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INTRODUCTION |
A number of mitogenic growth factors, growth-regulatory proteins,
and growth factor receptors have been reported to be localized in the
cell nucleus and its substructures: e.g.
platelet-derived growth factor, fibroblast growth
factor-1 (FGF-1),1 FGF-2, and
ciliary neurotrophic factor (1, 2). The data show that nuclear
localization is a general phenomenon for some growth factors,
suggesting nuclear functions independent of the function as
extracellular factors.
FGF-2 is a member of the FGF family, which has been shown to mediate a
variety of biological processes during development and in the adult
organism, including mitogenesis, angiogenesis, chemotaxis, mesoderm
induction, and differentiation of various mesoderm- and
neuroectoderm-derived cells. FGF-2 as an extracellular ligand is able
to bind to high affinity tyrosine transmembrane receptors (FGF
receptors, FGFR). FGF-2 has been shown to bind to all four known FGFR,
however, with distinct affinities (3).
FGF-2 exists in protein isoforms translated from a common messenger RNA
by alternative use of AUG (18,000 isoform) and CUG (high molecular
weight isoforms 21,000 and 23,000) start codons. The isoforms exert
different biological effects when overexpressed in different cell
types. Specific effects seen by the 23-kDa isoform but not the 18-kDa
isoform after overexpression are reduced spreading of pancreatic cancer
cells (4), the ability of NIH 3T3 cells to grow in low serum medium
(5), increased radioresistance in HeLa cells (6), growing in serum-free
medium of rat AR4-2J cells (7), and differential effects on
binucleation and nuclear morphology of neonatal rat cardiac myocytes
(8). We have previously shown that in PC12 cells and rat immortalized
Schwann cells, cell growth and morphology are altered after
transfection with constructs coding for 18- and 23-kDa FGF-2,
respectively (9, 10). In this study we extend our previous data by
analyses of subcellular localization and interaction of FGF-2 in
immortalized rat Schwann cells.
The FGF-2 isoforms are known to localize to the nucleus, however, the
18-kDa isoform has been found also in the cytoplasm (8, 10-14).
The N terminus of the 23-kDa isoform exhibits a nuclear localization
signal (NLS) (15). However, because the 18-kDa isoform localizes also
to the nucleus, a second NLS in the 18-kDa "core" sequence can be
expected. The individual isoforms were reported to localize to the
nucleoli (18, 16). The aim of the present study was to analyze the
localization of FGF-2 isoforms in more detail in other nuclear
substructures by means of tagging with fluorescent proteins (EGFP and
DsRed) and to elucidate the molecular mechanisms responsible for the
nuclear localization of FGF-2 isoforms. Our results revealed a
differential localization of the isoforms in nuclear substructures of
rat immortalized Schwann cells and, in addition, identified two nuclear
localization sequences in the 23-kDa isoform. A further important and
interesting result in our study is the first presentation of a
co-immunoprecipitation and co-localization of the 23-kDa FGF-2 high
molecular weight isoform with the survival of motor neuron protein
(SMN), which is mutated in patients with spinal muscular atrophy. This
study provides new insights into the physiological functions of FGF-2 in the nucleus.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs and Subcloning--
The FGF-2 isoforms were
cloned by PCR using the vector RSVp
metFGF comprising the full-length
23-kDa isoform as a template. In this vector the ATG start codon of the
18-kDa isoform is mutated and replaced by a HindIII linker
(17). PCR was used for cloning of the respective isoforms. Because the
5' region of the 23-kDa FGF-2 coding sequence is very G/C-rich, a PCR
optimized for G/C-rich sequences was employed using 40 µM
of each dNTP, 20 pmol of each primer, PCR buffer containing 1.5 mM MgCl2 (Qiagen), 1× Q-Solution (Qiagen), and
2 units of KlenThermPlatinum Taq polymerase (Genecraft). The
forward primers introduced an ATG start codon (replacing the CTG
alternative start codons) and additional restriction sites for cloning
purposes (NheI for the 23-kDa isoform construct and HindIII for the 18-kDa construct). The reverse primer for
both isoforms introduced an additional EcoRI site. PCR
products were purified and cloned into the pGEM-T vector (Promega),
sequenced, and cut with the appropriate restriction enzymes. For
C-terminal tagging with EGFP and red fluorescent protein DsRed,
respectively, the vectors pEGFP-N1 and pDsRed-N1
(Clontech) were used for subsequent in-frame
cloning of the PCR products, resulting in pEGFP-23, pEGFP-18, pDsRed-23, and pDsRed-18. A N-terminal construct of the 23-kDa isoform
(coding for residues 1-61 of the 23-kDa isoform) and a C-terminal
construct (acid residues 141 to 188 of the 23-kDa isoform) were PCR
cloned as stated above. All constructs were checked by sequencing.
Site-directed mutageneses of FGF-2 were performed by using the
GeneEditor site-directed mutagenesis system (Promega) according to the
protocol of the manufacturer. Rat SMN cDNA comprising the complete
cDNA was cloned by PCR, introduced into
EcoRI/BamHI cut pEGFP-N1 (resulting in clone
pEGFP-SMN), and sequenced. For co-localization experiments with
Coilin-p80 a cDNA clone (I.M.A.G.E. Consortium Clone ID 4275993 (18)) comprising the complete coding sequence was digested with
SmaI/HindIII and cloned into pEGFP-N1.
Cell Culture and Transfection--
Immortalized rat Schwann
cells (19) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% final concentration of fetal calf serum, 2 mM glutamine, and 100 units/ml penicillin/streptomycin at
37 °C in humidified 5% CO2, 95% air incubator.
Transfections were repeated at least four times and performed using
calcium phosphate, Effectene (Qiagen), or Metafectene (Biontex). Cell lines stably expressing the FGF-2 isoforms were selected by addition of
Geniticin (450 µg/ml) to the culture medium 48 h after
transfection. Analysis of endogenous FGF-2 expression was performed by
Western blotting of nuclear and cytoplasmic fractions extracted with
NE-PER reagents (Pierce) according to the manufacturer's protocol.
Controls of nuclear protein enrichment were performed by Western blot
with an antibody against the nucleolar protein anti-Nopp140 (Santa Cruz
Biotechnology; data not shown).
Fluorescence and Imaging--
Epifluorescence of transfected
cells was observed using fluorescence microscopes BX60 and BX70
(Olympus) with the appropriate filter sets. Images were collected with
a cooled Slowscan CCD Colorview 12 camera and Analysis (Soft Imaging,
Münster, Germany) software. Confocal images were collected on
a custom built microscope with a Bio-Rad krypton argon primary laser.
Immunoprecipitations--
Schwann cells were grown to
subconfluency (about 80%), washed with 1× phosphate-buffered saline,
and 1 ml of IP buffer added per 10-cm cell culture dish (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 2 mM EDTA, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 1%
desoxycholate, 1× Complete protease inhibitors (Roche Molecular
Biochemicals)). Lysate was collected, incubated on ice for 30 min, and
centrifuged for 15 min at 4 °C at 14,000 × g. 50 µl of the supernatant was saved as input material. For immunoprecipitations rabbit polyclonal anti-FGF-2 (Sigma, F-3393, lot
number 086H8868, fractionated antiserum) or monoclonal anti-SMN (BD
Biosciences) antibodies, respectively, were added at concentrations of
4.2 and 0.4 µg/ml lysate, respectively, and incubated on ice overnight. Control immunoprecipitations were performed with Protein A/Protein G-agarose or rabbit/mouse IgG only. Subsequently, Protein A-
and Protein G-agarose, respectively, were added and incubated for
1 h on a rotator. The gel slurry was subsequently extensively washed with 1× lysis buffer and 4× ice-cold phosphate-buffered saline. Pellets were incubated with 1× Laemmli sample buffer
containing
-mercaptoethanol for 5 min in a cooking water bath. After
SDS-PAGE, proteins were detected by Western blot. As input control
0.25% of cell lysates was used per lane. For immunoprecipitations with anti-SMN antibody Schwann cells stably overexpressing the FGF-2 isoforms were used.
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RESULTS |
FGF-2 Isoforms Localize Differentially in the Nuclei of Schwann
Cells--
Immortalized Schwann cells express the 18-, 21-, and 23-kDa
FGF-2 isoforms in the nucleus and the cytoplasm (10, 14) (Fig. 1c). Immunolocalization
studies of FGF-2 revealed nuclear localization of FGF-2 in Schwann
cells, according to studies in different cell types (8, 11-13).
However, the available antibodies did not allow distinction between
localization patterns of single FGF-2 isoforms or certain deletion and
single-point mutations. In the approach applied here, we cloned the
isoforms (Fig. 1a) in-frame into red fluorescent protein
vector (pDsRed), transfected immortalized rat Schwann cells with the
constructs, and observed directly the in vivo localization
of the proteins by fluorescence microscopy. Cell lines stably
expressing the 18- and 23-kDa isoforms, respectively, were selected and
the localization patterns were studied. The isoforms were cloned by
means of PCR with reverse primers deleting the stop codons for in-frame
ligations into pDsRed1-N1. In the 23-kDa construct the natural ATG
start codon of the 18-kDa core sequence (Fig. 1a) was
replaced by an HindIII linker sequence (14, 17) and an ATG
codon introduced at the 5' end replacing the alternative start codon
CTG in the cDNA. Because it is known that DsRed can aggregate
resulting in artificial spots in the cell, we performed controls by
using a new mutagenized version of the vector coding for DsRed2
(Clontech) and molecularly cloned the cDNAs of
the 18- and 23-kDa isoforms into it. This DsRed2 carries the mutations
R2A, K5E, K9T, V105A, I161T, and S197A, preventing the protein from
nonspecific aggregating and resulting in a faster developing
fluorescence. However, no changes of the localization patterns of FGF-2
in the nuclei could be observed (data not shown). In other controls,
Schwann cells were co-transfected with 18-kDa GFP/18-kDa DsRed and
23-kDa GFP/23-kDa DsRed to determine localization changes dependent on
the nature of the fluorescent tag. The localizations were nearly
identical, but for EGFP with a stronger nucleoplasmic labeling in
relation to e.g. nucleolar label. Although cytoplasmic
labeling could be observed in a number of cells according to
biochemical data (Fig. 1c), many cells displayed low
staining of the cytoplasm arguing for a dynamic and regulated distribution in the cell.

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Fig. 1.
FGF-2 constructs, sequence of the N terminus
of the 23-kDa FGF-2 isoform, and endogenous expression of FGF-2 in
Schwann cells. a, FGF-2 can be observed in cells
because of alternatively used start codons (AUG for the 18-kDa isoform
and CUG for the 23-kDa isoform). In this study, FGF-2 isoforms and
N-terminal constructs were fused to DsRed, respectively, with ATG start
codons from the 18-kDa core sequence deleted in the 23-kDa FGF-2 and
NT-(1-61) constructs. b, the primary structure of the
N terminus of the 23-kDa isoform comprises RGR motifs with hydrophobic
spacer sequences. c, endogenous expression of FGF-2
isoforms in nuclear and cytoplasmic extracts from Schwann cells
separated on a 15% SDS-polyacrylamide gel. The Western blot (protein
amounts not normalized) shows expression of the 18-, 21-, and 23-kDa
isoforms. NE, nuclear extract; CE, cytoplasmic
extract.
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The 18- and 23-kDa FGF-2 isoforms displayed distinct localizations. The
18-kDa isoform was clearly enriched in the nuclei of Schwann cells
where it localized to the nucleoli and nucleoplasm (Fig.
2, a and b). This
demonstrates that the 18-kDa FGF-2 isoform core sequence possesses a
previously not described nuclear localization signal (see below).
Cytoplasmic staining could be observed to some extent. The nucleolar
localization was verified by phase-contrast microscopy (Fig.
2b) showing prominent nucleoli of the Schwann cells. In
addition, cells displayed red-labeled nuclear bodies in a dynamic
pattern, probably dependent on the physiological state (Fig.
3, a-c). In the transfected
Schwann cell line about 35% of the cells displayed a pattern with
distinct nuclear bodies visible. These nuclear bodies were in spatial
relationship to the nucleoli (Fig. 3a), sometimes
demonstrating continuous staining from the nuclear body to the nucleoli
(Fig. 3b) or no nuclear bodies (Fig. 3c). The
observed continuous staining pattern is in agreement with a transport
phenomenon between these nuclear structures. This has been observed
previously for proteins traveling between Cajal bodies (formerly coiled
bodies; Ref. 20) and nucleoli as in the case of Nopp140 (21). To verify
that the 18-kDa isoform of FGF-2 is indeed enriched in Cajal bodies we
performed double labeling experiments with Coilin-p80 (20) fused to
green fluorescent protein (GFP) as a marker protein for this nuclear
structure. The 18-kDa isoform and Coilin-p80 partially co-localize in
Cajal bodies (Fig. 3, d-f). The co-transfection analysis
also revealed Coilin-p80 enriched in nucleoli.

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Fig. 2.
Localization of FGF-2 isoforms and deletion
mutants in transfected rat Schwann cells.
a, 18-kDa FGF-2 isoform localizes to nucleoli and
nucleoplasm. b, same cell in phase contrast to demonstrate
the nucleoli. c and d, the 23-kDa isoform
localizes to nucleoplasm, the nucleolar periphery, and displayed an
additional punctuate distribution (most prominent in the nuclear
periphery as shown in e (confocal microscopy image) and
f. g and h, localization of the
N-terminal deletion mutants. During mitosis the 23-kDa isoform
(k) is associated with chromosomes as compared with the
stain for AT-rich DNA Hoechst 33258 (l), whereas the 18-kDa
isoform (i) is not associated with chromosomes
(j). In interphase the 23-kDa isoform is chromatin
associated (m and n). o, control
cells transfected with DsRed.
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Fig. 3.
Co-localization of FGF-2 isoforms with Coilin
and SMN. a-c, images of cells transfected with the 18-kDa
FGF-2 isoform/DsRed construct showing dynamic nuclear body
(arrows) associations. A continuum of protein is visible
between these nuclear bodies and the nucleoli in a subset of cells.
d-f, Schwann cells were co-transfected with 18-kDa
FGF-2/DsRed and Coilin-p80/GFP as a marker for Cajal bodies. The
superimposed images are shown in f. g-i, the
23-kDa isoform co-localizes with SMN in nuclear gems. Schwann cells
were co-transfected with the 23-kDa FGF-2/DsRed and SMN/GFP construct.
SMN-GFP is expressed in the cytoplasm (h) as expected, where
it exerts its function as an assembly factor for spliceosomal
complexes. In the nucleus it is enriched in nuclear gems
(arrows). Superimposition of the images (i)
displays the co-localization of 23-kDa FGF-2 and SMN in nuclear
gems.
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In contrast, the 23-kDa FGF-2 isoform was distributed in a punctuate
pattern, in the periphery of nucleoli and in the nucleoplasm (Fig. 2,
c-f) with significantly less red-labeled 23-kDa FGF-2 in
the nucleoli than it was the case for the 18-kDa isoform. This pattern
was observed in almost all cells not undergoing mitosis in the stably
transfected Schwann cell line. With regard to nucleoli, confocal
microscopy revealed localization of the 23-kDa isoform with the
nucleolar periphery (Fig. 2e). Most of the fusion protein also localized close to the nuclear membrane (Fig. 2f)
suggesting a chromatin-associated pattern. This was verified by
labeling cells with Hoechst 33258 (Fig. 2, m and
n), which stained AT-rich DNA, demonstrating a
colocalization of both 23-kDa FGF-2 and DNA patterns. In cells
undergoing mitosis the 23-kDa isoform displayed a redistribution and
associated with mitotic chromosomes (Fig. 1, k and
l). In strong contrast, association of the 18-kDa isoform with mitotic chromosomes could not be observed (Fig. 2, i
and j). The observed putative chromatin or DNA-associated
distribution pattern of the 23-kDa isoform is probably because of its
N-terminal extension including several arginine-glycine-argine motifs
in comparison to the 18-kDa core sequence (Fig. 1b). This
motif can also be found in the chromatin-associated high-mobility group protein A1 (formerly HMGI), which is known to bind to AT-rich DNA (22)
and co-localize with 23-kDa FGF-2 (data not shown).
The N Terminus of the 23-kDa FGF-2 Isoform Confers Nuclear
Localization--
The difference between both FGF-2 isoforms is the
N-terminal extension of the 23-kDa isoform. This N-terminal region has
been shown to contain a nuclear localization signal (15), however, the
localization pattern had to be determined. Using the same approach as
above we cloned this N-terminal fragment comprising the 34-amino acid
residues of the N-terminal extension (NT-(1-34); Fig.
1a) and additionally a fragment with the consecutive 27 residues of the 18-kDa core sequence up to a BamHI site in
the cDNA in-frame into pDsRed (NT-(1-61); Fig.
1a). The N-terminal extension contains several RGR motifs,
surrounded by mainly hydrophobic residues (Fig. 1b). This
structure constitutes a positively charged N terminus of the 23-kDa
FGF-2 isoform.
The N-terminal extension of the 23-kDa FGF-2 isoform bears a NLS
in agreement with previously published data (15). In addition, we could
demonstrate that the transfected cells revealed a nuclear localization
pattern of the N terminus (Fig. 2, g and h) like after transfection with the complete 23-kDa isoform, but with no clear
labeling in the nucleolar periphery. This suggests a nucleolar
localization signal located in the 18-kDa core sequence. Additionally,
the N terminus is able to associate with mitotic chromosomes similar to
the 23-kDa isoform (data not shown).
The 18-kDa FGF-2 Isoform Contains an Additional Nuclear
Localization Signal--
Both 18- and 23-kDa FGF-2 isoforms localized
to the nuclei of Schwann cells. The 18 kDa lacks the N-terminal
extension of the 23-kDa isoform, which we demonstrated to serve as the
nuclear localization signal, and does not display any canonical NLS. To identify sequences responsible for nuclear localization in the 18-kDa
core isoform, we performed site-directed mutageneses of clusters of
basic residues that could serve as putative NLS (Fig. 4a). Two amino acid residues
in each cluster were mutagenized together to nonpositively charged
amino acid residues, cloned into pDsRed, and the localization was
analyzed after transfection of Schwann cells (Fig. 4b). A
Western blot analysis after transfection of Schwann cells with the
23-kDa DsRed isoform revealed a 5-10-fold higher expression of the
tagged protein compared with the total amount of endogenous FGF-2
isoforms (Fig. 4d). No changes in the nuclear localization
pattern for the mutants K63G/R64G and R86G/K88E could be observed.
However, the more C-terminal mutant R149G/R151G displayed a cytoplasmic
localization (Fig. 4b). Both the 18- and 23-kDa FGF-2
isoforms completely failed to localize to the nuclei when carrying this
mutation (Fig. 4b; shown for the 23-kDa isoform). The
observed localization pattern is not the result of partial proteolysis
of the DsRed-tagged protein as shown by the Western blot in Fig.
4c. The results demonstrate an important role for residues Arg149/Arg151 for nuclear
localization of both isoforms. These residues are surrounded by
putative phosphorylation sites at residues Thr147 and
Ser150 for protein kinase C and Ser154 for cAMP
or cGMP-dependent protein kinase (Fig. 4a). The
residues are putative targets for regulated localization by introducing negatively charged phosphate groups in close proximity to the positively charged guanidino groups of the arginine residues and therefore neutralizing charges.

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Fig. 4.
Mutation analyses of FGF-2. Double point
mutations of FGF-2 were generated from the 23-kDa isoform at positions
with clustered basic amino acid residues (a). Schwann cells
were transfected with the respective DsRed fusion constructs. The
R149G/R151G mutation failed to localize to the nucleus indicating a
second sequence motif responsible for nuclear localization
(b). Putative phosphorylation sites (P) are close
to the mutated residues indicating possible sites for a regulative
localization mechanism. c, total cell lysates from cells
transfected with mutated FGF-2 plasmids and FGF-2 23-kDa control
plasmid were probed on a Western blot (samples not normalized) with
anti-FGF-2 antibody to demonstrate that the observed patterns are not
the result of partial proteolysis. The blot was slightly overexposed to
show that no significant differences between the lanes even in the
background exist. d, nuclear extract of Schwann cells
transfected with the 23-kDa FGF-2/DsRed construct (23 tag)
display a 5-10-fold overexpression compared with the total amount of
protein of the endogenous isoforms (18, 21, and 23 kDa). Note that also
the 34-kDa FGF-2 isoform is detectable in this blot.
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The 23-kDa Isoform, but Not the 18-kDa Isoform of FGF-2
Co-immunoprecipitates with the SMN and Co-localizes with SMN in Nuclear
Gems--
The N-terminal extension of high molecular weight FGF-2
contains arginine residues reported to be dimethylated (23, 24). The
biological function of arginine methylation in FGF-2 still remains
unclear. The SMN has been reported to bind to methylarginine sequences
in proteins (25) exhibiting with this post-translational modification
an interface for protein-protein interactions. We performed
immunoprecipitation experiments to test the hypothesis, that SMN could
possibly bind to the 23-kDa FGF-2 isoform through its N-terminal extension.
Anti-FGF-2 antibody was able to co-precipitate SMN protein from Schwann
cell extract (Fig. 5, b and
c). Immunoprecipitation experiments performed
with anti-SMN antibody in extracts from cells overexpressing either the
DsRed-tagged 18- or 23-kDa FGF-2 isoform revealed precipitation of the
23-kDa isoform only (Fig. 5c, lanes 3 and
4). We conclude that SMN is in a common complex or is able
to interact specifically with the 23-kDa isoform by possibly binding to
the N-terminal extension of this isoform. In transfected Schwann cells
a SMN-GFP fusion construct localizes to the cytoplasm, nucleoplasm, and
to certain nuclear bodies (Fig. 3h), known as nuclear gems
(gemini of Cajal bodies; Ref. 26), identifying these nuclear structures
as the putative interaction sites with 23-kDa FGF-2. To elucidate a
possible co-localization of 23-kDa FGF-2 and SMN in nuclear gems we
performed co-transfections (Fig. 3, g-i). Superposition of
the images reveals a partial co-localization in nuclear gems.

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Fig. 5.
Co-immunoprecipitation analyses of FGF-2
isoforms with survival of motor neuron protein. a, the
fractionated anti-FGF-2 antiserum ( -FGF-2 pol.; polyclonal
anti-FGF-2 antibody; reactive with human, bovine, and rat FGF-2) was
tested on Western blots of extracts from a human hepatocellular
carcinoma cell line (HepG2; provided by Transduction Laboratories as a
positive control for SMN) and rat Schwann cells transfected with the
23-kDa FGF-2/DsRed plasmid. The position of the respective
proteins, 23-kDa FGF-2/DsRed (23 tag), SMN, and the
endogenous FGF-2 isoforms (18, 21, and 23 kDa) are indicated. The
fractionated antiserum showed no cross-reactivity with SMN (lanes
1 and 3 and 4 and 6) in both
extracts and was used in subsequent immunoprecipitation analyses as
well as the anti-SMN monoclonal antibody ( SMN).
FGF-2 mon., monoclonal FGF-2 antibody used as a detection
antibody for Western blots of immunoprecipitated proteins.
b, immunoprecipitation analyses in Schwann cells (SC;
lane 1) and with the 23-kDa FGF-2/DsRed-tagged
isoform-transfected Schwann cells (SC-transf.23; lane
2) show immunoprecipitation of 40-kDa SMN with anti-FGF-2
antibody, but not with rabbit IgG as a control (lane 3).
Total protein content was not normalized between individual extracts,
which accounts for the differences seen between signal strengths.
Lanes 4 and 5 show input controls. Lanes
6 and 7 show no change in expression of endogenous
FGF-2 in untransfected (SC) and transfected
(SC-transf.23) Schwann cells after
immunoprecipitation with anti-FGF-2 antibody. Note that this separation
was on 10% SDS-polyacrylamide gels and therefore no separation of the
individual isoforms could be observed. c, in Schwann
cells transfected with DsRed-tagged 23- and 18-kDa constructs, the
23-kDa but not the 18-kDa isoform can be immunoprecipitated by anti-SMN
antibody (lanes 3 and 4). Controls show the individ ual isoforms in the
input material (lanes 1 and 2) and
immunoprecipitated tagged FGF-2 isoforms (lanes 5 and
6). Lanes 7-12 demonstrate SMN in the
input (lanes 7 and 8), as control in the
immunoprecipitation with anti-SMN antibody (lanes 9 and
10) and immunoprecipitated with anti-FGF-2 antibody
(lanes 11 and 12). Co-immunoprecipitated SMN in
lane 12 is probably because of the presence of endogenous
23-kDa FGF-2 in the 18-kDa FGF-2 overexpressing cell line.
HC, IgG heavy chain; 23 tag, 18 tag, 18- and
23-kDa FGF-2 isoforms tagged with DsRed. d, model of
FGF-2 18- and 23-kDa isoforms demonstrating the N terminus of the
23-kDa isoform with RGR motifs as a putative interaction domain with
SMN (SMN-Int) and the determined NLS.
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DISCUSSION |
Immunolocalization studies and Western blotting after cell
fractionation have previously demonstrated the existence of FGF-2 in
the nucleus (10-13, 27). In this study we labeled FGF-2 isoforms by
fusion constructs with the red fluorescent protein DsRed. Because no
specific antibodies against the individual isoform are available, we
used this approach, although the possibility of artificial localization
patterns because of overexpression of the tagged proteins cannot be
completely excluded. One previous study shows a 18-kDa FGF-2/GFP fusion
protein localizing to the nuclei in corneal endothelial cells, however,
the nuclear substructures enriched in this isoform have not been
described explicitly (28). Here we demonstrate differential
localization of FGF-2 isoforms in the nucleus, localization in nuclear
bodies, and a specific protein-protein interaction of 23-kDa FGF-2. For
several growth factors, apart from FGF-2, localization in the cell
nucleus is well known including platelet-derived growth factor (29),
FGF-1 (30), FGF-3 (31), and EGF (32, 33). However, only limited information about their specific nuclear roles exists. Additionally, nuclear localization of the respective receptors has been described as
in the case of FGFR-1 (34, 35), which is part of a transcription complex in the nucleus (36). Nuclear EGF receptor acts as a putative
transcription factor and binds to the promoter region of cyclin D1
in vivo (37).
In this study, the 23-kDa FGF-2 isoform has been found to be putatively
chromatin-associated. At present, it is unclear if specific chromatin
components can be bound by FGF-2 in vivo, however, an
association with histone H1 and an influence on chromatin compaction of
the FGF-2 high molecular weight isoforms has been reported previously
(12, 38). For the 18-kDa FGF-2 isoform we demonstrate in this study for
the first time the localization in Cajal bodies. Recent data suggest a
role of Cajal bodies in the maturation and transport of small nuclear
ribonucleoprotein particles (39). Sm proteins are transported in the
nucleus, where they localize first to Cajal bodies, before they are
transported through the nucleolus to splicing factor compartments
(40).
Many growth factors display sequences required for nuclear localization
(1). Removal of the NLS from FGF-1
leads to decreased mitogenic
activity, whereas receptor binding and activation is not influenced.
Addition of a heterologous NLS from histone H2 is able to restore the
mitogenic function (41). We show in this study that both FGF-2 isoforms
localize to the nucleus, with the 18-kDa isoform possessing one and the
23-kDa displaying a second NLS: the N-terminal extension exclusively in
the 23-kDa isoform and a common C-terminal sequence element in the
18-kDa core primary structure. This is in agreement with data showing
that the N-terminal extension is able to target
-galactosidase to
the nucleus (15). Interestingly, mutations of the second NLS in the
23-kDa sequence (R149G/R151G) override the effect of the N-terminal
extension NLS, suggesting an effect on the overall structure of this
isoform. It is possible, of course, that the other mutants also had an influence on FGF-2 structure, but they retained nuclear localization. Our data indicate that the 18-kDa FGF-2 core sequence is able to confer
nucleolar localization, because the N-terminal constructs displayed no
clear enrichment in the nucleoli. For FGF-3 it has been shown recently
that a nucleolar binding partner NoBP is essential for its
nucleolar localization (42). The localization of the nucleolar localization sequence in FGF-2 has to be
determined in further studies.
With regard to putative functions distinct effects of the isoforms have
been shown, e.g. on neurite formation and survival of
dopaminergic neurons after exogenous application of the isoforms (43).
Examination of cell death rates and proliferation in Schwann cells
stably overexpressing either the 18- or 21/23-kDa isoforms resulted in
differential effects of both isoforms: the 18-kDa cells exhibited a
significant increase of cell death but no higher proliferation rate in
contrast to the high molecular weight isoforms (14).
For the understanding of nuclear functions of FGF-2, identification of
interacting proteins is a crucial step. A previously unknown protein,
called FIF (FGF-2-interacting factor), has been found in a two-hybrid
assay and the exclusive interaction with high molecular weight FGF-2
was determined in a co-immunoprecipitation analysis (44). Recently, it
was shown that the N terminus of the 23-kDa high molecular weight
isoform of FGF-2 contains arginine residues that are
post-translationally dimethylated (24). The RG repeats of the
N-terminal extension are substrates for protein-arginine methyltransferases (23, 45). The product of the spinal muscular atrophy
gene SMN is able to bind preferentially to dimethylarginine containing
proteins, as shown for SmD1 and SmD3 (25). These proteins possess
C-terminal RG domains. Here we show specific binding of 23-kDa FGF-2 to
SMN by co-immunoprecipitation (Fig. 5, b and c)
and, in addition, co-localization in nuclear gems (Fig. 3,
g-i). This indicates a putative interaction with the N-terminal extension of the 23-kDa isoform or an indirect interaction in a common complex in nuclear gems. The majority of SMN is a cytoplasmic protein, where it is part of a complex with spliceosomal Sm
proteins, Gemin-2 (formerly SIP1), and Gemin-3 and -4 (for review see
Refs. 46 and 47). In the nucleus, SMN can be detected in gems and Cajal
bodies (26, 48). Despite the presence of both the 18-kDa FGF-2 isoform
and SMN in Cajal bodies, we could not detect co-immunoprecipitation of
these proteins. Therefore, direct or indirect interaction via bridging
proteins of SMN with FGF-2 is probably confined to the 23-kDa isoform
in nuclear gems. Different functional domains of SMN are responsible
for known protein-protein interactions. The C terminus is responsible
for Sm-protein binding (49) and oligomerization of SMN (50), whereas an
N-terminal region is responsible for SIP1 binding. For the putative
direct interaction with FGF-2 the responsible domains have to be
elucidated in further studies. Spinal muscular atrophy is associated
with deletion and mutations of the SMN1 gene. Spinal muscular atrophy patients suffer from severe symptoms of motoneuron degeneration. However, SMN is ubiquitously expressed in most cell types
examined. The reasons for specific symptoms only in motoneurons are not
yet clear. The severity of the disease is correlated with the amount of
functional SMN protein from the second (SMN2) gene. Blocking
of the central interaction domain of SMN, the Tudor domain, prevents
binding of small nuclear ribonucleoprotein particle core factors (Sm
proteins) and abolishes assembly of small nuclear ribonucleoprotein.
Deletion of 27 residues from the N terminus of SMN leads to inhibition
of small nuclear ribonucleoprotein assembly, splicing, and
transcription (51, 52).
The specific interaction of the 23-kDa FGF-2 isoform with SMN suggests
a new role for nuclear FGF-2 in RNA metabolism. Although the
physiological relevance of this interaction is not clear, it is worth
noting in this context that FGF-2 mediates neurotrophic effects on
motoneurons in vivo and in vitro (53). In
addition, the endogenous FGF-2 is up-regulated in motoneurons after
peripheral nerve lesion (54) and iodinated FGF-2 is specifically
transported by motoneurons (55). In a recent study SMN could be linked
to a growth factor signal transduction pathway by interacting with the
zinc finger protein ZPR1 (56). ZPR1 binds to EGFR and redistributes to
the nucleus and Cajal bodies together with SMN in mitogen-treated cells.
FGF-2 bound to the extracellular side by FGF receptors can be
internalized as a receptor-ligand complex, escape the desensitization processes, and can be transported to the nucleus (11). In this study we
cannot exclude the possibility that FGF-2, which lacks a classical
export signal, is transported outside the cell and subsequently
redirected to the nucleus. However, in addition to previous studies our
data suggest the existence of independent nuclear growth factor functions.