Formation of Disulfide Bridges Drives Oligomerization, Membrane Pore Formation, and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces

Abstract

Background: FGF2 translocation across plasma membranes depends on phosphoinositide-dependent oligomerization and membrane pore formation. Results: Two unique surface cysteines are critical for efficient FGF2 oligomerization, membrane pore formation, and FGF2 secretion from cells. Conclusion: Formation of intermolecular disulfide bridges drives phosphoinositide-dependent FGF2 oligomerization at plasma membranes. Significance: A new cis element critical for unconventional secretion of FGF2 was identified and validated.

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

“…FGF2 exits cells by direct translocation across the plasma membrane (18,19). This process involves (i) membrane recruitment at the inner leaflet mediated by the phosphoinositide PI(4,5)P 2 (1, 2, 21, 22), (ii) FGF2 oligomerization and membrane pore formation (9,11,23,24,30), and (iii) extracellular trapping mediated by membraneproximal heparan sulfate proteoglycans (25,26). In addition, Tec kinase (9, 31) and ATP1A1 (12,(32)(33)(34), two additional factors physically associated with the plasma membrane, have been shown to play critical roles in the unconventional secretory pathway of FGF2 (12).…”

“…Again, the inactive compounds 18 and 19 were taken as negative controls. To quantify FGF2 secretion, a well established biotinylation assay was used to detect FGF2 bound to heparan sulfates on cell surfaces (23,26,51). Briefly, following doxycycline-induced expression of FGF2-GFP, proteins present on cell surfaces were modified using a membrane-impermeable biotinylation reagent.…”

“…FGF2 was expressed in Escherichia coli, purified to homogeneity (23,24), and covalently coupled to epoxy beads (34). Various constructs of Tec kinase were expressed and purified from insect cells (Fig.…”

“…It was further shown that a phosphomimetic variant form of FGF2 is secreted from cells in a Tec kinase-independent manner (9,31). Furthermore, based upon biochemical reconstitution experiments, a phosphomimetic form of FGF2 was found to be characterized by an increased ability to form PI(4,5)P 2 -dependent membrane pores (23,24), the intermediates of FGF2 membrane translocation in cells (12).…”

“…It is based upon direct translocation of folded species of FGF2 across plasma membranes (9,(17)(18)(19)(20). Hallmarks of this process are (i) FGF2 recruitment at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P 2 2 (9, 21, 22), (ii) FGF2 oligomerization and membrane pore formation (11,12,23,24), and (iii) extracellular trapping of FGF2 mediated by membrane-proximal heparan sulfate proteoglycans (1,2,25,26). Recently, these hallmarks of FGF2 secretion have also been implicated in unconventional secretion of HIV-Tat (HIV trans-activator of transcription) (12, 27-30).…”