The whole cell lysate is shown for comparison, the immunoglobulin heavy chain (IgG) is indicated, and molecular weights are indicated in kD. (b–e) Nuclear translocation of FGFR1 in Swiss 3T3 fibroblasts was examined using an in vitro nuclear import assay (Adam et al. 1990), and cells were analyzed by FGFR1 immunostaining and confocal microscopy. Unpermeabilized cells (b) were untreated or incubated for 30 min with FGF-2. Digitonin-permeabilized cells (c–e) were incubated for 30 min with FGF-2 in the absence or presence of exogenous cytosol (c), with mock- or importin β–depleted exogenous cytosol (d) or with exogenous cytosol plus mouse IgG or a neutralizing antibody against importin β (e). Depletion of importin β from exogenous cytosol was confirmed by immunoblotting (d). Bar, 10 μm.

Mentions: Participation of nuclear FGFR1 in FGF-induced signal transduction events requires regulated transport of the receptor into the nucleus. In examining the energy dependence of FGF-2–induced nuclear accumulation of FGFR1, we observed that ligand-induced nuclear translocation occurred in fibroblasts depleted of ATP using a combination of oligomycin B and 2′-deoxyglucose (Fig. 1). Interestingly, ATP depletion also induced the nuclear translocation of FGFR1 in the absence of FGF-2. Confocal microscopy demonstrated the localization of FGFR1 in untreated cells to the plasma membrane and in vesicle-like structures within the cytoplasm (Fig. 1 a; see Fig. 4 b for higher magnification). The low level of plasma membrane staining is a result of the technique used to permeabilize the cells (Maher 1996b), and cytoplasmic localization of FGFR1 has been described previously (Maher 1996a). In FGF-2–treated cells, FGFR1 was located within the nucleus and not associated with the nuclear envelope (Fig. 1 a). ATP depletion caused morphological changes in the cells, including shrinkage of the cell body and nucleus. Localization of FGFR1 in the nucleus of ATP-depleted cells was confirmed by differential interference contrast microscopy, and FGFR1 immunoreactivity was coincident with a fluorescent nuclear counterstain (data not shown). These data were confirmed by biochemical isolation of membrane-depleted nuclei (Fig. 1 b). As ATP concentrations within cells decreased, a progressive increase in the nuclear accumulation of FGFR1 was observed, and this effect was potentiated at all time points by pretreatment with FGF-2 (Fig. 1 c).


Importin beta-mediated nuclear import of fibroblast growth factor receptor: role in cell proliferation.

Reilly JF, Maher PA - J. Cell Biol. (2001)

Nuclear import of FGFR1 is mediated by importin β. (a) Coimmunoprecipitation of importin β with FGFR1. Cells were untreated or subjected to ATP depletion by treatment with oligomycin B and 2-deoxyglucose for 2 h, in the absence or presence of FGF-2, and separated into cytosolic (C) and nuclear (N) fractions. FGFR1 was immunoprecipitated from equal amounts of protein, and complexes were separated by SDS-PAGE and immunoblotted for importin β. The whole cell lysate is shown for comparison, the immunoglobulin heavy chain (IgG) is indicated, and molecular weights are indicated in kD. (b–e) Nuclear translocation of FGFR1 in Swiss 3T3 fibroblasts was examined using an in vitro nuclear import assay (Adam et al. 1990), and cells were analyzed by FGFR1 immunostaining and confocal microscopy. Unpermeabilized cells (b) were untreated or incubated for 30 min with FGF-2. Digitonin-permeabilized cells (c–e) were incubated for 30 min with FGF-2 in the absence or presence of exogenous cytosol (c), with mock- or importin β–depleted exogenous cytosol (d) or with exogenous cytosol plus mouse IgG or a neutralizing antibody against importin β (e). Depletion of importin β from exogenous cytosol was confirmed by immunoblotting (d). Bar, 10 μm.
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Figure 4: Nuclear import of FGFR1 is mediated by importin β. (a) Coimmunoprecipitation of importin β with FGFR1. Cells were untreated or subjected to ATP depletion by treatment with oligomycin B and 2-deoxyglucose for 2 h, in the absence or presence of FGF-2, and separated into cytosolic (C) and nuclear (N) fractions. FGFR1 was immunoprecipitated from equal amounts of protein, and complexes were separated by SDS-PAGE and immunoblotted for importin β. The whole cell lysate is shown for comparison, the immunoglobulin heavy chain (IgG) is indicated, and molecular weights are indicated in kD. (b–e) Nuclear translocation of FGFR1 in Swiss 3T3 fibroblasts was examined using an in vitro nuclear import assay (Adam et al. 1990), and cells were analyzed by FGFR1 immunostaining and confocal microscopy. Unpermeabilized cells (b) were untreated or incubated for 30 min with FGF-2. Digitonin-permeabilized cells (c–e) were incubated for 30 min with FGF-2 in the absence or presence of exogenous cytosol (c), with mock- or importin β–depleted exogenous cytosol (d) or with exogenous cytosol plus mouse IgG or a neutralizing antibody against importin β (e). Depletion of importin β from exogenous cytosol was confirmed by immunoblotting (d). Bar, 10 μm.
Mentions: Participation of nuclear FGFR1 in FGF-induced signal transduction events requires regulated transport of the receptor into the nucleus. In examining the energy dependence of FGF-2–induced nuclear accumulation of FGFR1, we observed that ligand-induced nuclear translocation occurred in fibroblasts depleted of ATP using a combination of oligomycin B and 2′-deoxyglucose (Fig. 1). Interestingly, ATP depletion also induced the nuclear translocation of FGFR1 in the absence of FGF-2. Confocal microscopy demonstrated the localization of FGFR1 in untreated cells to the plasma membrane and in vesicle-like structures within the cytoplasm (Fig. 1 a; see Fig. 4 b for higher magnification). The low level of plasma membrane staining is a result of the technique used to permeabilize the cells (Maher 1996b), and cytoplasmic localization of FGFR1 has been described previously (Maher 1996a). In FGF-2–treated cells, FGFR1 was located within the nucleus and not associated with the nuclear envelope (Fig. 1 a). ATP depletion caused morphological changes in the cells, including shrinkage of the cell body and nucleus. Localization of FGFR1 in the nucleus of ATP-depleted cells was confirmed by differential interference contrast microscopy, and FGFR1 immunoreactivity was coincident with a fluorescent nuclear counterstain (data not shown). These data were confirmed by biochemical isolation of membrane-depleted nuclei (Fig. 1 b). As ATP concentrations within cells decreased, a progressive increase in the nuclear accumulation of FGFR1 was observed, and this effect was potentiated at all time points by pretreatment with FGF-2 (Fig. 1 c).

Bottom Line: Here, we show that the nuclear translocation of fibroblast growth factor receptor (FGFR)1 occurs via a mechanism distinct from classical nuclear import but dependent on importin beta, a component of multiple nuclear import pathways.Furthermore, we show that nuclear FGFR1 induces c-Jun and is involved in the regulation of cell proliferation.These data are the first description of a nuclear import pathway for transmembrane growth factor receptors and elucidate a novel signal transduction pathway from the cell surface to the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA. jreilly@neurome.com

ABSTRACT
Although growth factor receptors are generally thought to carry out their role in signal transduction at the cell surface, many of these transmembrane proteins translocate to the nucleus after ligand stimulation. Here, we show that the nuclear translocation of fibroblast growth factor receptor (FGFR)1 occurs via a mechanism distinct from classical nuclear import but dependent on importin beta, a component of multiple nuclear import pathways. Furthermore, we show that nuclear FGFR1 induces c-Jun and is involved in the regulation of cell proliferation. These data are the first description of a nuclear import pathway for transmembrane growth factor receptors and elucidate a novel signal transduction pathway from the cell surface to the nucleus.

Show MeSH
Related in: MedlinePlus