Fibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is exported from cells by an unconventional secretory pathway. This process is based on direct translocation of FGF2 across the plasma membrane. FGF2 membrane translocation depends on PI(4,5)P2-induced formation of membrane-inserted FGF2 oligomers followed by extracellular trapping of FGF2 at the outer leaflet mediated by cell surface heparan sulfate proteoglycans. Beyond the well-characterized core mechanism of FGF2 membrane translocation, the Na,K-ATPase has been proposed to play a so far unknown role in unconventional secretion of FGF2. Here, we define a direct physical interaction of FGF2 with a subdomain of the cytoplasmic part of the α1 subunit of the Na,K-ATPase. Employing NMR spectroscopy and molecular dynamics simulations, we identified two lysine residues on the molecular surface of FGF2 that are shown to be essential for its interaction with α1. In intact cells, the corresponding lysine-to-glutamate variants of FGF2 were characterized by inefficient secretion and reduced recruitment to the inner plasma membrane leaflet as shown by single molecule TIRF microscopy. Our findings suggest that α1 acts upstream of PI(4,5)P2 facilitating efficient membrane translocation of FGF2 to the cell surface of tumor cells.

The α1 subunit of the Na,K-ATPase acts upstream of PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2 from tumor cells