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

Abstract

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 Cu 2؉ -binding protein and Cu 2؉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 Cu 2؉ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu 2؉ with similar affinity and to interact in the presence of Cu 2؉ 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 Cu 2؉ 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 Cu 2؉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 Cu 2؉ 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 Cu 2؉ 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 cotransfectants 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...

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

“…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].…”

Copper chelation represses the vascular response to injury

“…Both IL-1␣ (19) and FGF1 (20) use intracellular Cu 2ϩ to force the assembly of a multiprotein complex near the inner surface of the plasma membrane (19,20). Although both IL-1␣ and FGF1 form Cu 2ϩ -dependent heterotetrameric complexes with S100A13 to facilitate their release, the FGF1 release pathway also requires the function of the extravesicular domain of synaptotagmin (Syt)1 for export (21).…”

Synergistic Ca2+ and Cu2+ requirements of the FGF1–S100A13 interaction measured by quartz crystal microbalance: An initial step in amlexanox-reversible non-classical release of FGF1

“…is reported to be homodimerized through the Cu 2+ -catalyzed oxidation of Cys30 of FGF1, and released upon stress in a non-classical manner LaVallee et al, 1998;Landriscina et al, 2001a). Thus, it is interesting to speculate that Cu 2+ bound to S100A13 may stabilize the interaction between FGF1 and S100A13 through oxidation and homodimerization of FGF1 (Fig.…”

“…Although the static role of Cu 2+ in non-classical FGF1 release is proven LaVallee et al, 1998;Landriscina et al, 2001a), its dynamic regulation is unlikely to be important, since the cellular level may not change upon the stress. However, some genetic diseases are closely related to the deficiency or excess of Cu 2+ .…”

“…This unique nature may be related to the fact that S100A13 has only two Ca 2+ -binding sites, unlike other S100 family proteins. It should be noted that FGF1 also has Cu 2+ -binding affinity and that the depletion of intracellular Cu 2+ by a specific chelating agent prevents stress-induced FGF1 release (Landriscina et al, 2001a;Matsunaga and Ueda, 2006a and b). Furthermore, it is also reported that the homodimerization and non-classical release of FGF1 are closely related to the Cu 2+ -mediated oxidation of cysteine (Cys) 30 Tarantini et al, 1995).…”