Role of RalGPS2, a new possible oncogene, in tunneling nanotubes formation

Ral proteins are members of the Ras small GTPase superfamily and are involved in multiple cellular events including cytoskeletal organization and tumorigenesis “in vitro” and in animal models. RalA overexpression is associated with bladder and prostate cancer. RalGPS2 is a guanine exchange factor (GEF) for RalA belonging to RalGPS family that contains a GEF domain, a PxxP motif and a PH domain (Ceriani et al., 2007). Previous experiments have demonstrated that RalGPS2 activates RalA in vivo, while the overexpression of PH-PxxP region acts as a dominant negative for RalA activation in NIH3T3 and PC12 cells (Ceriani et al., 2010). These data suggest that the PH-PxxP region of RalGPS2 could inhibit RalA activation in tumor cell lines. The aim of this work is to analyze the role of RalGPS2 and of its PH-PxxP region in human bladder cancer cells (5637). The overexpression of PH-PxxP region of RalGPS2 reduced the level of RalA-GTP also in 5637 cells and confocal microscopy analyses revealed that there is a partial co-localization between RalA and truncated proteins containing only the PH domain or the PH-PxxP region of RalGPS2 at plasma membrane and in thin membrane protrusions. Besides the overexpression of PH domain and PH-PxxP region induced a marked cytoskeleton re-organization: the PH domain caused formation of thin vesiculating protrusion while the PH-PxxP domains caused formation of long inter-cellular structure. Morphological characterization of protrusions induced by PH domain overexpression reveled that they were tunnelling nanotubes (TNTs). TNTs are a new type of cell-cell communication between remote cells and through TNTs cells can exchange various cellular components and signals (Hase K. et al., 2009). Finally, immunoprecipitation assays reveal the presence of a complex between RalA, RalGPS2 and LST1, a transmembrane protein that induces TNTs formation.