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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 FGFR1 induces c-Jun expression and potentiates FGF-induced cyclin D1 expression. (a) NIH 3T3 fibroblasts were transfected with constructs encoding full-length wild-type FGFR1, FGFR1 with the signal peptide replaced by the SV-40 large T antigen NLS (NLS-R1), or with a construct encoding NLS-R1 with a kinase-inactivating point mutation (NLS-R1kd), each with an epitope tag at the COOH terminus. Cells were separated into cytosolic (C) and nuclear (N) fractions, and equal amounts of protein were immunoblotted for the epitope tag. The molecular weight of wild-type FGFR1 (140 kD) is greater than that of NLS-R1 (110 kD) due to glycosylation. (b) Basal and FGF-induced immediate early gene expression in transfected cells. Cells were transiently transfected with the indicated constructs, starved for 24 h in low-serum medium, and then treated for 1 h with 1 ng/ml FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for c-Jun, c-Fos, and c-Myc. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated FGFR1-transfected cells and represent the mean ± SEM of triplicate experiments. *P < 0.01 versus FGFR1-transfected cells treated with FGF-2. (c) FGF-2–induced cyclin D1 and p27Kip1 expression in transfected cells. Transiently transfected cells were starved for 24 h in low-serum medium and then treated for 16 h with FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for cyclin D1 and p27Kip1. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated cells and represent the mean ± SEM of triplicate experiments.
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Figure 5: Nuclear FGFR1 induces c-Jun expression and potentiates FGF-induced cyclin D1 expression. (a) NIH 3T3 fibroblasts were transfected with constructs encoding full-length wild-type FGFR1, FGFR1 with the signal peptide replaced by the SV-40 large T antigen NLS (NLS-R1), or with a construct encoding NLS-R1 with a kinase-inactivating point mutation (NLS-R1kd), each with an epitope tag at the COOH terminus. Cells were separated into cytosolic (C) and nuclear (N) fractions, and equal amounts of protein were immunoblotted for the epitope tag. The molecular weight of wild-type FGFR1 (140 kD) is greater than that of NLS-R1 (110 kD) due to glycosylation. (b) Basal and FGF-induced immediate early gene expression in transfected cells. Cells were transiently transfected with the indicated constructs, starved for 24 h in low-serum medium, and then treated for 1 h with 1 ng/ml FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for c-Jun, c-Fos, and c-Myc. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated FGFR1-transfected cells and represent the mean ± SEM of triplicate experiments. *P < 0.01 versus FGFR1-transfected cells treated with FGF-2. (c) FGF-2–induced cyclin D1 and p27Kip1 expression in transfected cells. Transiently transfected cells were starved for 24 h in low-serum medium and then treated for 16 h with FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for cyclin D1 and p27Kip1. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated cells and represent the mean ± SEM of triplicate experiments.

Mentions: To examine the functional role of nuclear FGFR1, we transfected mouse fibroblasts with a construct encoding full-length FGFR1 with the signal peptide replaced by the SV-40 large T antigen NLS. This protein (NLS-R1) was constitutively localized to the nucleus (Fig. 5 a), as determined by biochemical fractionation of the cells. Cells expressing NLS-R1 showed elevated expression of c-Jun compared with cells transfected with wild-type FGFR1 (Fig. 5 b) or with a construct encoding FGFR1 lacking the signal peptide (ΔSP-R1) or with vector alone (data not shown). A construct encoding NLS-R1 with a point mutation that inactivates the tyrosine kinase (NLS-R1kd) failed to stimulate c-Jun expression, demonstrating that the observed responses depend on receptor kinase activity. The basal levels of two other immediate early gene products, c-Fos and c-Myc, were unaffected by NLS-R1 when compared with the expression in cells transfected with wild-type FGFR1 (Fig. 5 b). Treatment with FGF-2 induced the expression of c-Jun, c-Fos, and c-Myc in FGFR1 and NLS-R1–transfected cells, and FGF-2–induced c-Jun expression was potentiated in NLS-R1–transfected cells compared with cells transfected with wild-type FGFR1 (Fig. 5 b). The level of c-Jun expression in unstimulated FGFR1–transfected cells was similar to that in vector-transfected controls and most likely represents incomplete quiescence due to the transfection procedure or the duration of the serum deprivation. This increased baseline expression may be masking part of the stimulatory effects of NLS-R1. In addition to immediate early gene expression, several other FGF-induced signal transduction events were examined in cells transfected with vector, FGFR1, ΔSP-R1, or NLS-R1. No significant differences were observed in basal or FGF-stimulated phosphorylation of ERK1/2, p38 MAPK, CREB, ATF-2, Akt/PKB, or p70S6K (data not shown). This is consistent with the activation of these kinase modules and their downstream effectors by cell surface FGFR1 and indicates that the induction of c-Jun by NLS-R1 is a specific result of the nuclear localization of the receptor.


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

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

Nuclear FGFR1 induces c-Jun expression and potentiates FGF-induced cyclin D1 expression. (a) NIH 3T3 fibroblasts were transfected with constructs encoding full-length wild-type FGFR1, FGFR1 with the signal peptide replaced by the SV-40 large T antigen NLS (NLS-R1), or with a construct encoding NLS-R1 with a kinase-inactivating point mutation (NLS-R1kd), each with an epitope tag at the COOH terminus. Cells were separated into cytosolic (C) and nuclear (N) fractions, and equal amounts of protein were immunoblotted for the epitope tag. The molecular weight of wild-type FGFR1 (140 kD) is greater than that of NLS-R1 (110 kD) due to glycosylation. (b) Basal and FGF-induced immediate early gene expression in transfected cells. Cells were transiently transfected with the indicated constructs, starved for 24 h in low-serum medium, and then treated for 1 h with 1 ng/ml FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for c-Jun, c-Fos, and c-Myc. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated FGFR1-transfected cells and represent the mean ± SEM of triplicate experiments. *P < 0.01 versus FGFR1-transfected cells treated with FGF-2. (c) FGF-2–induced cyclin D1 and p27Kip1 expression in transfected cells. Transiently transfected cells were starved for 24 h in low-serum medium and then treated for 16 h with FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for cyclin D1 and p27Kip1. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated cells and represent the mean ± SEM of triplicate experiments.
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Figure 5: Nuclear FGFR1 induces c-Jun expression and potentiates FGF-induced cyclin D1 expression. (a) NIH 3T3 fibroblasts were transfected with constructs encoding full-length wild-type FGFR1, FGFR1 with the signal peptide replaced by the SV-40 large T antigen NLS (NLS-R1), or with a construct encoding NLS-R1 with a kinase-inactivating point mutation (NLS-R1kd), each with an epitope tag at the COOH terminus. Cells were separated into cytosolic (C) and nuclear (N) fractions, and equal amounts of protein were immunoblotted for the epitope tag. The molecular weight of wild-type FGFR1 (140 kD) is greater than that of NLS-R1 (110 kD) due to glycosylation. (b) Basal and FGF-induced immediate early gene expression in transfected cells. Cells were transiently transfected with the indicated constructs, starved for 24 h in low-serum medium, and then treated for 1 h with 1 ng/ml FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for c-Jun, c-Fos, and c-Myc. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated FGFR1-transfected cells and represent the mean ± SEM of triplicate experiments. *P < 0.01 versus FGFR1-transfected cells treated with FGF-2. (c) FGF-2–induced cyclin D1 and p27Kip1 expression in transfected cells. Transiently transfected cells were starved for 24 h in low-serum medium and then treated for 16 h with FGF-2. Equal amounts of whole cell lysate protein were immunoblotted for cyclin D1 and p27Kip1. Expression levels were quantified by densitometry of immunoblots, and values are normalized to untreated cells and represent the mean ± SEM of triplicate experiments.
Mentions: To examine the functional role of nuclear FGFR1, we transfected mouse fibroblasts with a construct encoding full-length FGFR1 with the signal peptide replaced by the SV-40 large T antigen NLS. This protein (NLS-R1) was constitutively localized to the nucleus (Fig. 5 a), as determined by biochemical fractionation of the cells. Cells expressing NLS-R1 showed elevated expression of c-Jun compared with cells transfected with wild-type FGFR1 (Fig. 5 b) or with a construct encoding FGFR1 lacking the signal peptide (ΔSP-R1) or with vector alone (data not shown). A construct encoding NLS-R1 with a point mutation that inactivates the tyrosine kinase (NLS-R1kd) failed to stimulate c-Jun expression, demonstrating that the observed responses depend on receptor kinase activity. The basal levels of two other immediate early gene products, c-Fos and c-Myc, were unaffected by NLS-R1 when compared with the expression in cells transfected with wild-type FGFR1 (Fig. 5 b). Treatment with FGF-2 induced the expression of c-Jun, c-Fos, and c-Myc in FGFR1 and NLS-R1–transfected cells, and FGF-2–induced c-Jun expression was potentiated in NLS-R1–transfected cells compared with cells transfected with wild-type FGFR1 (Fig. 5 b). The level of c-Jun expression in unstimulated FGFR1–transfected cells was similar to that in vector-transfected controls and most likely represents incomplete quiescence due to the transfection procedure or the duration of the serum deprivation. This increased baseline expression may be masking part of the stimulatory effects of NLS-R1. In addition to immediate early gene expression, several other FGF-induced signal transduction events were examined in cells transfected with vector, FGFR1, ΔSP-R1, or NLS-R1. No significant differences were observed in basal or FGF-stimulated phosphorylation of ERK1/2, p38 MAPK, CREB, ATF-2, Akt/PKB, or p70S6K (data not shown). This is consistent with the activation of these kinase modules and their downstream effectors by cell surface FGFR1 and indicates that the induction of c-Jun by NLS-R1 is a specific result of the nuclear localization of the receptor.

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