RESUMEN
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.
Asunto(s)
Membrana Celular/enzimología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Células CHO , Cricetulus , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Sistemas de Mensajero Secundario , Vías Secretoras , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induced angiogenesis. Here, we describe a novel time-resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose-response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2-dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.