S100A13 Participates in the Release of Fibroblast Growth Factor 1 in Response to Heat Shock in Vitro

Abstract

S100A13, a member of the S100 gene family of Ca 2؉ -binding proteins has been previously characterized as a component of a brain-derived heparin-binding multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells results in the constitutive release of S100A13 into the extracellular compartment at 37°C, co-expression of S100A13 with FGF1 represses the constitutive release of S100A13 and enables NIH 3T3 cells to release S100A13 in response to temperature stress. S100A13 release in response to stress occurs with kinetics similar to that observed for the stress-induced release of FGF1, but S100A13 expression is able to reverse the sensitivity of FGF1 release to inhibitors of transcription and translation. The release of FGF1 and S100A13 in response to heat shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate saturation, and the expression of a S100A13 deletion mutant lacking its novel basic residue-rich domain acts as a dominant negative effector of FGF1 release in vitro. Surprisingly, the expression of S100A13 also results in the stress-induced release of a Cys-free FGF1 mutant, which is normally not released from NIH 3T3 cells in response to heat shock. These data suggest that S100A13 may be a component of the pathway for the release of the signal peptide-less polypeptide, FGF1, and may involve a role for S100A13 in the formation of a noncovalent FGF1 homodimer. FGF11 and FGF2 are the prototype members of a large family of heparin-binding growth factor genes that regulate numerous biological processes such as neurogenesis, mesoderm formation, and angiogenesis (1, 2). FGF1 and FGF2 lack a classical signal peptide sequence that provides access to the conventional endoplasmic reticulum (ER)-Golgi secretion pathway, a characteristic that led to the hypothesis that the release of these polypeptides may proceed through novel release/export pathways (2). Our laboratory previously demonstrated that FGF1, but not FGF2, is released as a latent homodimer by a transcription-and translation-dependent mechanism in response to a variety of cellular stresses including heat shock (3), hypoxia (4), and serum starvation (5). Conversely, the disruption of communication between the ER and Golgi apparatus by brefeldin A does not prevent the release of FGF1 from NIH 3T3 cells, confirming that FGF1 release may occur through a nonconventional pathway (6).FGF1 is released in vitro as a reducing agent-and denaturant-sensitive complex, which contains the p40 extravesicular domain of the Ca 2ϩ -binding protein, p65 synaptotagmin (Syt)1 (7). The release of FGF1 in response to stress is dependent on Syt1 expression, since the expression of either a deletion mutant lacking 95 amino acids from the extravesicular portion of Syt1 or an antisense-Syt1 gene is able to repress FGF1 release in NIH 3T3 cells (7,8). In addition, FGF1 purified from ovine brain as a high molecular weight aggregate exists as a component of a noncovalent heparin-binding complex wit...

Release of FGF1 and p40 synaptotagmin 1 correlates with their membrane destabilizing ability

“…The plasmids for eukaryotic expression of FGF1 (pXZ38) and p40 Syt1 (p40-Syt1:Myc in pMEX hygro) were prepared as previously described [14,21,22]. The K326,327,331Q mutant of p40 Syt1 was produced by mutagenesis from the p40 Syt1:Myc pMEX Hygro and GST-p40 Syt1-pGEX-KG original plasmids [22,34] for eukaryotic and prokayotic expression, respectively.…”

“…FGF1 K126,127A and FGF1 K114,115A mutants were produced by mutagenesis of the FGF1:HA pCR3.1 construct (gift of Andrew Baird, Human BioMolecular Research Institute, San Diego, CA) for eukaryotic expression, and the FGF1 pET3C construct [35] for prokaryotic expression. Recombinant FGF1, S100A13, and p40 Syt1 were produced and purified by HPLC as previously described [21,34,35].…”

“…Released p40 Syt1 was isolated using heparin Sepharose chromatography as described [22]. Released FGF1:HA was immunoprecipitated using anti-HA monoclonal antibodies (Covance) as described [21]. FGF1 and p40 Syt1 were resolved by SDS PAGE and detected by immunoblotting as described [14,22].…”

“…FGF1 is secreted from cells upon stress stimulation such as heat shock [14], hypoxia [17], serum starvation [18], and treatment with oxidized LDL [19]. The availability of free intracellular copper ions is necessary for FGF1 release, and in vitro data suggest the formation of a copper-and stress-dependent multiprotein export complex [20], which contains the calcium-binding proteins, S100A13 and p40 Syt1 [21,22]. p40 Syt1 is a non-transmembrane isoform of the integral component of secretory vesicles, synaptotagmin (Syt)1, that is involved in the fusion of exocytotic vesicles with the plasma membrane [23].…”

“…Their constitutive release is blocked when they are cotransfected into the cells along with FGF1; however upon stress, they are released in a complex with FGF1 [21,22].…”