Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Rasmediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Rasmediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.Mutational activation of the KRAS oncogene is the most prevalent genetic alteration associated with essentially 100% of pancreatic ductal adenocarcinomas (PDACs) 3 (1). Therefore, considerable effort has been made to develop inhibitors of K-Ras for PDAC treatment. Currently, most of these efforts are focused on inhibitors of K-Ras downstream effector signaling, with numerous inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)-AKT pathway currently under clinical evaluation (2). However, recent studies suggest that additional Ras effector pathways may also play significant roles in Ras-mediated oncogenesis. In particular, there is increasing evidence for the importance of Ral guanine nucleotide exchange factors (RalGEFs), activators of the Ral (Ras-like) small GTPases (3), in cancer growth (4).Ral GTPases function as GDP/GTP-regulated binary switches that regulate extracellular stimulus-regulated signaling networks that control a diversity of cellular processes, including exocytosis, endocytosis, activation of transcription factors, and actin cytoskeletal reorganization (4). Surprisingly, despite their significant sequence identity (82%) and interaction with a common set of effectors, the related RalA and RalB isoforms serve very distinct functions in oncogenesis. Importantly, Chien and White (5) found that RalA is necessary for tumor cell anchorage-independent growth, whereas RalB is necessary for tumor but no...