Dissecting the role of the Na,K-ATPase in unconventional secretion of Fibroblast Growth Factor 2

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Fibroblast Growth Factor 2 (FGF2) is a survival factor involved in tumor-induced angiogenesis. FGF2 further mediates chemoresistance of tumor cells towards anti-cancer therapies based on an autocrine signaling cascade blocking apoptosis. The initiation of these signaling pathways occurs at cell surfaces requiring transport of FGF2 into the extracellular space. This process is mediated by unconventional protein secretion (UPS) with FGF2 belonging to the Type I UPS cargo family that is secreted by direct translocation across plasma membranes. A key component of this secretory mechanism is the phosphoinositide PI(4,5)P2, a membrane lipid at the inner leaflet of the plasma membrane that triggers FGF2 to oligomerize concomitant with the formation of a lipidic membrane pore. Membrane-inserted FGF2 oligomers are then removed and disassembled at the outer leaflet of the plasma membrane mediated by heparan sulfate proteoglycans. This process results in the translocation of FGF2 molecules into the extracellular space where they engage in FGF receptor activation and signaling.While the molecular mechanism of the core process of FGF2 membrane translocation is understood in quite some detail, the precise function of additional components of this pathway remained elusive. The participation of the Na,K-ATPase in this process has been demonstrated by three different methodological approaches. First, ouabain, a pharmacological inhibitor of the Na,K-ATPase impairs FGF2 secretion. Second, RNAi-mediated down-regulation of the α subunit of the Na,K-ATPase inhibits FGF2 secretion. Third, FGF2 has been demonstrated to engage in a physical interaction with the cytoplasmic domain of the α subunit of the Na,K-ATPase. Variant forms of FGF2 that cannot bind to this part of the Na,K-ATPase are impaired in secretion efficiency. However, while these findings established a role of the Na,K-ATPase in unconventional secretion of FGF2, they did not directly reveal its mechanism of action.The overarching goal of this research proposal is to test the hypothesis for the existence of a coupling mechanism by which FGF2 regulates the activity of the Na,K-ATPase at localized events of membrane translocation, a process that involves the transient formation of lipidic membrane pores in the plasma membrane. In this way, potential perturbations of the membrane potential that may occur during this process could be compensated through an FGF2-induced activation of the enzymatic activity of the Na,K-ATPase. Thus, through a highly interdisciplinary approach with a broad range of methodological approaches including structural biology, biochemical reconstitution, theoretical approaches such as molecular dynamics simulations and advanced imaging techniques as well as supported by several international collaborations, we aim at elucidating the function of the Na,K-ATPase in unconventional secretion of FGF2.