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(Received for publication, December 13,
1995) From the
Fibroblast growth factor-1 (FGF-1) is a potent mitogen for
mesoderm- and neuroectoderm-derived cell types in vitro.
However, a mutant FGF-1 with deletion in its nuclear localization
sequence (NLS, residues 21-27) is not mitogenic in
vitro. We demonstrated that synthetic peptides containing this NLS
were able to stimulate DNA synthesis in a FGF receptor-independent
manner after they were delivered into living NIH 3T3 cells by a
cell-permeable peptide import technique. The stimulation of maximal DNA
synthesis by these peptides required the presence of peptides during
the entire G Mitogenic signaling for many growth factors is triggered by
their binding to the transmembrane receptor tyrosine kinases, for
example, those for epidermal growth factor and platelet-derived growth
factor. Upon ligand-receptor binding, these receptors are dimerized and
autophosphorylated. The activated receptors further phosphorylate the
receptor substrates to initiate intracellular kinase signaling cascades (1, 2, 3) . It is evident, however, that
there may be alternative signaling pathways for some growth factors
involving their nuclear transport and signaling (for review see (4) ). In this context, subsequent to receptor-ligand
internalization, growth factor ligands may translocate to the nucleus
and directly function in mitogenic processes(5, 6) .
FGF-1 ( To examine
directly the functional role of the NLS in FGF-1-stimulated
mitogenesis, we have delivered the peptide encompassing this sequence
into living cells by using our recently developed cell-permeable
peptide import method (CPPI) (see (11) ). We demonstrated in
this report that cell membrane-permeable peptides containing this NLS
sequence can stimulate DNA synthesis in NIH 3T3 cells in a FGF
receptor-independent manner. Our results together suggest the nuclear
transport of FGF-1 plays an important role in the mitogenic pathway
initiated by exogenous FGF-1. This may represent an important signaling
mechanism for certain growth factors.
Figure 1:
Sequences of cell membrane-permeable
peptides containing the NLS of FGF-1 and control peptides
(single-letter amino acid code). The cell membrane-translocating
sequence (11) is single underlined, the NLS of FGF-1
is double underlined, and the residues mutated in the NLS
sequence are in boldface. These two regions are separated by a
spacer region of A-A-A.
Figure 2:
A, demonstration of the intracellular
SA
The mitogenic
effect of the SA peptide was verified by flow cytometric analysis of
the DNA distribution in each phase of the cell cycle in the SA
peptide-treated 3T3 cells. As shown in Table 1and Fig. 2C, the DNA content in the S-phase, which
reflected the cell fractions in this phase, was significantly increased
when the cells were treated for 20 h with the SA peptide at 100
µg/ml, which coincided with the fully effective concentration in
the thymidine incorporation assay (Fig. 2B). A similar,
but stronger, stimulation was observed in the cells treated with FGF-1
containing the same NLS ( Table 1and Fig. 2C).
These results support the important role of the NLS region of FGF-1 in
inducing DNA synthesis.
Figure 3:
A, the effect of NLS mutations on SA
In this report, we suggest a dissociation of FGF-1-stimulated
mitogenesis from its receptor tyrosine kinase activation in NIH 3T3
cells. We have demonstrated that the peptide containing the NLS of
FGF-1 can stimulate DNA synthesis after it is delivered into NIH 3T3
cells by using a cell-permeable peptide import method(11) . Our
finding is supported by a recent observation that a mutant FGF-1 with
deletion in its NLS is not mitogenic in vitro(5) . We
thus propose that the NLS of FGF-1 may play two functional roles in
exogenous FGF-1-stimulated mitogenesis. First, in mediating nuclear
translocation of FGF-1, internalization of FGF-1 following its receptor
binding may allow the association of the partitioned growth factor
through the NLS with the intracellular machinery that facilitates
nuclear transport of FGF-1. A number of cytosolic proteins have been
known to mediate nuclear translocation of various NLS-containing
proteins (for review see (23) ). This role of the NLS of FGF-1
may not be crucial (24) because internalized FGF-1 with a
molecular size of 16.5 kDa should enter the cell nucleus by free
diffusion. However, the NLS could be important if FGF-1 is transported
to the nucleus in the form of FGF The mitogenic effect of SA peptides
is not limited to NIH 3T3 cells. We found that SA peptides could also
induce DNA synthesis in bovine hamster kidney-21 cells. However, the
same peptides, unlike full-length FGF-1, were inactive in murine LE-II
endothelial cells despite their good cell membrane permeability in this
cell line (data not shown). These results thus suggest that different
mechanisms may be involved in FGF-1-stimulated mitogenesis in various
cell types.
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5305-5308
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
phase of the cell cycle. The mitogenic effect
was specific for the NLS of FGF-1 because a peptide with double point
mutations at lysine residues was inactive in stimulating DNA synthesis.
Our results suggest that the NLS plays an important role in the
mitogenic pathway initiated by exogenous FGF-1 by its direct
involvement in the nuclear transport and signaling of internalized
FGF-1.
)is one of two prototype members of the fibroblast
growth factor family. It is a potent mitogen for many cell types and is
involved in embryogenesis, angiogenesis, and neurite
outgrowth(7, 8) . The mechanism by which FGF-1
transmits mitogenic signals is still not entirely clear. It has been
shown, however, that a mutant FGF-1 with deletion in its nuclear
localization sequence (NLS) Asn-Tyr-Lys-Lys-Pro-Lys-Leu (residues
21-27) failed to stimulate DNA synthesis and cell proliferation in vitro although it could still bind to the FGF receptor and
induce intracellular receptor-mediated tyrosine phosphorylation and
c-fos expression(5) . The fact that
FGF-1-(21-27) was able to direct 
-galactosidase into the
nucleus, as well as the evidence of nuclear localization of
FGF-1(9, 10) , suggest that nuclear transport of FGF-1
following receptor-mediated internalization might be important for
stimulating DNA synthesis by FGF-1 in vitro.
Peptides and Antibodies
Peptides listed in Fig. 1were synthesized by a stepwise solid-phase peptide
synthesis method and purified by C
reverse-phase high
pressure liquid chromatography as
described(11, 12, 13) . The molecular weights
of the purified peptides were verified by mass spectrometry analysis
and were shown to agree with the calculated molecular masses. A
polyclonal anti-SM peptide antibody raised against the SM
peptide-keyhole limpet hemocyanin conjugate in rabbits (11) recognized in enzyme-linked immunosorbent assay not only
SM peptide but also SA
peptide analogs.
Indirect Immunofluorescence Assay
Confluent NIH
3T3 cells grown on the chamber slides (Nunc) were treated with 0.5 ml
of SA peptide solution in Dulbecco's modified Eagle's
medium (DMEM) containing 10% fetal bovine serum (FBS) under the
conditions indicated in the figures. The intracellular peptide was
detected by an indirect immunofluorescence assay using anti-SM peptide
antibody and rhodamine-labeled anti-rabbit antibody as
described(11) . Coverslips with stained cells were mounted in
Poly/Mount (Polyscience) and analyzed in an Olympus fluorescence
microscope using a 
100 oil immersion lens.Mitogenic Assays
Confluent 3T3 cells grown
initially in DMEM containing 10% FBS were transferred to a low serum
medium (DMEM containing 0.5% FBS) for 2 days. FGF-1 in the presence of
heparin (5 units/ml) or the test peptides was added to a fresh low
serum medium at the indicated concentrations. In thymidine
incorporation assay, [
H]thymidine was added after
16 h, and 4 h later, the cells were washed with phosphate-buffered
saline, treated with trichloroacetic acid, and solubilized with 0.15 M NaOH, and the radioactivity was determined in a liquid
scintillation counter. In a DNA quantitative assay, after 20 h of
incubation with peptide or FGF-1, cells were harvested, spun down, and
washed with serum-free phosphate-buffered saline three times. The cells
were fixed with methanol precooled to -20 °C for DNA analysis
by the flow cytometric method.Translocation of
Both SAI-Labeled Peptides into
NIH 3T3 Cells
and SAM4 peptides were
radiolabeled with I by the IODO-GEN method (Pierce). The
specific activities of both peptides were similar (1.44
10
and 1.66 10 cpm/ng, respectively).
The confluent NIH 3T3 monolayers (1.6 10
cells) on
each dish were treated with 30 ng of I-labeled peptide at
37 °C for 30 min. The cells were washed and lysed as
described(11) . The radioactivity in the cell lysate was
counted in a Packard Auto-Gamma counter.
Tyrosine Phosphorylation Studies
To examine the
tyrosine phosphorylation induced by FGF-1 and SA peptides, confluent
3T3 cells grown initially in DMEM containing 10% FBS were starved in
DMEM containing 0.5% FBS for 2 days. FGF-1 in the presence of heparin
(5 units/ml) or SA peptide was added to cells. After incubation for the
indicated time periods, cells were washed and lysed in lysis buffer (10
mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM EDTA,
50 mM NaF, 30 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride,
and 1% Triton X-100). Cell lysates were electrophoresed on 5-12%
gradient SDS-polyacrylamide gels and transferred to Hybond-ECL. The
blot was then analyzed for immunoreactive phosphotyrosine-containing
proteins with monoclonal anti-Tyr(P) antibody (Upstate Biotechnology,
Inc.) followed by horseradish peroxidase-linked anti-mouse antibody
according to ECL protocol(11) .
Cellular Import of Synthetic Peptides Containing the
NLS of FGF-1
To study the role of the NLS in FGF-1-stimulated
mitogenesis, we delivered a 28-residue cell membrane-permeable peptide
(referred to as SA, see Fig. 1) containing the NLS of FGF-1
into living NIH 3T3 cells. This SA peptide encompassed a cell
membrane-translocating sequence (11) at its amino-terminal end
and the NLS of FGF-1 at its carboxyl-terminal region, separated by a
middle spacer region of Ala-Ala-Ala. In addition, a Met-Pro dipeptide
sequence was attached to the carboxyl terminus of the peptide to form
an epitope tag (Leu-Met-Pro) that can be recognized by an available
anti-peptide antibody (11) in enzyme-linked immunosorbent assay
and in an immunofluorescence assay. We examined whether the SA
peptide can be taken up efficiently by living NIH 3T3 cells. As shown
in Fig. 2A, intracellular SA peptide was observed
in a punctate staining pattern in NIH 3T3 cells treated with the
SA peptide in an indirect immunofluorescence assay using the
peptide antibody. A considerable amount of fluorescence was located in
the perinuclear areas. Immunodetection of the SA peptide was
specific because cells incubated with the antibody preabsorbed with the
peptide showed no intracellular fluorescence signals (data not shown).
Likewise, cells not exposed to the SA peptide were negative (Fig. 2A).
peptide in NIH 3T3 cells by fluorescence microscopy analysis.
Confluent NIH 3T3 cells were treated at 37 °C with 100 µg/ml of
SA or diluent for 30 min. Intracellular SA peptide deposits
were detected by indirect immunofluorescence assay using anti-SM
peptide IgG and rhodamine-labeled anti-rabbit antibody (11) and
analyzed in an Olympus fluorescence microscope using a 100 oil
immersion lens. B, [H]thymidine
incorporation by NIH 3T3 cells stimulated by synthetic peptides (left panel) or by FGF-1 (right panel). Serum-starved
NIH 3T3 cells were incubated with various amounts of peptides or
FGF-1/heparin (5 units/ml). After 16 h,
[H]thymidine was added, and 4 h later, the cells
were washed, solubilized, and the [H]thymidine
incorporation was determined. Data are the mean ± S.D. of
triplicate samples and calculated as multiplicity of counts in the
tested sample over the control sample (untreated cells). The experiment
was repeated three times with similar results. C, cell cycle
distribution of NIH 3T3 cells treated with SA peptide or FGF-1.
Serum-starved cells are untreated (control) or treated with SA peptide
(100 µg/ml) or FGF-1 (15 ng/ml) for 20 h, and DNA content in each
phase of the cell cycle was determined by flow cytometric analysis
stained with propidium iodide. This is the analysis of a single
representative experiment. A statistical analysis of the results of six
experiments can be seen in Table 1.
Mitogenic Activities of Cell-permeable Peptides Carrying
the NLS of FGF-1
Having demonstrated that the SA peptide
can be readily introduced into cells, we examined its potential
activity in functional assays. In a thymidine incorporation assay, we
found that SA peptide induced a mitogenic response of NIH 3T3
cells in a manner similar to the full-length FGF-1 (Fig. 2B). [H]Thymidine
incorporation stimulated by the peptides was concentration-dependent,
reaching maximal induction (about 6.3-fold) at 100 µg/ml
(extracellular peptide concentration). It should be noted that
approximately 4% of extracellular SA peptide was imported into
cells according to the peptide import assay using I-labeled SA
peptide (see the results below). In the
same thymidine assay, the full-length FGF-1 in vitro induced
approximately a 7.5-fold stimulation (Fig. 2B). As
controls for the SA peptide, we used a non-cell-permeable peptide
comprising only the NLS of FGF-1 (ANL peptide, see Fig. 1) and
an available cell-permeable peptide containing a non-FGF-1 sequence (SM
peptide) (11) . In contrast to SA peptide, both control
peptides did not show any measurable functional activities when tested
within comparable concentration ranges (Fig. 2B). These
results suggest that neither the cell membrane-translocating sequence
alone (SM peptide) nor the nuclear localization sequence alone (ANL
peptide) was sufficient for stimulating DNA synthesis. Cell-permeable
SA peptide therefore was active because it carried the functional
NLS of FGF-1 into cells. To clarify whether the functional activity of
SA peptide was contributed exclusively by the NLS of FGF, we also
prepared and tested a cell-permeable analog of the SA peptide
(referred to as SA, see Fig. 1), which contained only the NLS of
FGF-1 but not the carboxyl-terminal epitope tag. Fig. 2B indicates the functional importance of the NLS because the SA and
SA peptides were identical in their ability to stimulate thymidine
incorporation. Both SA peptide analogs within the concentration range
used were not cytotoxic as determined by staining with fluorescein
diacetate/ethidium bromide(11, 14) .Functional Importance of Lysine Residues in the NLS of
FGF-1
The NLS of FGF-1 sequence contains 3 lysine residues
(positions 22, 23, and 25 in SA peptide in Fig. 1). To
determine the functional importance of these basic residues, a series
of cell-permeable peptides containing single or double point mutations
of 3 lysine residues was prepared (Fig. 1) and examined in a
thymidine incorporation assay. As shown in Fig. 3A, the
peptides (SAM1, SAM2, and SAM3) with a single point
mutation in each of 3 lysine residues (Lys
Thr,
Lys
Thr, and Lys
Thr,
respectively) were still able to stimulate the
[
H]thymidine incorporation in a manner similar to
that of the SA peptide, although the SAM3 peptide was
slightly less active. However, the peptide SAM4 with double point
mutations (Lys
Thr and Lys
Thr)
exhibited impaired activity, suggesting that these basic residues are
involved in the pathway leading to DNA synthesis. To exclude the
possibility that the inactivity of the SA
M4 peptide was due to its
lower cell membrane permeability, NIH 3T3 cells were incubated with
either I-SA
or I-SA
M4, and the
amount of cell-associated I-labeled peptides was measured
and compared. No significant difference between
I-SA
- and I-SA
M4-treated cells
was observed in cell-associated radioactivity counts (18,120 ±
1,933 versus 16,730 ± 2,747 cpm/1.6 10 cells, p > 0.05, n = 6,
Student's t test). Therefore, our results from these
assays indicate that the loss of mitogenic activity of SAM4
peptide is due to the lack of the two functional lysine residues in
this FGF-1 sequence. Interestingly, subcellular fractionation of these
cells showed that about 90% of the I-labeled SA
peptide was associated with the nuclear fraction (data not shown).
Therefore, lack of strong nuclear staining in the immunofluorescence
assay (Fig. 2A) might have resulted from modification
and/or association of the SA peptide with specific nuclear
constituents, which prohibited peptide recognition by the antibody.
peptide-stimulated [H]thymidine incorporation by
NIH 3T3 cells. Serum-starved NIH 3T3 cells were treated with various
amounts of SA or its mutant peptides. The rest of the procedure
was the same as described in the legend to Fig. 2B. Bars represent the mean ± S.D. of six samples and are
calculated as multiplicity of counts in the tested sample over the
control sample (untreated cells). The differences between SA
peptide and SAM4 peptide and between SA peptide and SAM3
peptide at 75 and 100 µg/ml were significant (p < 0.01
and 0.05, respectively) by analysis of variance. The experiment was
repeated three times with similar results. B, long term
peptide exposure required for the SA peptide-stimulated
[H]thymidine incorporation by NIH 3T3 cells.
Serum-starved cells were untreated (control) or treated with SA peptide
(100 µg/ml) or FGF-1 (15 ng/ml)/heparin (5 units/ml) for the
indicated time periods. The rest of the procedure was the same as
described in the legend to Fig. 2B. Bars represent the mean ± S.D. of triplicate samples and are
calculated as multiplicity of counts in the tested sample over the
control sample. The experiment was repeated three times with similar
results. C, the induction of tyrosine phosphorylation by SA
peptide or FGF-1 in NIH 3T3 cells. Serum-starved cells were untreated (lane C) or treated with SA peptide (100 µg/ml) or FGF-1
(15 ng/ml) for the indicated time periods. The immunoblot of the
equivalent amount of whole cell lysates was obtained with
anti-phosphotyrosine antibody. The tyrosine-phosphorylated 90-kDa
protein and 150-kDa PLC- protein affirmed by anti-PLC
antibody were indicated.
Stimulation of DNA Synthesis by the SA Peptide Required a
Long Incubation Time
We further examined whether the mitogenic
stimulation by SA peptides was cell cycle-dependent. As shown in Fig. 3B, [H]thymidine uptake by
NIH 3T3 cells was not observed in the cells treated for 6 h (G phase) with SA peptide at 100 µg/ml. However, it became
substantial after cells were treated for 20 h (S phase). A similar
pattern of stimulation was seen in cells treated with the full-length
FGF-1 in vitro (Fig. 3B). It has also been
shown that stimulation of maximal DNA synthesis by FGF-1 in Balb/c 3T3
cells requires the presence of FGF-1 during the entire G phase of the cell cycle(15) .The Mitogenic Effect of SA Peptides Is FGF
Receptor-independent
It is known that exogenous FGF-1 binds to
FGF receptors on NIH 3T3 cells and induces the tyrosine phosphorylation
of a number of intracellular
proteins(15, 16, 17, 18, 19, 20) .
Among them, a 90-kDa protein and a 150-kDa phospholipase C-
(PLC-
) are rapidly and strongly phosphorylated in FGF-1-stimulated
NIH 3T3 cells and are often used as the indicators of FGF receptor
activation(18, 19, 20) . However, recent
studies have suggested that phosphorylation of PLC-
and the
resulting hydrolysis of phosphatidylinositol are not required for
FGF-stimulated mitogenesis(21, 22) . In Fig. 3C, we demonstrated that unlike FGF-1, SA peptide
did not stimulate the early tyrosine phosphorylation of the 90-kDa
protein and PLC-
in NIH 3T3 cells even at concentrations
sufficient to induce DNA synthesis, suggesting that the mitogenic
effect of SA peptides is FGF receptor-independent. Instead, SA peptide
triggered the slight tyrosine phosphorylation of several unknown
proteins at the late G
phase of the cell cycle (Fig. 3C). It is still not clear whether this low level
phosphorylation is important for the SA peptide-induced activity.
FGF receptor complex. As
concerns the second role of the NLS of FGF-1, the functional ability of
SA peptides in stimulating DNA synthesis suggests that the NLS is
directly involved in the FGF-1-induced nuclear mitogenic signaling.
Such signaling may be initiated by the binding of the positively
charged NLS to specific molecules in the nucleus or on the nuclear
membrane. The importance of the basic residues as demonstrated by our
mutagenesis study is buttressed by our recent finding that
cell-permeable peptides containing the NLS of nuclear factor 
B p50
protein or the NLS of v-Rel protein can also stimulate DNA synthesis in
NIH 3T3 cells in a manner similar to SA peptides (data not shown).
Because the basic cores of the two NLS sequences, KRQK (p50) and KRQR
(v-Rel), are similar to that of the NLS of FGF-1, KKPK (Fig. 1),
a 4-residue sequence motif, K-K(R)-X-K(R), may be functionally
important. It is expected that this proposed sequence motif can be
found in many intracellular NLS-containing proteins. However, it may
become physiologically relevant only when a significant amount of
molecules containing this motif is translocated into the nucleus, for
example by receptor-mediated FGF-1 internalization or by cell-permeable
peptides as shown in this study.
)
We thank C. Walter for editorial assistance and Dr. J.
Donahue for review of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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