Characterization of the role of RalGPS2, a RalA GEF, in transformed and cancer cells

Ral proteins are members of the Ras GTPase superfamily and localize at the plasma membrane, in endocytic and exocytic vesicles and in synaptic vesicles. These GTPases are involved in multiple cellular events including proliferation, migration, differentiation, cytoskeletal organization, vesicular transport, exocytosis and receptor endocytosis. They are also implicated in tumorigenesis, invasion and metastasis. RalGPS2 is a guanine nucleotide exchange factor for RalA belonging to RalGPS family. It is a 590 aminoacids polypeptide that contains a well conserved Ras-GEF domain, a PxxP motif and a PH (Pleckstrin Homology) domain. Previous experiments have demonstrated that RalGPS2 can activate RalA in vivo, while the PH-PxxP domain acts as a dominant negative for RalA activation in both NIH3T3 and PC12 cells. These data suggest that the PH-PxxP domain of RalGPS2 could inhibit RalA activation, thus influencing cytoskeleton rearrangement. The principal aim of this work is to analyze the role of RalGPS2 and of its PH-PxxP domain in human and rodent cells transformation. In particular our attention was been focused on transformed murine fibroblasts (NIH3T3 k-Ras) and on human bladder cancer cells (5637 cell line). We found that RalA and RalGPS2 were highly expressed in 5637 human bladder cancer cells and that the overexpression of PHPxxP domain reduced the level of active RalA bound at GTP. Furthermore the expression of PH and PH-PxxP domains in both NIH3T3 k-ras and 5637 cell line, induced a marked cytoskeleton re-organization. In particular PH domain caused formation of thin protrusions with the presence of vesicles. PH-PxxP domain caused formation of long inter-cellular structure that are probably involved in the exchange of signals and cellular components between different cells. It will be interesting in the future to understand the molecular nature of these ‘‘hairy’’ structures that could be nanotubes.