Ral-Specific Guanine Nucleotide Exchange Factor Activity Opposes Other Ras Effectors in PC12 Cells by Inhibiting Neurite Outgrowth

Abstract

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only ϳ20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.

The Rgr oncogene (homologous to RalGDS) induces transformation and gene expression by activating Ras, Ral and Rho mediated pathways

“…This eect is consistent with previous reports that demonstrated that members of the subfamily of GEFs for Ral are able to induce c-fos promoter activity (Wolthuis et al, 1997;Goi et al, 1999). However, Ral gave little, if any, activation of the c-fos promoter by itself (Murai et al, 1997;Wolthuis et al, 1997).…”

Ral GTPases Contribute to Regulation of Cyclin D1 through Activation of NF-κB

“…This observation has led to the hypothesis that Ral may negatively regulate the activity of these GTPases. In support of this, studies using PC12 cells suggest that RalGEFs can interfere with neurite differentiation in a Rac-dependent fashion (19). However, a direct effect of Ral on Rac or Cdc42 regulation has not yet been demonstrated.…”

Galectin-3 Augments K-Ras Activation and Triggers a Ras Signal That Attenuates ERK but Not Phosphoinositide 3-Kinase Activity

“…Third, the types of effectors required for Ras transformation of rodent cells were found to differ from those required in human cells (12). An analogous distinct effector-type requirement was documented in PC-12 cells, in which Raf and PI3-K signaling promote cell cycle arrest, whereas RalGEF signaling promotes cell proliferation (13). Last is the observation that activated K-Ras in human pancreatic tumor cell lines does not correlate with persistent activation ERK (14).…”