Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Sharon L. Campbell,Dominico Vigil,Falina Williams,Jen Jen Yeh,Channing J. Der,Timothy D. Martin

doi:10.1074/jbc.m110.116756

Abstract

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-Ras-mediated 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 Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.

Highlights

Pancreatic ductal adenocarcinomas (PDACs)3 [1]
Because mutationally activated K-Ras is found in essentially all pancreatic ductal adenocarcinoma (PDAC), a logical mechanism would involve one or more of the four Ras association (RA) domain-containing Ral guanine nucleotide exchange factors (RalGEFs) (RalGDS, Rgl1, Rgl2, and Rgl3) that function as effectors of Ras (Fig. 1)
Activated Ras promotes RalGEF activation by promoting RalGEF association with the plasma membrane, and previous studies showed that the addition of a Ras C-terminal plasma membrane targeting sequence results in constitutively activated RalGDS [34], Rgl2 [24], and Rgl3 [13]

Summary

Introduction

Pancreatic ductal adenocarcinomas (PDACs)3 [1]. considerable effort has been made to develop inhibitors of K-Ras for PDAC treatment. We demonstrated that RalB subcellular localization to the plasma membrane at the leading edge was dependent on Rgl2 expression, suggesting that RalGEFs may regulate spatially restricted Ral activation.

Results

Conclusion