Modulation of phospholipase D by Ras proteins mediated by its effectors Ral-GDS, PI3K and Raf-1

Transformation by ras oncogenes induces the deregulation of intracellular signalling cascades that are critical elements in cell growth control. Ras proteins are molecular switches with the ability to interact and activate several effector molecules. Among those, Raf-1 kinase, PI3K and Ral-GDS are the best characterised. Raf activates the mitogenic MEK/ERK kinases pathway, while PI3K regulates the PKB/Akt cascade, involved in the control of proliferation, metabolism and apoptotic responses. Finally, Ral-GDS belongs to a family of guanine nucleotide exchange factors that activate Ral GTPases. While Raf and PI3K have emerged as critical elements in regulating cell growth and apoptosis, little is known about the role of the Ral-GDS family. We have previously reported that Ras proteins are critical elements in the regulation of phospholipase D (PLD), a proposed target for the Ral-GDS/RalA pathway. Physiological regulation of PLD by growth factors requires the simultaneous activation of the endogenous, wild-type Ras proteins, and a PKC-dependent mechanism. Transformation by ras oncogenes induces drastic alterations in PLD activity and the usual response to external stimuli, through a PKC-independent mechanism. Here we provide further evidence on the mechanisms by which oncogenic Ras proteins induces the deregulation of PLD and here we try to identify the specific effectors involved. A complex system for PLD regulation is unravelled which implies the existence of two positive regulatory pathways, mediated by Ral-GDS and PI3K, and two negative feedback mechanisms mediated by Raf and Ral-GDS. These results strongly support participation of PLD in Ras-mediated signalling. Furthermore, we provide evidence that oncogenic Ras proteins constitutively activate PLD by mechanisms different to those used by normal Ras proteins.