From the Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
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ABSTRACT |
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Acidic fibroblast growth factor (aFGF) contains a
phosphorylation site recognized by protein kinase C. A non-mitogenic
mutant growth factor is devoid of this phosphorylation site. We have changed amino acids in and close to the phosphorylation site and studied the consequences of this for binding of the growth factor to
high affinity receptors as well as to heparin. We have also studied the
ability of the mutants to stimulate DNA synthesis and cell
proliferation as well as phosphorylation of mitogen-activated protein
kinase and the ability of the growth factor mutants to be transported
to the nucleus. The results indicate that while the mutations strongly
affect the ability of the growth factor to bind to heparin, they do not
affect much the binding to the specific FGF receptors, activation of
mitogen-activated protein kinase or transport of the growth factor to
the nucleus. The mutations affect to various extents the ability of the
growth factor to stimulate DNA synthesis and to induce cell
multiplication. We find that phosphorylation of aFGF is not required
for mitogenic activity. The data suggest that altered interaction of
the growth factor with a cellular component different from the
receptor, possibly a component in the nucleus, is the reason for the
different mitogenicity of the different growth factor mutants.
Acidic fibroblast growth factor (aFGF or
FGF-1)1 belongs to large
family of growth factors that bind to four closely related receptors,
FGFRs 1-4 (1). The receptors contain, in their cytoplasmic part, a
split tyrosine kinase domain that is activated upon binding of FGF to
the extracellular receptor domain (2). Signaling through this tyrosine
kinase with the activation of downstream signaling molecules is an
important part in the signal transduction from FGF (3-7). However,
there is also evidence that some growth factors of this family, in
particular aFGF and basic FGF (bFGF), enter the cytosol and the nucleus
and act directly on intracellular targets (8-18).
aFGF contains an exposed loop that is involved in binding to heparin
(19). Mutation in this region of lysine 132 to glutamic acid (K132E)
reduces heparin affinity and essentially abolishes the ability of the
growth factor to stimulate DNA synthesis in cells (15, 20). aFGF(K132E)
binds to FGFR with similar affinity as wild-type aFGF (15) and
activates the tyrosine kinase of the receptor (15, 20) and stimulates
MAP kinase.2 Furthermore, the
K132E mutant stimulates expression of several immediate-early genes
(15, 20, 21), and it is as potent as the wild-type aFGF in inducing
mesoderm formation in Xenopus animal caps (22). The mutant
was found to be transported to the nuclear fraction similarly as the
wild-type (15). We recently cloned a novel, intracellular protein,
FIBP, which binds the wild-type aFGF, but not aFGF(K132E) (23).
The K132E mutation destroys a consensus phosphorylation site for
protein kinase C (PKC) in aFGF, Ser130-Cys-Lys. aFGF was
previously shown to be phosphorylated by PKC on serine in
vitro, but not by protein kinase A or casein kinase I and only
negligibly by casein kinase II (24). We found that wild-type aFGF was
phosphorylated in vitro in a cell lysate while aFGF(K132E)
was not. In vitro phosphorylation in cell lysate was augmented in the presence of diacylglycerol and phosphatidylserine and
inhibited by staurosporine (15). Furthermore, externally added
wild-type aFGF, but not aFGF(K132E), was phosphorylated in intact cells
(15). Phosphorylation in vivo depended on translocation of
aFGF to the cytosol or nucleus, because phosphorylation was not
observed when aFGF was incubated with cells transfected with FGFR4
constructs incapable of mediating translocation to these locations.3 Taken together,
these data indicate that after translocation to the cytosol or nucleus
aFGF becomes phosphorylated on serine 130 by PKC.
In attempts to elucidate the role of the phosphorylation site in the
exposed loop, we made several mutations in this region and measured the
ability of the mutated growth factors to bind to heparin and FGFR and
to stimulate MAP kinase, DNA synthesis, and cell proliferation.
Plasmid Construction and Protein Purification--
Constructs
for in vitro transcription and translation encoding aFGF
with different mutations (Fig. 1A) were generated by
polymerase chain reaction-directed mutagenesis with the plasmid
paFGF(K132E) (15) as template. Polymerase chain reaction products were
cut with StuI and EcoRI and ligated into
paFGF(K132E) between the same sites. Each construct contained a new
restriction site for colony screening, and the sequence of each
construct was verified by DNA sequencing. Constructs encoding
maltose-binding protein (MBP) fusion proteins were generated by
inserting the SphI-EcoRI fragment from each of
these plasmids between the same sites in pMal-cN-aFGFCAAX, which had
been previously constructed by inserting the
NcoI-EcoRI fragment of pHBGF-cax (13) into the
polylinker of pMal-cN (25). In some of the constructs a stop-codon had not been generated by polymerase chain reaction and was instead made by
cutting the plasmids with EcoRI, filling in with T4 DNA polymerase and religating the plasmids. This procedure introduced an
in-frame stop-codon and an AseI restriction site. MBP fusion proteins were affinity-purified on an amylose resin column as described
by the manufacturer (New England Biolabs, Beverly, MA).
Cell Culture and Polyacrylamide Gel Electrophoresis in the
Presence of Sodium Dodecyl Sulfate (SDS-PAGE)--
Cells were
propagated in Dulbecco's modified essential medium with 7.5% (v/v)
fetal calf serum in a 5% CO2 atmosphere at 37 °C.
SDS-PAGE was carried out with 7.5 or 10% gels as described by Laemmli
(26) and processed as described (8).
In Vitro Phosphorylation--
MBP fusion proteins, cleaved with
factor Xa to separate MBP from mutant or wild-type aFGF, at 0.3 mg/ml
(final concentration) or pure aFGF or aFGF(K132E) at 0.1 mg/ml (final
concentration) were incubated with purified PKC (0.6 unit/ml) (Sigma)
in kinase buffer (20 mM Tris, pH 7.6, 10 mM
MgCl2, 2 mM MnCl, 1 mM
dithiothreitol, 1 mM EGTA, 0.1 mM
Na3VO4, 1 µg/ml aprotinin, 0.1 mM
ATP, 40 µCi/ml [ In Vitro Transcription and Translation--
Plasmids were
linearized with EcoRI and transcribed in vitro
with T3 RNA polymerase. Ethanol-precipitated transcripts were translated for 1 h 30 min at 30 °C in a nuclease-treated rabbit reticulocyte lysate system (Promega, Madison, WI) in the presence of 1 µM [35S]methionine or unlabeled methionine.
To be able to compare concentrations of translated growth factor in
different unlabeled translation mixtures, labeled translations were
always run in parallel. After translation the lysates were dialyzed
against dialysis buffer (20 mM HEPES, pH 7.0, 140 mM NaCl, 2 mM CaCl2) (27).
Measurement of DNA Synthesis and Cell Proliferation--
DNA
synthesis was measured by incorporation of [3H]thymidine
in serum-starved NIH 3T3 cells stimulated with in vitro
translated, unlabeled growth factors for 24 h. Cell proliferation
was measured in serum-starved NIH 3T3 cells after stimulation for
48 h with in vitro translated growth factors by
counting the cells in a cell counter.
Heparin Affinity--
In vitro translated,
[35S]methionine-labeled growth factors diluted in
PBS:water = 1:1 were incubated under rotation with 100 µl of
heparin-Sepharose at 4 °C for 2 h. The beads were washed, and
bound proteins were eluted with stepwise increasing concentrations of
NaCl (1 ml of each concentration), and radioactivity in each fraction
(1 ml) was measured.
Binding of Wild-type and Mutant aFGF to Cells--
Calf
pulmonary artery endothelial cells were incubated for 2 h at
4 °C in HEPES medium supplemented with 10 units/ml heparin and 5 ng/ml [35S]methionine-labeled wild-type or mutant aFGF in
the absence or presence of excess unlabeled wild-type aFGF. The cells
were washed three times, lysed, and the postnuclear supernatant was
analyzed by SDS-PAGE and fluorography. In other experiments,
125I-labeled wild-type aFGF (5 ng/ml) was incubated with
NIH 3T3 cells for 3 h at 4 °C in HEPES medium containing 10 units/ml heparin in the absence or presence of increasing
concentrations of MBP fusion proteins with aFGF mutants or wild-type.
In some cases, the fusion proteins had been cleaved with factor Xa. The
cells were washed three times, lysed in 0.1 M KOH, and the
radioactivity was measured.
Fractionation of Cells into Cytosol/Membrane and Nuclear
Fraction--
After lysis in lysis buffer (0.1 M NaCl, 10 mM Na2HPO4, 1% Triton X-100, 1 mM EDTA, pH 7.4, 1 mM phenylmethylsulfonyl
fluoride, 4 µg/ml aprotinin) cells were centrifuged for 15 min at
720 × g. The supernatant was centrifuged again for 5 min at 15,800 × g and designated the cytosol/membrane
fraction. The pellet was washed once by resuspension in lysis buffer
and once by resuspension in lysis buffer containing 0.3 M
sucrose that was layered on lysis buffer containing 0.8 M
sucrose and centrifuged at 720 × g for 15 min at
4 °C, then sonicated in lysis buffer containing additional 0.5 M NaCl and centrifuged for 5 min at 15,800 × g. The supernatant of this centrifugation was designated the
nuclear fraction.
Transport of Externally Added Growth Factor to the Nuclear
Fraction--
Serum-starved NIH 3T3 cells were incubated in Dulbecco's
modified essential medium containing 1 mM unlabeled
methionine, 10 units/ml heparin, and 2 µl/ml
[35S]methionine-labeled mutant or wild-type growth
factors for different periods of time. In some experiments 50 µM LY294002 was also present. Cells were washed and
treated with 5 mg/ml Pronase at 37 °C for 5 min, transferred to an
Eppendorf tube, and 1 mM phenylmethylsulfonyl fluoride and
4 µg/ml aprotinin were added. Cells were washed once by resuspension
in HEPES medium with the same protease inhibitors, lysed, and
fractionated. Trichloroacetic acid precipitable material was analyzed
by SDS-PAGE and fluorography.
MAP Kinase Activity--
Serum-starved NIH 3T3 cells were
incubated at 37 °C for different periods of time in HEPES medium
with different concentrations of in vitro translated
wild-type or mutant aFGF or with a translation mixture without added
mRNA in the presence of heparin (5 units/ml). The cells were washed
twice with HEPES medium on ice and lysed in P-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% Triton
X-100) with protease inhibitors. The postnuclear supernatant was
subjected to immunoprecipitation with an anti-MAP kinase (ERK1 and
ERK2) antibody (Zymed Laboratories Inc.), and the
immunoprecipitates were subsequently incubated for 15 min at 37 °C
in 25 µl of kinase buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 2 mM MnCl2, 1 mM dithiothreitol) with 1 µCi of
[ Generation of Mutants in the Exposed Loop of aFGF--
Since
aFGF(K132E) exhibits strongly reduced ability to stimulate DNA
synthesis in cells (15, 20, 22) and is incapable of being
phosphorylated in vitro and in vivo (15), we
carried out a number of additional mutations in this region of the
molecule (Fig. 1A). Serine 130 in the consensus sequence for phosphorylation by PKC was mutated to
glutamic acid (S130E) or to alanine (S130A) to mimic phosphorylated and
unphosphorylated growth factor, respectively. Similar mutations have
been made previously in other proteins to study the role of
phosphorylation (29-32). To study if lysine 132 as such is required,
we mutated it to alanine (K132A). Since this mutation destroys the PKC
phosphorylation site, we also made the double mutant S130E/K132A as a
"phosphorylated" control. We further made the conservative mutation
of lysine 132 into arginine (K132R). Burgess et al. (22)
made the double mutant cysteine 131 into serine and lysine 132 into
glutamic acid (C131S/K132E) and reported that it was mitogenic to the
same extent as the bovine wild-type aFGF. In this double mutant a new,
potential PKC phosphorylation site is created
(Ser131-Glu-Arg), and therefore we also made this double
mutant to investigate if it would become phosphorylated.
In Vitro Phosphorylation and Heparin Affinity of the
Mutants--
Some of the mutants were expressed as fusion proteins
with maltose-binding protein in bacteria and affinity-purified. These fusion proteins were then cleaved with the protease, factor Xa, to
separate the MBP part from the growth factor part, and analyzed by an
in vitro kinase assay with purified PKC. As shown in Fig. 1B, lower panel, lane 4, the wild-type
growth factor was strongly phosphorylated. The mutants where serine 130 had been mutated to alanine (lane 1) or glutamic acid
(lane 2) were not or negligibly phosphorylated, consistent
with the notion that serine 130 is the major phosphorylation site in
aFGF. The mutant C131S/K132E, where we had introduced a possible PKC
site one amino acid away from the normal site, was also not
phosphorylated (lane 3). In lanes 5 and
6 we used growth factor expressed as such in bacteria and
purified as described previously (14, 15) as controls. The pure,
wild-type growth factor was phosphorylated to a similar extent as
wild-type growth factor expressed as MBP fusion protein (compare
lanes 4 and 6). With pure aFGF(K132E) a weak
phosphorylated band was detectable (lane 5) similarly as
with the other mutants. Since aFGF(K132E) is not phosphorylated
in vitro in a cell lysate or in vivo in intact
cells (15), the weak phosphorylation observed in lanes 1-3
and 5 probably represents background phosphorylation in the
in vitro assay with pure PKC. In aFGF(K132R) the PKC
consensus site is intact, since both lysine and arginine are tolerated
at this position, and we found that aFGF(K132R) was indeed substrate for PKC (not demonstrated).
aFGF(K132E) exhibits strongly reduced heparin affinity (20) as compared
with wild-type aFGF. To measure heparin affinity of the different aFGF
mutants, we bound in vitro translated,
[35S]methionine-labeled growth factors to
heparin-Sepharose columns, washed, and eluted with stepwise increasing
concentrations of NaCl. The results, shown in Fig. 1C,
demonstrate that the different mutants varied considerably in their
affinity for heparin. The S130E mutant exhibited virtually identical
heparin affinity as the wild-type, while the S130A mutant was eluted in
a broad peak. This result was reproducible in four different
experiments, but the reason for the unusual broadness of the peak is
unclear. The mutants where lysine 132 had been changed all exhibited
reduced heparin affinity. The most conservative mutation, K132R,
reduced the affinity least, while the K132A mutation reduced heparin
affinity considerably. We found that aFGF(K132E) eluted at 0.3 M NaCl (not demonstrated). It appears that the heparin
affinity of aFGF is very sensitive to mutations of lysine 132 and that
more drastic mutations have a more pronounced effect than conservative ones.
Ability of Mutant Growth Factors to Bind to Cells--
To test the
ability of the different mutants to bind to cells, in vitro
translated, [35S]methionine-labeled growth factors were
incubated with calf pulmonary artery endothelial cells at 4 °C in
the presence of heparin and in the absence or presence of excess
unlabeled wild-type aFGF. All mutants were bound to a similar extent
and the binding could be completely competed out with excess aFGF in
all cases (Fig. 2A).
To measure relative binding affinities to cells of the wild-type and
mutant growth factors, we incubated NIH 3T3 cells at 4 °C with
125I-labeled, pure wild-type aFGF in the presence of
heparin and different concentrations of unlabeled wild-type or mutant
aFGF as MBP fusion proteins. After washing, the cell-associated
radioactivity was measured. Most of the tested mutants were able to
compete with 125I-labeled wild-type aFGF for binding to
cells with a similar potency as the wild-type itself (Fig.
2B). However, it appears that the S130E mutant was somewhat
more potent than the wild-type in this respect. Similar data were
obtained when the MBP fusion proteins had been cleaved off with factor
Xa prior to the experiment (not demonstrated).
Ability of the Different Mutants to Stimulate DNA Synthesis and
Cell Proliferation--
To study the mitogenic activity of the
different mutants, we added increasing amounts of unlabeled, in
vitro translated growth factors to serum-starved NIH 3T3 cells and
measured the ability of the cells to incorporate
[3H]thymidine during 24 h of stimulation (Fig.
3A). The concentration of
unlabeled growth factor was for each mutant estimated from parallel
translation reactions containing [35S]methionine, which
were analyzed by SDS-PAGE and densitometric scanning. The data in Fig.
3A are corrected for differences in translation efficiency.
The different mutants varied strongly in their ability to stimulate DNA
synthesis. Wild-type aFGF and the S130E and S130A mutants were equally
potent in this matter. The K132A and the S130E/K132A mutants were both
approximately 10-fold less potent, while the K132R mutant was 3-fold
less potent than wild-type to stimulate DNA-synthesis. For comparison,
we also tested the K132E mutant, which was at least 100-fold less active than wild-type aFGF. aFGF(K132E) could not elicit a full mitogenic response within the limits of this assay using in
vitro translated growth factors. As control, addition of
translation mixture to which no mRNA had been added did not
stimulate [3H]thymidine incorporation at all (not
demonstrated).
To study the ability of the different mutants to support proliferation,
we incubated serum-starved NIH 3T3 cells for 48 h with different
concentrations of wild-type and mutant growth factors that were made
in vitro and then counted the cells in a cell counter (Fig.
3B). In this assay, one µl/ml of translation mixture gave optimal stimulation of proliferation for all constructs tested, and
increasing the concentration to 3 µl/ml did not increase the effect
reproducibly. Therefore, only results with the concentration of 1 µl/ml is shown in Fig. 3C. Wild-type aFGF and the S130E
mutant stimulated proliferation most effectively, while the S130A and the K132R mutants were somewhat less effective. The K132A and the
S130E/K132A mutants gave the weakest stimulation of proliferation. Together, these data indicate that phosphorylation of aFGF is not
required for stimulation of DNA synthesis. It appears that lysine 132 is important for the mitogenic effect of aFGF, independent of
phosphorylation of serine 130.
Stimulation of MAP Kinase by the Mutant Growth Factors--
Upon
binding of aFGF to the FGFR, the tyrosine kinase of the receptor is
activated and several signal transduction cascades are initiated
(33-35). Activation of the Ras/Raf/Mek/MAP kinase cascade has been
suggested to be important for stimulation of cell proliferation by FGF
(34, 36, 37). Therefore, we measured the ability of the different
mutants to stimulate MAP kinase. Serum-starved NIH 3T3 cells were
stimulated with different concentrations of in vitro
translated wild-type or mutant growth factor for 5, 30, or 60 min,
lysed, and MAP kinase (ERK1 and ERK2) was immunoprecipitated. MAP
kinase activity in the immunoprecipitates was measured in an in
vitro kinase assay with myelin basic protein as substrate.
There was virtually no MAP kinase activity immunoprecipitated from
cells treated with a translation mixture to which no mRNA had been
added (Fig. 4, lane 1). Ten
percent fetal calf serum (lane 2) and the higher
concentration used of wild-type aFGF (lane 4) stimulated MAP
kinase activity to a similar extent. The aFGF induced MAP kinase
activation was completely prevented when the cells were treated with
genistein, a tyrosine kinase inhibitor (lanes 15 and
16, upper panel). Importantly, no detectable
differences in MAP kinase activity could be measured when the cells
were stimulated with the different mutant or wild-type growth factors
(lanes 3-14). The apparent differences after 5-min
stimulation in the experiment shown were not reproducible between
experiments. Also the kinetics of MAP kinase activation was similar for
the different aFGF constructs (compare lanes 3-14 in
upper, middle, and lower panels).
Together, all the aFGF constructs tested stimulated MAP kinase
activation to a similar extent.
Transport of Mutant Growth Factors to the Nuclear Fraction--
We
and others have previously provided evidence suggesting a role of aFGF
in the nucleus for stimulation of DNA synthesis (8, 10, 14-18, 38).
Therefore, we examined the ability of the different aFGF mutants to be
transported to the nuclear fraction. [35S]Methionine-labeled, in vitro translated
wild-type and mutant growth factors were added to cells together with
heparin and incubated at 37 °C for different periods of time. The
cells were washed, treated with Pronase to digest growth factor at the
cell surface or bound to the plastic, then the cells were lysed and
fractionated into cytosol/membrane and nuclear fractions as described
under "Experimental Procedures." In Fig.
5 only the nuclear fractions are shown.
After 9-h incubation of the cells with growth factor, all mutants as
well as the wild-type aFGF were found in the nuclear fraction (Fig.
5A). Transport to the nuclear fraction could be completely
inhibited by the phosphoinositide 3-kinase inhibitor LY294002 (Fig.
5B), which we have found recently, is able to inhibit transport of externally added aFGF to the cytosol and nucleus of
cells.4 It was reported
recently that in retinal cells, phosphorylated aFGF was more stable
intracellularly than unphosphorylated aFGF (39). We also tested the
different mutants for presence in the nucleus after longer incubation
times, but no reproducible differences could be detected. However, in
the cytosol/membrane fraction a rather stable degradation product of
approximately 14 kDa was reproducibly obtained from cells incubated
with wild-type aFGF and with the S130E mutant, but not from cells
incubated with the other mutants (not demonstrated). The degradation
product was probably enclosed within a vesicular compartment, since it
was also obtained in the presence of LY294002. The degradation product was never obtained from the nuclear fraction.
Based on the ability to bind to wild-type aFGF, but not to the K132E
mutant, we recently cloned a novel intracellular protein, which we
designated FIBP (aFGF Intracellular
binding protein) (23). We tested the ability of
several of the aFGF mutants as MBP fusion proteins to bind to FIBP
in vitro (23). In contrast to the K132E mutant, all of the
other mutants that we tested in this way (S130E, K132A, S130E/K132A)
bound to FIBP, although somewhat less effectively than did the
wild-type (not demonstrated).
In this paper we report the generation and characterization of
several mutations in an exposed loop of aFGF, known to be of importance
for heparin affinity and mitogenic activity of the growth factor. The
mutants varied 10-fold in mitogenic activity, but were equally potent
in stimulating MAP kinase. A correlation, however, could be observed
between mitogenic activity and affinity to heparin.
High affinity receptor binding varied little among the constructs, with
the exception of the S130E mutant, which apparently bound with higher
affinity. Interestingly, bFGF phosphorylated by protein kinase A on
threonine 112 (corresponding to Thr120 according to the
numbering used in Ref. 1) was found to be three to eight times more
potent at displacing 125I-labeled FGF from the high
affinity receptors (24). This threonine is located in a part of bFGF
reported to be involved in both heparin binding and high affinity
receptor binding (40). Possibly, phosphorylation of aFGF may modulate
its binding to FGFR.
The Ras/Raf/Mek/MAP kinase cascade is considered to be important for
mitogenic activity of several growth factors, FGF included (41).
Microinjection of an antibody against FRS2, a tyrosine-phosphorylated protein that links FGFR activation to activation of the Ras/Raf/Mek/MAP kinase cascade, inhibited stimulation of DNA synthesis by aFGF (34). A
cell-permeable peptide derived from the Grb2-binding sequence of
epidermal growth factor receptor was shown to inhibit MAP kinase
activation in cells stimulated with epidermal growth factor or with
platelet-derived growth factor, but not in cells stimulated with bFGF.
The peptide also inhibited the mitogenic response of the cells to
epidermal growth factor and to platelet derived growth factor, but not
to bFGF (42). However, even though the different aFGF mutants described
in the present paper differed 10-fold in mitogenic activity, they were
equally potent in stimulation of MAP kinase. We have also found that
the K132E mutant, which exhibits at least a 100-fold lower mitogenic
activity than wild-type aFGF, shows a similar potency as the wild-type
to induce MAP kinase activity.2 It therefore appears that
activation of the Ras/Raf/Mek/MAP kinase cascade is not sufficient for
stimulation of DNA synthesis by aFGF.
We previously provided data suggesting that the strongly reduced
mitogenic activity of the K132E mutant is due to a defect in its
intracellular action (15). We also found that the K132E mutation
rendered the growth factor incapable of being phosphorylated in
vivo (15). One of the aims of the present study was to investigate whether there could be a cause-effect relationship between these observations. To use phosphorylated wild-type growth factor in experiments that last for 24 h or more, may not be optimal (24). Therefore, we took the strategy of mutating the serine in the phosphorylation site to the negatively charged glutamic acid or to the
uncharged alanine to mimic phosphorylated and unphosphorylated growth
factor, respectively. Similar strategies have previously been used
successfully with other proteins (29-32). However, we have no positive
control to tell us whether the S130E mutant really mimics
phosphorylated aFGF. Anyhow, we can conclude that inability of being
phosphorylated can not be the only reason for the mitogenic defect of
the K132E mutant. Rather, it appears that lysine 132 as such is
important, independent of phosphorylation.
Interaction of aFGF (5) and bFGF (43) with heparin or heparan sulfate
proteoglycans has been proposed to play a pivotal role in stimulation
of FGFR. In this study, we have found a correlation between mitogenic
potency and heparin affinity of the different mutants. The leading
hypothesis on how heparin or heparan sulfate proteoglycans exert their
effect is that these molecules bind several FGF molecules at the same
time as each FGF molecule binds to an FGFR molecule, thereby dimerizing
or oligomerizing FGFR molecules (5, 44, 45). If the reason for the
reduced mitogenic activity of the mutants found in this and previous
studies (15, 20) were reduced heparin affinity, a proportional
reduction in activation of FGFR and its downstream effectors should be
expected from this hypothesis. This is not the case. One possible
explanation could be that interaction with intracellular targets of
aFGF also depends on the ability of aFGF to interact with negatively
charged (parts of) molecules. In this context, it is interesting that glypican, a heparan sulfate proteoglycan, and biglycan, a chondroitin sulfate proteoglycan, have been found to contain functional nuclear localization sequences and to exist in the nucleus (46).
In conclusion, phosphorylation of aFGF is not required for its
mitogenic activity. Lysine 132 of aFGF is important both for mitogenic
effect and for heparin affinity. We cannot detect a correlation between
mitogenic activity and ability to activate MAP kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP) at 37 °C for 1 h.
The reaction mixtures were analyzed by SDS-PAGE and autoradiography
(15).
-32P]ATP and 20 µg of myelin basic protein.
SDS-PAGE sample buffer was added, and the samples were analyzed by
SDS-PAGE and autoradiography (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ability of different mutants of aFGF to be
phosphorylated by protein kinase C and to bind to heparin.
A, schematic overview of amino acids 130-133 of constructs
used. Amino acids differing from the wild-type are in bold,
and sequences that conform to a PKC consensus phosphorylation site are
underlined. B, constructs as indicated were
expressed as fusion protein with MBP, affinity-purified on amylose
resin, and cleaved with factor Xa between MBP and aFGF (lanes
1-4) or expressed alone and purified as described (15)
(lanes 5 and 6). The purified proteins were used
as substrates for in vitro kinase reactions containing
[ -32P]ATP and purified PKC and then analyzed by
SDS-PAGE. The Coomassie-stained gel is shown in upper panel
and autoradiography of the same gel in lower panel. C,
in vitro translated, [35S]methionine-labeled
mutant and wild-type growth factors were bound to heparin-Sepharose,
washed, and eluted with stepwise increasing salt concentrations. The
radioactivity in fractions of 1 ml was measured. For easier comparison,
the data are plotted as percent of the value for the peak fraction of
each construct. Essentially identical results were obtained in two to
five experiments.
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Fig. 2.
Ability of the different mutants to bind to
cells. A, calf pulmonary artery endothelial cells were
incubated with in vitro translated,
[35S]methionine-labeled mutant and wild-type growth
factors for 2 h at 4 °C in the presence of heparin and in the
absence or presence of excess unlabeled wild-type aFGF. The cells were
then washed, lysed, and the postnuclear supernatant was analyzed by
SDS-PAGE and fluorography. B, NIH 3T3 cells were incubated
with 125I-labeled wild-type aFGF (5 ng/ml) for 3 h at
4 °C in medium containing 10 units/ml heparin in the absence or
presence of increasing concentrations of aFGF constructs expressed and
purified as MBP fusion proteins. The cells were washed three times,
lysed in 0.1 M KOH, and the cell-bound radioactivity was
measured. The data shown are mean of three experiments ± S.D.
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Fig. 3.
Ability of the different mutants of aFGF to
stimulate DNA synthesis and cell proliferation. A,
serum-starved NIH 3T3 cells were stimulated with different amounts of
in vitro translated, unlabeled growth factors for 24 h
in the presence of heparin (10 units/ml) and
[3H]thymidine. The cell-associated, trichloroacetic
acid-precipitable radioactivity was then measured. The experiments in
A were repeated four to six times with similar results. The
data shown are from a representative experiment. B,
serum-starved NIH 3T3 cells were treated with 1 µl of in
vitro translated, unlabeled growth factors for 48 h in the
presence of heparin (10 units/ml), trypsinized, and counted in a cell
counter. The data are plotted as percent of the cell number in the
culture treated with wild-type growth factor. Data shown are mean
(±S.E.) of four experiments with two or three parallels in each.
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Fig. 4.
Ability of aFGF mutants to stimulate MAP
kinase activity. Serum-starved NIH 3T3 cells were stimulated with
1 and 5 µl of in vitro translated, unlabeled growth
factors (lanes 3-14), with 5 µl/ml of a translation
mixture without added mRNA (lane 1) or with 10% fetal
calf serum (lane 2) for 5 min (upper panel), 30 min (middle panel), or 60 min (lower panel). To
be able to compare the intensity of the bands after 5 min of
stimulation with the intensity of the bands after longer stimulation
times, in lanes 15 and 16, middle and
lower panels, the cells were treated for 5 min with in
vitro translated wild-type aFGF, the samples were processed as
described below and then run on the same gel as the other samples in
the same panel. In lanes 15 and 16, upper
panel, 50 µg/ml genistein was present during 15 min of
preincubation and 5 min of stimulation. In all cases heparin (5 units/ml) was present. The cells were lysed, and the postnuclear
supernatant was subjected to immunoprecipitation with anti-ERK1 and
anti-ERK2 antibodies and kinase activity in the immunoprecipitates was
measured with myelin basic protein as substrate for 15 min at 37 °C
in kinase buffer containing [ -32P]ATP. SDS-PAGE sample
buffer was added, and the samples were analyzed by SDS-PAGE and
autoradiography.
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Fig. 5.
Transport of the different mutants to the
nuclear fraction. A, serum-starved NIH 3T3 cells were
incubated at 37 °C for 9 h in the presence of 10 units/ml
heparin and 2 µl/ml [35S]methionine-labeled growth
factors, washed, and treated with 5 mg/ml Pronase at 37 °C for 5 min. The cells were washed in the presence of protease inhibitors,
lysed, and the nuclear fraction was collected by centrifugation. After
sonication, the trichloroacetic acid-precipitable material in the
nuclear fraction was analyzed by SDS-PAGE and fluorography.
B, serum-starved NIH 3T3 cells were treated with wild-type
aFGF as in A for 10 h or 28 h. In lanes
1 and 2, 50 µM LY294002 was present. The
cells were further processed and analyzed as in A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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The expert work with the cell cultures by Jorunn Jacobsen and the skillful technical assistance of Mette Sværen are gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by The Norwegian Cancer Society, Novo Nordisk Foundation, The Norwegian Research Council for Science and Humanities, Blix Legat, Rachel and Otto Kr. Bruun's Legat, and by The Jahre Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Fellow of The Norwegian Cancer Society.
2
O. Klingenberg, A. Wid
ocha, and S. Olsnes, our unpublished data.
3
O. Klingenberg, A. Wid
ocha, A. Rapak,
D. Khnykin, L. Citores, and S. Olsnes, submitted for publication.
4
O. Klingenberg, A. Wid
ocha, and S. Olsnes, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; FGFR, fibroblast growth factor receptor; MAP kinase, mitogen-activated protein kinase; PKC, protein kinase C; MBP, maltose-binding protein; FIBP, aFGF intracellular binding protein; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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