From the Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074 and the Center for the Biophysical Sciences, University of Maine, Orono, Maine 04469
Received for publication, April 3, 2001, and in revised form, May 4, 2001
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ABSTRACT |
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Fibroblast growth factor (FGF) 1 is known
to be released in response to stress conditions as a component of a
multiprotein aggregate containing the p40 extravescicular domain of p65
synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a
Cu2+-binding protein and Cu2+ is known to
induce its dimerization, we evaluated the capacity of recombinant FGF1,
p40 Syt1, and S100A13 to interact in a cell-free system and the role of
Cu2+ in this interaction. We report that FGF1, p40 Syt1,
and S100A13 are able to bind Cu2+ with similar affinity and
to interact in the presence of Cu2+ to form a multiprotein
aggregate which is resistant to low concentrations of SDS and sensitive
to reducing conditions and ultracentrifugation. The formation of this
aggregate in the presence of Cu2+ is dependent on the
presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1.
Interestingly, S100A13 is also able to interact in the presence of
Cu2+ with Cys-free FGF1 and this observation may account
for the ability of S100A13 to export Cys-free FGF1 in response to
stress. Lastly, tetrathiomolybdate, a Cu2+ chelator,
significantly represses in a dose-dependent manner the heat
shock-induced release of FGF1 and S100A13. These data suggest that
S100A13 may be involved in the assembly of the multiprotein aggregate
required for the release of FGF1 and that Cu2+ oxidation
may be an essential post-translational intracellular modifier of this process.
FGF11 and FGF2, the
prototype members of a family of heparin-binding growth factors
involved in the regulation of neurogenesis, mesoderm formation, and
angiogenesis (1, 2), function as extracellular mitogens for diverse
populations of target cells, yet they both lack the presence of a
classical signal peptide sequence that enables their secretion through
the endoplasmic reticulum-Golgi apparatus (1, 2). FGF1, but not
FGF2 (3), is released from NIH 3T3 cells in response to heat shock (4), hypoxia (5), and serum starvation (6) in a transcription- and
translation-dependent manner, as a biologically inactive
homodimer with decreased affinity for heparin (4). Recent studies have suggested that FGF1 may be released as a multiprotein aggregate containing FGF1 and the p40 extravesicular domain of p65 synaptotagmin (Syt)1 (7, 8). These studies are consistent with the observation that
FGF1 is present in neuronal tissue as a component of a non-covalent aggregate containing p40 Syt1 and S100A13 (9). It is also known that
(i) both p40 Syt1 and S100A13 are constitutively released at 37 °C
from NIH 3T3 (8, 10), (ii) the expression of FGF1 is able to repress
the constitutive release of S100A13 but not p40 Syt1 at 37 °C (8,
10), and (iii) FGF1 and S100A13 NIH 3T3 cell co-transfectants are able
to release FGF1 and S100A13 in response to temperature stress in a form
which alters the solubility of FGF1 at 100% (w/v) ammonium sulfate
(10). Moreover, S100A13 appears to play a role in the regulation of
FGF1 release since (i) a deletion mutant of S100A13, lacking the
carboxyl-terminal basic residue-rich domain, acts as a
dominant-negative repressor of FGF1 release, (ii) the expression of
S100A13 is able to revert the requirement for transcription and
translation in FGF1 release, and (iii) the expression of S100A13 is
able to induce the release of Cys-free FGF1, a mutant that is unable to
access the extracellular compartment in response to heat shock
(10).
The FGF prototypes have been previously characterized as
Cu2+-binding proteins and Cu2+ oxidation is
able to induce the formation of a FGF1 homodimer in a cell-free system
(11). In addition, Cu2+ is able to stimulate the migration
(12) and the proliferation (13) of endothelial cells in
vitro and D-penicillamine, a Cu2+
chelator, inhibits human endothelial cell proliferation in
vitro and angiogenesis in vivo (14). Recently, it has
also been shown that Cu2+ potentiates human angiogenin
binding to its endothelial cell receptor (15) and that SPARC, an
extracellular matrix-binding protein known to be involved in the
regulation of endothelial cell proliferation, is a source of
Cu2+-binding polypeptides that stimulate angiogenesis (16).
Furthermore, Cu2+ also appears to play a role in the
remodeling of the extracellular matrix since it is able to increase
fibronectin synthesis in vitro (12) and act as a cofactor
for proteins involved in extracellular matrix formation (17).
Because FGF1 is released in response to heat shock as a FGF1 homodimer
(4, 18) and Cu2+ oxidation is able to facilitate FGF1
homodimer formation (11), we examined the ability of the known members
of the FGF1 release pathway to associate in a cell-free system and
evaluated the Cu2+ dependence of this association. We
report that, like FGF1, the recombinant forms of p40 Syt1 and S100A13
are able to bind to Cu2+ and interact in the presence of
Cu2+ to form a S100A13-dependent multiprotein
aggregate. The ability of S100A13 to interact with FGF1 and p40 Syt1 is
independent of disulfide bond oxidation since S100A13 is devoid of
cysteines and is able to associate with Cys-free FGF1 in the presence
of Cu2+. Last, tetrathiomolybdate (TTM), a Cu2+
chelator, is able to inhibit the release of FGF1 and S100A13 in
response to temperature stress. These data suggest that S100A13 may
participate in the FGF1 release pathway by its ability to promote, in a
Cu2+-dependent manner, the assembly of a
multiprotein aggregate containing FGF1, p40 Syt1, and S100A13.
Evaluation of the Copper Affinity of Recombinant FGF1, S100A13,
and p40 Syt1--
The recombinant (r) forms of human FGF1, Cys-free
FGF1, Cys30 FGF1, S100A13, and p40 Syt1 were
obtained as previously described (7, 10, 11). The Cu2+
affinity of FGF1, S100A13, and p40 Syt1 was evaluated using the HiTrap
Chelating columns (Amersham Pharmacia Biotech) following the
manufacturer's instructions. Briefly, the column was washed with
deionized water, loaded with 0.1 M CuCl2, and
equilibrated with 20 mM sodium phosphate buffer, pH 7.2, containing 1 M NaCl. The individual recombinant proteins (2 µg) were adsorbed to the column, washed with 20 mM sodium
phosphate buffer, pH 7.2, containing 1 M NaCl and eluted
with increasing concentrations of imidazole (Sigma) in the
equilibration buffer. The eluted fractions were concentrated using
either Centricon 3 or Centricon 10 (Amicon Inc.), resolved by either
15% (w/v) or 10% (w/v) SDS-PAGE, transferred to nitrocellulose
membrane (Hybond C, Amersham Pharmacia Biotech), and immunoblotted with
either a rabbit anti-FGF1 (19), a rabbit anti-S100A13 (10), or a rabbit
anti-p40 Syt1 antibody (7). Specific bands were visualized by
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Evaluation of the Interactions among the Recombinant Forms of
FGF1, p40 Syt1, and S100A13--
The recombinant proteins were mixed
at the molar ratios described under "Results," lyophilized, and
resuspended in 50 µl of phosphate buffer, pH 7.2, containing 0.15 M NaCl (PBS) in the presence and absence of 1 mM CuCl2, unless otherwise indicated. Samples
were incubated for 30 min at 42 °C, unless otherwise indicated, diluted in either nonreducing SDS-PAGE loading buffer containing 0.1%
(w/v) SDS or in the conventional reducing SDS-PAGE loading buffer,
resolved by 15% (w/v) acrylamide SDS-PAGE and the electrophoretic behavior of the proteins visualized by Coomassie Brilliant Blue staining (11). Bands of interest were electroeluted using the Bio-Rad
Model 422 Electroeluter according to the instructions of the
manufacturer and the eluted proteins concentrated by Centricon 3, resolved by 15% (w/v) acrylamide SDS-PAGE, transferred to
nitrocellulose membranes, and immunoblotted using a rabbit anti-FGF1, a
rabbit anti-S100A13, or a rabbit anti-p40 Syt1 antibody. In some
experiments, after incubation at 42 °C in either the presence or
absence of CuCl2, proteins were centrifuged for 45 min at
10,000 × g and pellet and supernatant fractions
obtained. Pellet fractions were washed 3 times with PBS by
centrifugation (45 min at 10,000 × g), resolved by
15% (w/v) acrylamide SDS-PAGE, transferred to nitrocellulose
membranes, and immunoblotted with a rabbit anti-FGF1, a rabbit
anti-S100A13, or a rabbit anti-p40 Syt1 antibody. For the evaluation of
the interaction between S100A13 and either Cys30 FGF1 or
Cys-free FGF1, these recombinant proteins were mixed at the molar ratio
described under "Results," lyophilized, resuspended in 250 µl of
PBS in the presence and absence of 1 mM CuCl2,
incubated for 30 min at 42 °C, and centrifuged for 18 h at
280,000 × g. The pellet fractions were resolved by
15% (w/v) SDS-PAGE, transferred to nitrocellulose membranes and
immunoblotted with either a rabbit anti-FGF1 or a rabbit anti-S100A13
antibody. Recombinant human epidermal growth factor (EGF), ovalbumin,
bovine brain S100B, and recombinant human parathyroid hormone were
obtained from Sigma.
Exclusion Chromatography--
The recombinant proteins were
mixed, lyophilized, resuspended in PBS in the presence and absence of 1 mM CuCl2, incubated for 30 min at 42 °C, and
resolved by size-exclusion chromatography using a Biosep-Sec-S4000
column (Phenomenex).
Cell Culture, Temperature Stress, and Processing of Conditioned
Media--
Myc-S100A13 and FGF1 NIH 3T3 cell co-transfectants (10)
were grown in Dulbecco's modified Eagle's medium (Cellgro)
supplemented with 10% (v/v) bovine calf serum (HyClone), 1% (v/v)
antibiotic/antimycotic (Life Technologies, Inc.), 0.15 g/liter
hygromycin B (Life Technologies, Inc.), and 0.4 g/liter Geneticin
(G418, Life Technologies, Inc.) on human fibronectin (10 µg/cm2)-coated dishes and the heat shock was performed as
previously described (4, 7, 8). Briefly, NIH 3T3 cell co-transfectants were grown to 70-80% confluence and, prior to temperature stress, the
cells were washed with Dulbecco's modified Eagle's medium containing
5 units/ml heparin (Upjohn Co.). The heat shock was performed in
Dulbecco's modified Eagle's medium containing 5 units/ml heparin for
110 min at 42 °C and control populations were incubated for
110 min at 37 °C in Dulbecco's modified Eagle's medium containing 5 units/ml heparin. The effect of ammonium TTM on FGF1 and Myc-S100A13 release was evaluated after preincubation of cells at 37 °C for 18 h with 100, 200, and 500 µM TTM (Sigma), followed
by heat shock of the cells in presence of the same concentration of
TTM. For the analysis of FGF1 and Myc-S100A13 release, following heat
shock, the conditioned media were collected, filtered, treated with
0.1% (w/v) DTT (Sigma) for 2 h at 37 °C and adsorbed to a
heparin-Sepharose CL-6B column (Amersham Pharmacia Biotech), which was
pre-equilibrated with 50 mM Tris, pH 7.4, containing 10 mM EDTA (TEB). The adsorbed proteins were washed with TEB
and eluted with 2.5 ml of TEB containing 1.5 M NaCl. Eluted
proteins were concentrated using Centricon 10, resolved by 15% (w/v)
SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted
with an anti-Myc (clone 9E10) monoclonal antibody (Oncogene Research
Products) or an anti-FGF1 polyclonal antibody. Total cell lysates were
obtained by resuspending cell pellets for 20 min in 0.5 ml of cold
lysis buffer (20 mM Tris, pH 7.5 containing 300 mM sucrose, 60 mM KCl, 15 mM NaCl,
5% (v/v) glycerol, 2 mM EDTA, 1% (v/v) Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, 2 µg/ml leupeptin, and 0.2% (w/v) deoxycholate),
centrifuged at 2,000 × g for 10 min and the
supernatant fraction evaluated by immunoblot analysis for either FGF1
or Myc-S100A13. The activity of lactate dehydrogenase (Sigma) in
conditioned media was utilized as an assessment of cell lysis as
previously reported (20).
FGF1, p40 Syt1, and S100A13 Are Able to Bind Copper with Similar
Affinity--
It is well established that FGF1 is able to bind
Cu2+ and Cu2+ is able to induce FGF1 homodimer
formation in vitro (11). Furthermore, a few members of the
S100 gene family have been characterized as Cu2+-binding
proteins (21-23) and S100B has been described as an inhibitor of
Cu2+-mediated L-ascorbate oxidation (22). Since
S100A13 and the p40 extravesicular domain of p65 Syt1 appear to
associate with FGF1 under stress conditions in vitro and
S100A13 is involved in the stress-induced release of FGF1 (7, 10), we
evaluated the affinity of FGF1, S100A13, and p40 Syt1 for
Cu2+. As shown in Fig. 1, we
observed that the recombinant forms of S100A13, p40 Syt1, and FGF1 are
able to bind a chelating affinity column that has been previously
loaded with CuCl2 and are eluted at 40 mM
imidazole. These data suggest that S100A13, p40 Syt1, and FGF1 are able
to bind Cu2+ with similar affinities.
Copper Mediates the Interaction among FGF1, p40 Syt1, and S100A13
in a Cell-free System--
Since our data demonstrated that FGF1, p40
Syt1, and S100A13 are Cu2+-binding proteins and that
Cu2+ is able to induce in vitro the dimerization
of FGF1 (11), we questioned whether Cu2+ was able to
mediate an interaction among the three polypeptides. Thus, the
recombinant forms of S100A13, FGF1, and p40 Syt1 were mixed at an
equimolar ratio, lyophilized, resuspended in PBS in the absence and
presence of increasing concentrations of Cu2+, incubated
for 30 min at 42 °C and resolved by nonreducing SDS-PAGE in the
presence of a low concentration, 0.1% (w/v), of SDS. In the absence of
Cu2+, Coomassie staining revealed the presence of three
major bands corresponding to the molecular masses of FGF1,
S100A13, and p40 Syt1 and a 120-kDa band that was also present in the
lane containing only p40 Syt1. The incubation of the three proteins
with varied levels of Cu2+ did not modify this pattern
(Fig. 2A). When the three
polypeptides were incubated in the presence of 10 and 100 µM CuCl2, we observed a significant decrease
in the intensity of the monomeric FGF1 band and the appearance of (i) a
band at the top of the stacking gel, (ii) a triplet with an apparent
molecular mass between 55 and 65 kDa, and (iii) an increase in the
intensity of the 120-kDa band (Fig. 2A). In addition,
another band at the interface of the stacking and resolving gel was
noted but its presence was variable in its appearance. Interestingly,
however, the appearance of the band at the top of the stacking gel was
significantly reduced as a result of analyzing these samples using
conventional SDS-PAGE in the presence of 2% (w/v) SDS and under
nonreducing conditions (data not shown).
The Copper-mediated Interaction among FGF1, p40 Syt1, and S100A13
Is S100A13-dependent--
In order to characterize the
bands with novel electrophoretic mobility obtained in the presence of
Cu2+, the recombinant forms of FGF1, S100A13, and p40 Syt1
were resuspended in PBS separately or in different combinations at
equimolar ratios and incubated in the absence and presence of
Cu2+. As shown in Fig. 2B, the addition of
Cu2+ to recombinant FGF1 produced the appearance of a
doublet with an apparent molecular masses of 34 kDa, corresponding to
the FGF1 homodimer (11), whereas the addition of Cu2+ to
p40 Syt1 enhanced the appearance of the 120-kDa band and the addition
of Cu2+ to S100A13 did not yield the formation of any
additional bands. No additional bands were also observed when
Cu2+ was added to the equimolar combination of S100A13 and
p40 Syt1. In contrast, the addition of Cu2+ to S100A13 and
FGF1 enabled the appearance of the band at the top of the stacking gel
and a significant decrease in the presence of the FGF1 doublet (Fig.
2B). The addition of Cu2+ to the equimolar
combination of FGF1 and p40 Syt1 induced the appearance of the triplets
between 55 and 65 kDa and the FGF1 doublet (Fig. 2B). In
contrast, the addition of Cu2+ to the equimolar combination
of all three recombinant proteins further increased the intensity of
the band near the top of the stacking gel (4% (w/v) acrylamide) while
decreasing the presence of the 55-65 kDa triplet and FGF1 doublet
(Fig. 2B). The formation of these novel electrophoretic
products in the presence of Cu2+ was not significantly
modified by incubating the recombinant proteins either at different
temperatures (25-50 °C) or in the presence of 20 mM
Tris buffer, pH 7.4 (data not shown). While a 5-fold increase in the
molar ratio between S100A13 and any of the other two recombinant
proteins significantly enhanced the intensity of the band near the top
of the stacking gel, no significant difference was observed in the
intensity of the band near the top of the stacking gel by increasing
the molar ratio between either FGF1 or p40 Syt1 and any of the other
two proteins (data not shown). These data suggest that, in the presence
of Cu2+, (i) FGF1 is able to dimerize as previously
reported (11) and to associate with S100A13 forming an aggregate near
the top of the stacking gel, (ii) the addition of p40 Syt1 enhances the
intensity of the band near the top of the stacking gel, and (iii) the
formation of this multiprotein aggregate is dependent upon the presence of Cu2+, S100A13, and FGF1. FGF1 and p40 Syt1 also appear
to interact in the presence of Cu2+ and form a triplet with
the apparent molecular masses between 55 and 65 kDa. Furthermore, while
increasing the molar ratio of S100A13 in this
Cu2+-dependent system does result in the
potentiation of the presence of the band near the top of stacking gel
(data not shown), it is likely that the level of S100A13 that is able
to enter the multiprotein aggregate is limited.
In order to further characterize the aggregates formed by FGF1,
S100A13, and p40 Syt1 in the presence of Cu2+, the area of
the stacking gel corresponding to the multiprotein aggregate was
electroeluted, further resolved by conventional reducing SDS-PAGE, and
analyzed by FGF1, p40 Syt1, and S100A13 immunoblot analysis. As shown
in Fig. 2C, the area corresponding to the band near the top
of the stacking gel revealed the presence of FGF1, p40 Syt1, and
S100A13 only from those electroelution samples where the three
polypeptides were preincubated with Cu2+. Immunoblot
analysis of the post-electrophoresis glycerol sample fraction also
revealed the presence of FGF1 and p40 Syt1, suggesting that a portion
of the multiprotein aggregate was not able to enter the gel at all
(data not shown). The area corresponding to the 55-65-kDa triplet
demonstrated only the presence of p40 Syt1 in the absence of
Cu2+ and, following incubation with Cu2+,
resulted in a significant increase of both p40 Syt1 and FGF1 (Fig.
2C). Moreover, FGF1, p40 Syt1, and S100A13 immunoblot
analysis of the 120-kDa band after electroelution exhibited the
presence of only p40 Syt1 (data not shown). These data suggest that the Cu2+-induced multiprotein aggregate band resolved near the
top of the stacking gel contained FGF1, p40 Syt1, and S100A13 and that the appearance of FGF1 and p40 Syt1 in this band requires the presence
of S100A13. In contrast, it is likely that the appearance of the 55-65
kDa triplet may be the result of the formation of a FGF1:p40 Syt1
heterodimer, since FGF1 contains three cysteine residues (18), while
p40 Syt1 contains only a single cysteine residue (24). Furthermore, gel
exclusion chromatography of the Cu2+-induced multiprotein
aggregate revealed an apparent minimum molecular mass of ~4 × 103 kDa, although the majority of the multiprotein
aggregate was unable to penetrate the gel exclusion column (data not shown).
The Copper-dependent Interaction among FGF1, S100A13,
and p40 Syt1 Is Specific for These Three Proteins and Sensitive to
Reducing Agents--
In order to characterize the specificity of the
interaction among FGF1, S100A13, and p40 Syt1, we substituted
recombinant human EGF for FGF1, ovalbumin for p40 Syt1, and
either S100B or recombinant human parathyroid hormone for S100A13 in
the Cu2+ oxidation reaction. As shown in Fig.
3A, the substitution of FGF1
with EGF resulted in the disappearance of the 55-65-kDa triplet, as
well as the band resolved on the top of the stacking gel and the FGF1
doublet. When p40 Syt1 was substituted with ovalbumin, the
disappearance of the 55-65-kDa triplet was observed (Fig. 3A). However, the band resolved at the top of the stacking
gel was present at a reduced level, and this is consistent with the observation that FGF1 and S100A13 are able to form this band in a
Cu2+-dependent manner at a reduced level (Fig.
2B). When S100A13 was substituted with either S100B or
parathyroid hormone, the formation of the band near the top of the
stacking gel was significantly reduced. The substitution of FGF1 with
FGF2, a FGF prototype which is able to bind Cu2+ but does
not form homodimers in response to Cu2+ oxidation (11), in
the presence of p40 Syt1, S100A13, and 1 mM
CuCl2 also resulted in the disappearance of the band near
the top of the stacking gel (data not shown). These results suggest that the formation of the multiprotein aggregate resolved near the top
of the stacking gel requires the presence of FGF1, S100A13, and p40
Syt1 as individual components as well as the presence of
Cu2+. In addition, we did not observe the formation of the
multiprotein aggregate as well as the FGF1 doublet and the FGF1:p40
Syt1 triplet when FGF1, p40 Syt1, and S100A13 were incubated in the
presence of 1 mM ZnCl2, FeCl2,
CaCl2, and CdCl2 (data not shown).
In order to determine whether the formation of the multiprotein FGF1-,
p40 Syt1-, and S100A13-containing aggregate resolved near the top of
the stacking gel required the Cu2+ oxidation of disulfide
bonds, the recombinant forms of FGF1, p40 Syt1, and S100A13 were
subjected to Cu2+ oxidation and resolved by conventional
reducing SDS-PAGE. As shown in Fig. 3B, we observed only the
presence of the monomeric forms of these three polypeptides suggesting
that formation of disulfide bonds is important in the aggregation of
the multiprotein complex. In addition, Cu2+ oxidation of
Cys-free FGF1 in the presence of p40 Syt1 and S100A13 resulted in the
absence of both the multiprotein aggregate resolved at the top of the
stacking gel and the FGF1:p40 Syt1 heterodimer band, and
Cys30 FGF1, like the Cys-free FGF1, was also unable to form
both of these bands (Fig. 3C). This experiment (Fig.
3C) was performed at a molar ratio of S100A13:Cys-free
FGF/Cys30 FGF1:p40 Syt1 of 5:1/1:1 since we noted that
increasing the molar ratio of S100A13 to either wild type FGF1 or p40
Syt1 potentiated the appearance of the band resolved near the top of
the stacking gel (data not shown). However, unlike Cys-free FGF1,
Cys30 FGF1 is able to form a single Cys30
FGF1:p40 Syt1 band (Fig. 3C). Indeed, using the S100A13 Is Able to Interact with FGF1 and p40 Syt1 in the Presence
of Copper--
Because our data suggested that FGF1, p40 Syt1, and
S100A13 are able to interact with each other in the presence of
Cu2+ to form a multiprotein aggregate, we questioned
whether we could utilize centrifugation to further analyze this
association. Thus, FGF1, p40 Syt1, and S100A13 were individually
incubated for 30 min at 42 °C in PBS containing Cu2+,
centrifuged for 45 min at 10,000 × g, and pellet and
supernatant fractions obtained. The pellet fractions were washed 3 times with PBS by centrifugation (45 min at 10,000 × g), resolved by conventional 15% (w/v) acrylamide, reduced
SDS-PAGE, and analyzed by FGF1, p40 Syt1, and S100A13 immunoblot
analysis. As shown in Fig. 4A, both FGF1 and p40 Syt1 were present only in the pellet fractions incubated in the presence of Cu2+. However, S100A13 was not
precipitable either in the presence or absence of Cu2+
(Fig. 4A) and was resolved in the supernatant fractions
(data not shown). Interestingly, the ability of either FGF1 or p40 Syt1 to form a precipitable aggregate when incubated in the presence of
Cu2+ was not altered by the incubation of these proteins in
the presence of 0.1% (w/v) DTT (data not shown). In contrast, however,
the incubation of S100A13 with FGF1 and p40 Syt1 at the molar ratio of
10:1:1 in the presence of Cu2+ resulted in the presence of
S100A13 in the pellet fraction (Fig. 4A). These data suggest
that Cu2+ oxidation is able to precipitate FGF1 and p40
Syt1 but not S100A13 in their monomeric forms and S100A13 is
susceptible to the Cu2+-dependent precipitation
when incubated in the presence of FGF1 and p40 Syt1. The ability of
Cu2+ to precipitate FGF1 and p40 Syt1 as individual
proteins and FGF1, p40 Syt1, and S100A13 as an aggregate was not
modified by incubation of the three recombinant proteins with
Cu2+ at different temperatures (25 and 42 °C) (data not
shown).
Because these results agree with our previous observation that FGF1,
p40 Syt1, and S100A13 are able to form a
Cu2+-dependent multiprotein aggregate, we
evaluated the ability of either FGF1 or p40 Syt1 to interact with
S100A13. Increasing amounts of S100A13 were incubated with either FGF1
or p40 Syt1 in the presence of Cu2+ at 42 °C for 30 min,
centrifuged at 10,000 × g for 45 min, pellet and
supernatant fractions obtained and analyzed by S100A13 immunoblot analysis. As shown in Fig. 4, B and C, S100A13
was resolved in the pellet fraction when incubated with either FGF1 or
p40 Syt1 in the presence of Cu2+. Moreover, increasing the
molar ratio between S100A13 and either p40 Syt1 or FGF1 resulted in an
increase in the level of S100A13 associated with the pellet fractions
(Fig. 4, B and C). In contrast, S100A13 was not
resolved in the pellet fraction when incubated in the presence of
Cu2+ with either EGF or ovalbumin (Fig. 4, B and
C). These data suggest that S100A13 is able to interact with
either FGF1 or p40 Syt1 in a Cu2+-dependent
manner and these interactions may be involved in the formation of the
FGF1, p40 Syt1, and S100A13 multiprotein aggregate.
S100A13 Is Able to Interact with Cys-free FGF1 in a
Copper-dependent Manner--
Because (i) S100A13 is able
to interact with FGF1 in a Cu2+-dependent
manner, (ii) Cu2+ is able to induce the formation of a
DTT-sensitive FGF1 aggregate, (iii) the expression of S100A13 is able
to induce the release of Cys-free FGF1 in response to heat shock (10),
and (iv) FGF1 is released as a Cys30 homodimer in response
to temperature stress (26), we assessed whether S100A13 and Cys-free
FGF1 were able to associate in a Cu2+-dependent
manner since this could explain the S100A13-dependent export of Cys-free FGF1 in response to heat shock (10). Thus, recombinant S100A13 was incubated for 30 min at 42 °C in the
presence and absence of Cu2+ with either Cys30
FGF1 or Cys-free FGF1 at the molar ratio of 10:1, centrifuged for
18 h at 280,000 × g, pellet and supernatant
fractions obtained, the pellet fractions resolved by 15% (w/v)
acrylamide SDS-PAGE and subjected to FGF1 and S100A13 immunoblot
analysis. We used ultracentrifugation because (i) analysis by SDS-PAGE
did not reveal the formation of the multiprotein aggregate resolved at
the top of the stacking gel in the presence of either Cys-free FGF1 or Cys30 FGF1 (Fig. 3C) and (ii) low centrifugal forces used to
pellet the Cu2+-dependent FGF1:p40 Syt1:S100A13
aggregate were unable to pellet the Cys-free form of FGF1. We observed
that following ultracentrifugation, Cys30 and Cys-free FGF1
were present in pellet fractions in the absence of either S100A13 or
Cu2+ (Fig. 5, A
and B). However, the presence of S100A13 in the pellet fraction was dependent upon the addition of either Cys30
FGF1 or Cys-free FGF1 as well as the presence of Cu2+ (Fig.
5, A and B). Similar results were also obtained
at equimolar ratios and molar ratios of 5:1 between S100A13 and either
Cys-free FGF1 or Cys30 FGF1 (data not shown). Furthermore,
varying the molar ratio between S100A13 and Cys-free FGF1 revealed that
this interaction is dependent upon the concentration of S100A13 and
occurs maximally at a molar ratio of 5:1 (Fig. 5C). These
data suggest that the expression of S100A13 in a Cys-free FGF1
background in NIH 3T3 cells may be able to induce the release of
Cys-free FGF1 in response to temperature stress (10) as a result of the
ability of S100A13 to associate with Cys-free FGF1.
The Copper Chelator, Tetrathiomolybdate, Is Able to Inhibit the
Release of FGF1 and S100A13 in Response to Temperature Stress--
Our
results suggest that, in a cell-free system, FGF1, S100A13, and p40
Syt1 are able to interact in the presence of Cu2+ and form
a multiprotein aggregate. Because our data also suggest that
Cu2+ oxidation may mediate the intracellular interactions
that occur in vitro among FGF1, p40 Syt1, and S100A13 during
temperature stress, we examined whether the Cu2+ chelator,
TTM, could repress the export of FGF1 and S100A13 in response to heat
shock. TTM is used in the treatment of Wilson's disease (27), a
genetic condition characterized by a reduction in the release of
Cu2+ from cells as well as a reduction in the incorporation
of Cu2+ into ceruloplasmin (28). In addition, TTM is being
used in clinical trials for the treatment of several human tumors as a potential anti-angiogenic agent (29, 30). Thus, Myc-S100A13 and FGF1
NIH 3T3 cell co-transfectants were incubated for 18 h in the
presence and absence of 100, 200, and 500 µM TTM and
subjected to heat shock. Conditioned media were treated with DTT,
adsorbed to heparin-Sepharose, eluted with 1.5 M NaCl,
resolved by conventional 15% (w/v) acrylamide SDS-PAGE, and analyzed
by Myc and FGF1 immunoblot analysis. As shown in Fig.
6, pretreatment with TTM was able to significantly inhibit in a dose-dependent manner the
release of both FGF1 and Myc-S100A13 in response to temperature stress.
In contrast, FGF1 and Myc immunoblot analysis of cell lysates suggested that TTM was unable to influence the intracellular cytosolic levels of
either FGF1 or Myc-S100A13 (data not shown). Indeed, these results
suggest that intracellular Cu2+ oxidation play a role in
the stress-mediated release of FGF1.
FGF1 has been purified from the ovine brain as part of a
multiprotein noncovalent aggregate containing p40 Syt1 and S100A13 (5).
This observation correlates with our in vitro results showing that, in NIH 3T3 cells, FGF1 is released in response to temperature stress as an aggregate containing p40 Syt1 and the electrophoretic mobility of the FGF1 and p40 Syt1 aggregate was sensitive to reducing and to denaturating reagents (7). FGF1 and
S100A13 are also able to associate in conditioned media from temperature-stressed NIH 3T3 cells, since the expression of Myc-S100A13 is able to increase the solubility of extracellular FGF1 at 100% (w/v)
ammonium sulfate saturation (10). Furthermore, a deletion mutant of p40
Syt1, lacking the C2A domain (8), and a deletion mutant of S100A13,
lacking the carboxyl-terminal basic residue-rich domain (10), are both
able to repress the temperature-dependent release of FGF1,
suggesting a functional role for p40 Syt1 and S100A13 in the FGF1
release pathway.
The data presented here suggest that S100A13 is able to interact with
both FGF1 and p40 Syt1 in a cell free-system in a
Cu2+-dependent manner and the simultaneous
incubation of FGF1, p40 Syt1, and S100A13 in the presence of
Cu2+ results in the formation of a multiprotein aggregate
containing FGF1, p40 Syt1, and S100A13. The electrophoretic mobility of
this multiprotein aggregate is resistant to low concentrations of SDS, but is sensitive to reducing conditions. These interactions appear to
be specific for each of the three proteins. The formation of this
multiprotein aggregate (i) is dependent on the presence of S100A13,
(ii) involves the formation of disulfide bonds between FGF1 and p40
Syt1 but not S100A13, and (iii) is dependent upon the presence of
Cu2+. These results are consistent with the ability of
Cu2+ to induce the homodimer formation of FGF1 (11) and
with the observation that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity.
The observation that FGF1, p40 Syt1, and S100A13 interact, in the
presence of Cu2+, to form a multiprotein aggregate may
provide some insights into the potential role of S100A13 in the release
of FGF1 in response to stress. Indeed, previous results from our
laboratory have suggested that FGF1 and S100A13 are able to interact
in vitro since FGF1 expression is able to inhibit the
constitutive release of the Myc-S100A13 chimera at 37 °C (10) and
Myc-S100A13 expression is able to induce the release of Cys-free FGF1
in response to heat shock (10). In contrast, FGF1 expression is not
able to repress the constitutive release of p40 Syt1 (8) and p40 Syt1 is not able to facilitate the release of Cys-free
FGF1.2 Since S100A13 is a
Cys-free protein, we suggest that the role of Cu2+
oxidation in the interaction between S100A13 and FGF1, as well as
between S100A13 and p40 Syt1, may not involve the oxidation of cysteine
residues between S100A13 and either FGF1 or p40 Syt1. This suggestion
is supported by the observation that S100A13 is able to interact with
Cys-free FGF1 in a Cu2+-dependent manner.
Moreover, the requirement for Cu2+ oxidation for the
interaction between S100A13 and FGF1, as well as for the formation of
p40 Syt1, FGF1, and S100A13 multiprotein aggregate is also supported by
the observation that the incubation of S100A13 with dimeric forms of
FGF1 in the absence of Cu2+ does not result in the
formation of a multiprotein aggregate (data not shown). Furthermore,
the presence of multiple Cys residues in FGF1 may also account for the
stability of the multiprotein aggregate at low concentrations of SDS
which would explain why ultracentrifugation was required to pellet the
Cys-free and Cys30 forms of FGF1 in the presence of S100A13
and Cu2+ and the failure to observe the multiprotein
aggregate under the conditions of limited SDS-PAGE (Figs. 2 and 3).
Several studies have demonstrated that S100 proteins are able to form
stable homodimers in the presence or absence of Ca2+, thus
enabling the carboxyl-terminal domain of the polypeptide to interact
with other proteins (21, 31). It is also well established that, due to
a conformational change induced by Ca2+-mediated S100
protein dimer formation, S100A11 and annexin (Anx) 1 (31, 32) and
S100A10 and Anx2 (33, 34) are able to form stable heterotetramers
(S100A112:Anx12 and
S100A102:Anx22). We suggest that in the case of
FGF1, p40 Syt1, and S100A13, the presence of Cu2+, not
Ca2+, may facilitate the interaction between either S100A13
and FGF1 or between S100A13 and p40 Syt1 to form a putative
S100A132:FGF12 or S100A132:p40
Syt12 heterotetramer. The
Cu2+-dependent formation of disulfide bonds
among cysteine residues from the monomer forms of FGF1 and p40 Syt1 may
also induce the formation of the multiprotein aggregate by the
disulfide linked cross-bridging of several of the putative
S100A132:FGF12 and S100A132:p40 Syt12 heterotetramers. This hypothesis is supported by the
observation that Cys-free FGF1 is not able to form the
Cu2+-dependent multiprotein aggregate in the
presence of S100A13 and p40 Syt1 but is able to interact with S100A13
in a Cu2+-dependent manner.
This premise also correlates with our previous observation that a
deletion mutant of S100A13, lacking the carboxyl-terminal basic
residue-rich domain, acts as dominant negative repressor of the
temperature-dependent release of FGF1 (10). Since the basic
residue-rich domain is a feature specific for S100A13 as a S100 gene
family member, we suggest that this domain may be important for the
interaction between either S100A13 and FGF1 or between S100A13 and p40 Syt1.
Our data also suggest that FGF1 and p40 Syt1 may be able to interact in
response to Cu2+ oxidation and that this interaction may be
mediated by the formation of a FGF1:p40 Syt1 heterodimer triplet and
this is consistent with the observation that the Cys-free FGF1 is not
able to form a Cys-free FGF1:p40 Syt1 heterodimer. Indeed, p40 Syt1
contains a single Cys residue which is located within the carboxyl
terminus of Syt1 (24) and this structural feature suggests that the C2A and the C2B domains of p40 Syt1 may be involved in binding
Cu2+ in a manner which may facilitate the formation of the
FGF1:p40 Syt1 heterodimer. The observation that Cu2+
facilitates the oxidation of a p40 Syt1:FGF1 heterodimer, as well as
FGF1 homodimer formation but does not facilitate the formation of a p40
Syt1 homodimer, is consistent with this premise since the oxidation of
the single Cys residue in p40 Syt1 requires the presence of FGF1.
We do not know whether the capacity of FGF1, p40 Syt1, and S100A13 to
interact in the presence of Cu2+ in a cell-free system
reproduces the physiological interactions that occur in
vitro under stress conditions which may enable a similar
multiprotein aggregate containing FGF1, p40 Syt1, and S100A13 to be
released (7, 10). However, the capacity of S100A13 to interact with
Cys-free FGF1 in a cell-free system may account for the ability of
Myc-S100A13 to export, in response to stress, Cys-free FGF1 (10), that
is normally not able to be released (18) and for the ability of
Cys-free FGF1 to repress the constitutive release of S100A13 at
37 °C (10). Indeed, our data suggest that S100A13 may be able to
form a putative S100A132:Cys-free FGF12
heterotetramer and to force Cys-free FGF1 to enter the release pathway
in response to temperature stress in the absence of FGF1 disulfide bond
formation. In contrast, p40 Syt1 may be able to interact with FGF1 only
through formation of a disulfide bond and, may explain why the
expression of either p65 Syt1 (8) or p40 Syt12 is unable to
induce the release of the Cys-free FGF1 from NIH 3T3 cells in response
to heat shock. Similarly, FGF1 is able to repress the constitutive
release of Myc-S100A13 (10), but not the constitutive release of p40
Syt1 (8), suggesting that the interaction between FGF1 and p40 Syt1,
but not the interactions between either S100A13 and FGF1 or between
S100A13 and p40 Syt1, may require oxidative events.
These cell-free results also support the premise that S100A13 may play
an important role in the formation of the FGF1 homodimer by its ability
to assemble a multiprotein aggregate, thus facilitating the release of
FGF1. Indeed, the expression of S100A13 is not only able to induce the
release of Cys-free FGF1 (10), but the expression of S100A13 is able to
overcome the transcriptional and translational requirement for the
release of FGF1 (10). Thus, we suggest that requirement for cellular
stress may not involve the transcriptional and translational activation
of either a known or novel stress-induced gene; rather, the requirement for cellular stress may involve the activation of post-translational mechanisms of intracellular redox pathways that may be involved in the
cytosolic regulation and maintenance of a stress-induced oxidative
state (35).
We have previously described that the Cys30 is necessary
for the formation of FGF1 homodimer in vitro and for the
release of FGF1 in response to stress conditions (26). Moreover,
crystallographic studies of FGF1 have shown that the Cys30
is normally not exposed to the solvent (36), suggesting that FGF1 may
undergo conformational changes before being released. We suggest that
under stress conditions the Cu2+-dependent
interaction between FGF1 and S100A13 may be requisite for the unfolding
of FGF1 and the reorientation of Cys30 in order to form the
FGF1 homodimer which is required for its release in response to stress
(18, 26). The capacity of S100A13 to interact with p40 Syt1 may also
account for the presence of p40 Syt1 in the multiprotein aggregate and
further supports the premise that S100A13 and p40 Syt1 may play a
functional role in the formation of this multiprotein aggregate based
upon their ability to associate with phosphatidylserine (37, 38).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The ability of FGF1, p40 Syt1, and S100A13 to
bind copper. Recombinant (r) human forms of FGF1, p40 Syt1, and
S100A13 were obtained as previously reported (7, 10, 11), and 2 µg of
rp40 Syt1 (A), rFGF1 (B), and rS100A13
(C) were independently adsorbed to HiTrap chelating columns
previous loaded with 0.1 M CuCl2. The column
was washed with 20 mM sodium phosphate buffer, containing 1 M NaCl, pH 7.2, and eluted with imidazole as indicated. The
eluted fractions were concentrated by either Centricon 3 or Centricon
10, resolved by either 10 (w/v) (A) or 15% (w/v)
(B and C) SDS-PAGE, and analyzed by Syt1
(A), FGF1 (B), and S100A13 (C)
immunoblot analysis. Recombinant protein standards and the flow-through
fraction are also shown for each immunoblot.
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Fig. 2.
The ability of FGF1, p40 Syt1, and S100A13 to
interact in the presence of copper. A, the copper
dependence of FGF1, p40 Syt1, and S100A13 interactions. The recombinant
human forms of FGF1 (10 µg), p40 Syt1 (20 µg), and S100A13 (5 µg)
were resuspended in 50 µl of PBS in the absence or presence of
various concentrations of CuCl2, incubated for 30 min at
42 °C, diluted in nonreducing SDS-PAGE loading buffer containing
0.1% (w/v) SDS, and resolved by 15% (w/v) SDS-PAGE. Proteins were
visualized by Coomassie Brilliant Blue staining and the electrophoretic
mobility of the recombinant proteins are shown. Arrows
indicate the position of the multiprotein aggregate (top),
the 120-kDa band and the 55-65-kDa triplet band. B, the
S100A13 dependence of the interaction among FGF1, p40 Syt1, and
S100A13. The recombinant forms of FGF1 (10 µg), p40 Syt1 (20 µg),
and S100A13 (5 µg) were resuspended in PBS as either individual
samples or in different combinations and incubated for 30 min at
42 °C in the absence or presence of 1 mM
CuCl2, diluted in nonreducing SDS-PAGE loading buffer
containing 0.1% (w/v) SDS, and resolved by 15% (w/v) SDS-PAGE.
Proteins were visualized by Coomassie Brilliant Blue staining.
Arrows indicate the position of the multiprotein aggregate,
the 120-kDa band, the 55-65-kDa triplet band, and the 34-kDa FGF1
homodimer bands. C, electroelution of recombinant proteins
from the multiprotein aggregate and the 55-65-kDa triplet
band. After Coomassie staining, the proteins present in the
areas of the gel corresponding to the multiprotein aggregate band at
the top of the stacking gel and the 55-65-kDa triplet band were
electroeluted from the negative Cu2+ control
( Cu2+) and Cu2+-containing lanes
(+Cu2+), resuspended in reducing SDS-PAGE loading buffer,
boiled, resolved by 15% (w/v) SDS-PAGE, and analyzed by p40 Syt1
(upper panel), FGF1 (middle panel), and S100A13
(lower panel) immunoblot analysis.
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Fig. 3.
The specificity (A) and the
sensitivity to reducing conditions (B) of the
interaction among FGF1, p40 Syt1, and S100A13. A, 10 µg of either rFGF1 or recombinant EGF, 20 µg of either rp40 Syt1 or
ovalbumin (ova), and 5 µg of either rS100A13 or
recombinant parathyroid hormone (PTH) or S100B were mixed in
different combinations, lyophilized, resuspended in PBS, incubated for
30 min at 42 °C in the presence of 1 mM
CuCl2, diluted in nonreducing SDS-PAGE loading buffer
containing 0.1% (w/v) SDS, and resolved by 15% (w/v) SDS-PAGE.
Proteins were visualized by Coomassie Brilliant Blue staining.
Arrows indicate the position of the multiprotein aggregate
(top), the 120-kDa band and the 55-65-kDa triplet band.
B, 10 µg of rFGF1, 20 µg of rp40 Syt1, and 5 µg of
rS100A13 were mixed, lyophilized, resuspended in PBS, incubated for 30 min at 42 °C in the presence and absence of 1 mM
CuCl2, diluted with either nonreducing SDS-PAGE loading
buffer ( 2-ME (mercaptoethanol)) containing 0.1% (w/v) SDS or
with conventional reducing SDS-PAGE loading buffer (+2ME),
and resolved by 15% (w/v) SDS-PAGE. Proteins were visualized by
Coomassie Brilliant Blue staining. Arrows indicate the
position of the multiprotein aggregate, the 120-kDa band, and the
55-65-kDa triplet band. C, the inability of Cys-free
and Cys30 FGF1 to form the multiprotein aggregate in
presence of copper. The recombinant forms of S100A13 (25 µg) and p40
Syt1 (20 µg) were mixed with wild type FGF1, Cys-free FGF1, or
Cys30 FGF1 (10 µg) at a molar ratio of 5:1:1,
respectively, in the absence and presence of 1 mM
CuCl2, lyophilized, resuspended in PBS, incubated for 30 min at 42 °C in the presence and absence of 1 mM
CuCl2, diluted with nonreducing SDS-PAGE loading buffer
containing 0.1% (w/v) SDS, and resolved by 15% (w/v) SDS-PAGE.
Proteins were visualized by Coomassie Brilliant Blue staining.
Arrows indicate the position of the multiprotein aggregate
(top), the 70-kDa band and the 55-65-kDa triplet
band.
-form of
FGF1 (25) we resolved the
Cys30 FGF1:p40 Syt1
heterodimer as a band with the apparent molecular mass of ~70 kDa
(Fig. 3C). Because both Cys30 FGF1 and p40 Syt1
contain a single cysteine residue (24, 26), it is likely that this form
of the FGF1:p40 Syt1 heterodimer is unable to associate with S100A13 to
form the multiprotein aggregate. Thus, it is reasonable to suggest that
the cysteines at positions 97 and 131 in FGF1 may participate in the
formation of FGF1:p40 Syt1 heterodimers that are competent to associate
with S100A13. Because (i) the Cu2+-induced appearance of
the 55-65-kDa triplet is specific for p40 Syt1 and FGF1 and does not
require the presence of S100A13, (ii) the formation of the 55-65-kDa
triplet is sensitive to reducing agents, and (iii) the presence of the
55-65-kDa triplet, as well as the FGF1 doublet, is significantly
reduced in the Cu2+-induced oxidation of the p40 Syt1,
FGF1, and S100A13 multiprotein aggregate, we suggest that the band near
the top of the stacking gel is a composite of disulfide-linked FGF1
homodimer and p40 Syt1:FGF1 heterodimers complexed non-covalently to S100A13.
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Fig. 4.
The ability of S100A13 to interact with FGF1
and p40 Syt1 in a copper-dependent manner.
A, centrifugation of the multiprotein aggregate containing
FGF1, p40 Syt1, and S100A13. One µg of rFGF1, 2 µg of rp40 Syt1,
and 5 µg of rS100A13 as either individual samples or in different
combinations were resuspended in PBS, incubated for 30 min at 42 °C
in the presence and absence of 1 mM CuCl2, and
centrifuged for 45 min at 10,000 × g. Pellet fractions
were washed 3 times with PBS by centrifugation (45 min at 10,000 × g), diluted with reducing loading buffer, resolved by
15% (w/v) SDS-PAGE, and evaluated by p40 Syt1 (upper
panel), FGF1 (middle panel), and S100A13 (lower
panel) immunoblot analysis. B, the ability of S100A13
to interact with FGF1 in the presence of copper. Increasing amounts of
rS100A13 were mixed with either recombinant FGF1 or recombinant EGF,
lyophilized, resuspended in PBS, incubated for 30 min at 42 °C in
the absence or presence of 1 mM CuCl2, and
centrifuged for 45 min at 10,000 × g. Pellet fractions
were washed 3 times with PBS by centrifugation (45 min at 10,000 × g), diluted with reducing loading buffer, resolved by
15% (w/v) SDS-PAGE, and evaluated by S100A13 immunoblot analysis.
C, the ability of S100A13 to interact with p40 Syt1 in the
presence of copper. Increasing amounts of rS100A13 were mixed with rp40
Syt1 or ovalbumin, lyophilized, resuspended in PBS, incubated for 30 min at 42 °C in the absence or presence of 1 mM
CuCl2, and centrifuged for 45 min at 10,000 × g. Pellet fractions were washed 3 times with PBS by
centrifugation (45 min at 10,000 × g), diluted with
reducing loading buffer, resolved by 15% (w/v) SDS-PAGE, and evaluated
by S100A13 immunoblot analysis.
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Fig. 5.
The ability of S100A13 to interact with
Cys-free and Cys30 FGF1 in the presence of copper.
A and B, Cys-free FGF1 and Cys30
FGF1. rS100A13, rCys-free (A), and rCys30 FGF1
(B) were individually resuspended in PBS and S100A13 was
mixed with either Cys-free (A) or Cys30 FGF1
(B) in PBS at the molar ratio of 10:1, incubated for 30 min
at 42 °C in the absence or presence of 1 mM
CuCl2, and centrifuged for 18 h at 280,000 × g. Pellet fractions were diluted with reducing loading
buffer, resolved by 15% (w/v) SDS-PAGE, and evaluated by S100A13
(lower panel) and FGF1 (upper panel) immunoblot
analysis. C, the effect of varying the molar ratio of
S100A13 to Cys-free FGF1 and its susceptibility to ultracentrifugation.
Cys-free FGF1 (5 µg) was incubated with S100A13 at molar ratios of
1:1 (2.5 µg of S100A13), 1:5 (12.5 µg of S100A13), and 1:10 (25 µg of S100A13) in PBS for 30 min at 42 °C, centrifuged at
280,000 × g for 18 h at 4 °C, pellet fractions
obtained, solubilized in loading buffer, resolved by conventional 15%
(w/v) SDS-PAGE, and subjected to FGF1 (upper panel) and
S100A13 (lower panel) immunoblot analysis.
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[in a new window]
Fig. 6.
The ability of TTM to inhibit FGF1 and
Myc-S100A13 release in response to heat shock. Myc-S100A13 and
FGF1 NIH 3T3 cell co-transfectants were incubated for 18 h at
37 °C in the absence and presence of 100, 200, or 500 µM TTM and then either subjected to either heat shock
(110 min at 42 °C) or incubated for 110 min at 37 °C in the
absence and presence of the same concentration of TTM. Conditioned
media were treated with 0.1% (w/v) DTT, adsorbed to heparin-Sepharose,
and eluted at 1.5 M NaCl. The elution fractions were
concentrated, resolved by 15% (w/v) SDS-PAGE, and evaluated by FGF1
(upper panel) and Myc (lower panel) immunoblot
analysis. The intracellular levels of FGF1 and Myc-S100A13 did not vary
as a result of either temperature stress or TTM treatment and were
similar to those previously reported (10).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank B. W. Schafer for the murine S100A13 cDNA and N. Albrecht for expert administrative assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL32348 and AG98503 (to T. M.).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.
Supported in part by a fellowship from the Catholic University of
Rome, Italy.
§ Present address: Dept. of Geriatric Medicine, University of Florence, School of Medicine, Florence, Italy.
¶ To whom correspondence should be addressed: Center for Molecular Medicine, Maine Medical Center Research Inst., 81 Research Dr., Scarborough, ME 04074. Tel.: 207-885-8200; Fax: 207-885-8179; E-mail: maciat@mmc.org.
Published, JBC Papers in Press, May 10, 2001, DOI 10.1074/jbc.M102925200
2 T. M. La Vallee and T. Maciag, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; Ann, annexin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Syt, synaptotagmin; TTM, tetrathiomolybdate; EGF, epidermal growth factor.
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REFERENCES |
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