The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2.

FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces. Legrand et al. identify two lysine residues on molecular surface of Fibroblast Growth Factor 2 (FGF2) essential for its interaction with α1 subunit of the Na,K-ATPase. They further conclude that this interaction precedes interaction of the FGF2 with PI(4,5)P2 and facilitates its unconventional secretion across the membrane, which is impaired by Ouabain, an Na,K-ATPase inhibitor.