Copper Induces the Assembly of a Multiprotein Aggregate Implicated in the Release of Fibroblast Growth Factor 1 in Response to Stress*

Matteo LandriscinaDagger, Cinzia Bagalá, Anna Mandinova, Raffaella Soldi, Isabella Micucci§, Stephen Bellum, Igor Prudovsky, and Thomas Maciag

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (14K):
[in this window]
[in a new window]
 
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.

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).


View larger version (31K):
[in this window]
[in a new window]
 
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.

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).


View larger version (51K):
[in this window]
[in a new window]
 
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.

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 beta -form of FGF1 (25) we resolved the beta 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.

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).


View larger version (11K):
[in this window]
[in a new window]
 
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.

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.


View larger version (20K):
[in this window]
[in a new window]
 
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.

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.


View larger version (11K):
[in this window]
[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

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).

    ACKNOWLEDGEMENTS

We thank B. W. Schafer for the murine S100A13 cDNA and N. Albrecht for expert administrative assistance.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Burgess, W. H., and Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606[CrossRef][Medline] [Order article via Infotrieve]
2. Friesel, R., and Maciag, T. (1999) Thromb. Haemostasis 82, 748-754[Medline] [Order article via Infotrieve]
3. Shi, J., Friedman, S., and Maciag, T. (1997) J. Biol. Chem. 272, 1142-1147[Abstract/Free Full Text]
4. Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10691-10695[Abstract]
5. Carreira, C. M., Landriscina, M., Bellum, S., Prudovsky, I., and Maciag, T. (2001) Growth Factors, in press
6. Shin, J. T., Opalenik, S. R., Wehby, J. N., Mahesh, V. K., Jackson, A., Tarantini, F., Maciag, T., and Thompson, J. A. (1996) Biochim. Biophys. Acta 1312, 27-38[Medline] [Order article via Infotrieve]
7. Tarantini, F., LaVallee, T., Jackson, A., Gamble, S., Carreira, C. M., Garfinkel, S., Burgess, W. H., and Maciag, T. (1998) J. Biol. Chem. 273, 22209-22216[Abstract/Free Full Text]
8. LaVallee, T. M., Tarantini, F., Gamble, S., Carreira, C. M., Jackson, A., and Maciag, T. (1998) J. Biol. Chem. 273, 22217-22223[Abstract/Free Full Text]
9. Carreira, C. M., LaVallee, T. M., Tarantini, F., Jackson, A., Lathrop, J. T., Hampton, B., Burgess, W. H., and Maciag, T. (1998) J. Biol. Chem. 273, 22224-22231[Abstract/Free Full Text]
10. Landriscina, M., Soldi, R., Bagalá, C., Micucci, I., Bellum, S., Tarantini, F., Prudovsky, I., and Maciag, T. (2001) J. Biol. Chem. 276, 22544-22552[Abstract/Free Full Text]
11. Engleka, K. A., and Maciag, T. (1992) J. Biol. Chem. 267, 11307-11315[Abstract/Free Full Text]
12. Hannan, G. N., and McAuslan, B. R. (1982) J. Cell. Physiol. 111, 207-212[Medline] [Order article via Infotrieve]
13. Hu, G. F. (1998) J. Cell. Biochem. 69, 326-335[CrossRef][Medline] [Order article via Infotrieve]
14. Matsubara, T., Saura, R., Hirohata, K., and Ziff, M. (1989) J. Clin. Invest. 83, 158-167[Medline] [Order article via Infotrieve]
15. Soncin, F., Guitton, J. D., Cartwright, T., and Badet, J. (1997) Biochem. Biophys. Res. Commun. 236, 604-610[CrossRef][Medline] [Order article via Infotrieve]
16. Lane, T. F., Iruela-Arispe, M. L., Johnson, R. S., and Sage, E. H. (1994) J. Cell Biol. 125, 929-943[Abstract]
17. Rucker, R. B., and Wold, F. (1988) FASEB J. 2, 2252-2261[Abstract/Free Full Text]
18. Jackson, A., Tarantini, F., Gamble, S., Friedman, S., and Maciag, T. (1995) J. Biol. Chem. 270, 33-36[Abstract/Free Full Text]
19. Sano, H., Forough, R., Maier, J. A., Case, J. P., Jackson, A., Engleka, K., Maciag, T., and Wilder, R. L. (1990) J. Cell Biol. 110, 1417-1426[Abstract]
20. Bergmeyer, H. U. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed) , pp. 736-743, Academic Press, New York
21. Heizmann, C. W., and Cox, J. A. (1998) Biometals 11, 383-397[CrossRef][Medline] [Order article via Infotrieve]
22. Nishikawa, T., Lee, I. S., Shiraishi, N., Ishikawa, T., Ohta, Y., and Nishikimi, M. (1997) J. Biol. Chem. 272, 23037-23041[Abstract/Free Full Text]
23. Schafer, B. W., Fritschy, J. M., Murmann, P., Troxler, H., Durussel, I., Heizmann, C. W., and Cox, J. A. (2000) J. Biol. Chem. 275, 30623-30630[Abstract/Free Full Text]
24. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R., and Sudhof, T. C. (1990) Nature 345, 260-263[CrossRef][Medline] [Order article via Infotrieve]
25. Forough, R., Engleka, K., Thompson, J. A., Jackson, A., Imamura, T., and Maciag, T. (1991) Biochim. Biophys. Acta 1090, 293-298[Medline] [Order article via Infotrieve]
26. Tarantini, F., Gamble, S., Jackson, A., and Maciag, T. (1995) J. Biol. Chem. 270, 29039-29042[Abstract/Free Full Text]
27. Brewer, G. J., Johnson, V., Dick, R. D., Kluin, K. J., Fink, J. K., and Brunberg, J. A. (1996) Arch. Neurol. 53, 1017-1025[Abstract]
28. Cox, D. W. (1999) Br. Med. Bull. 55, 544-555[Abstract]
29. Brewer, G. J., Dick, R. D., Grover, D. K., LeClaire, V., Tseng, M., Wicha, M., Pienta, K., Redman, B. G., Jahan, T., Sondak, V. K., Strawderman, M., LeCarpentier, G., and Merajver, S. D. (2000) Clin. Cancer Res. 6, 1-10[Abstract/Free Full Text]
30. Brem, S. (1999) Cancer Control 6, 436-458[Medline] [Order article via Infotrieve]
31. Donato, R. (1999) Biochim. Biophys. Acta 1450, 191-231[Medline] [Order article via Infotrieve]
32. Seemann, J., Weber, K., and Gerke, V. (1996) Biochem. J. 319, 123-129[Medline] [Order article via Infotrieve]
33. Mailliard, W. S., Haigler, H. T., and Schlaepfer, D. D. (1996) J. Biol. Chem. 271, 719-725[Abstract/Free Full Text]
34. Kang, H. M., Kassam, G., Jarvis, S. E., Fitzpatrick, S. L., and Waisman, D. M. (1997) Biochemistry 36, 2041-2050[CrossRef][Medline] [Order article via Infotrieve]
35. Lum, H., and Roebuck, K. A. (2001) Am. J. Physiol. Cell Physiol. 280, C719-741[Abstract/Free Full Text]
36. Blaber, M., DiSalvo, J., and Thomas, K. A. (1996) Biochemistry 35, 2086-2094[CrossRef][Medline] [Order article via Infotrieve]
37. Marqueze, B., Berton, F., and Seagar, M. (2000) Biochimie (Paris) 82, 409-420[CrossRef][Medline] [Order article via Infotrieve]
38. Schafer, B. W., and Heizmann, C. W. (1996) Trends Biochem. Sci. 21, 134-140[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.