Regulation of choline kinase activity by Ras proteins involves Ral ± GDS and PI3K Ana RamõÂrez de Molina 1 , VeroÂnica Penalva 1 , Luisa Lucas 1 and Juan Carlos Lacal* ,1 1 Instituto de Investigaciones BiomeÂdicas, CSIC, Arturo Duperier 4, 28029 Madrid, Spain Ras proteins are molecular switches that control signaling pathways critical in the onset of a variety of human cancers. The signaling pathways activated by Ras proteins are those controlled by its direct eectors such as the serine-threonine protein kinase Raf-1, the exchange factor for other GTPases Ral ± GDS, and the lipid kinase PI3K. As a consequence of Ras activation, a number of additional enzymes are aected, including several members of the serine-threonine intracellular proteins kinases as well as enzymes related to phospholipid metabolism regulation such as phospholi- pases A2 and D, and choline kinase. The precise mechanisms by which ras oncogenes impinge into these later molecules and their relevance to the onset of the carcinogenic process is still not fully understood. Here we have investigated the mechanism of regulation of choline kinase by Ras proteins and found no direct link between PLD and choline kinase activation. We provide evidence that Ras proteins regulate the activity of choline kinase through its direct eectors Ral ± GDS and PI3K, while the Raf pathways seems to be not relevant in this process. The importance of Ras-dependent activation of choline kinase is discussed. Oncogene (2002) 21, 937 ± 946. DOI: 10.1038/sj/onc/ 1205144 Keywords: ras; oncogenes; choline kinase; Ras eectors; Raf; Ral ± GDS; PI3K Introduction Ras GTPases constitute a central point in signal transduction pathways that lead to the regulation of cell growth (Malumbres and Pellicer, 1999; HernaÂndez- Alcoceba et al., 2000; RamõÂrez de Molina et al., 2001a) and have been widely implicated in the carcinogenic process in humans (Barbacid, 1987; Bos, 1989; Rodenhuis, 1992; Bernhared et al., 2000). The mechanisms by which Ras GTPases subvert the normal regulation in cells is still not completely understood. A great eort has been devoted to identify the signaling pathways governed by Ras proteins. As a result, evidence has been provided that supports a role of lipid-derived second messengers in the transforming properties of Ras oncoproteins. Phosphatidylcholine (PC), the major component of the plasma membrane, is hydrolyzed by phospholipase D (PLD) to yield phosphatidic acid (PA) and choline. PA is then either hydrolyzed to generate diacylglycerol (DAG), or deacylated to form lyso-PA (LPA). On the other hand, choline is phosphorylated by choline kinase (ChoK) to generate phosphorylcholine (PCho). Both PLD and ChoK have been demonstrated to play a role in malignant transformation. PLD has been implicated in the metastatic process (Reich et al., 1995; Aguirre-Ghiso et al., 1999), and increased levels of ChoK activity and PCho production in human cancers have been found (Bhakoo et al., 1996; Nakagami et al., 1999; Ruiz-Cabello and Cohen, 1992), keeping with previous reports using nuclear magnetic resonance techniques (NMR) (de Certaines et al., 1993; Smith et al., 1993). These observations have resulted in the development of dierent anti- tumoral strategies focused on PLD and ChoK (Rameh and Cantley, 1999; Gratas and Powis, 1993; Lucas et al., 2001; Herna ndez-Alcoceba et al., 1999; Lacal, 2001; RamõÂrez de Molina et al., 2001a). Nevertheless, not much is known about the way these lipid-derived second messengers are mediating the process of transformation, and work aimed to understand this mechanism will help to improve the design of an ecient anticancer therapy. In this sense, ras oncogenes have been shown to activate PLD and ChoK, resulting in an increase in PCho production (Lacal et al., 1987; Lacal, 1990; Macara, 1989; Rameh and Cantley, 1999; Cuadrado et al., 1993; JimeÂnez et al., 1995; Lucas et al., 2000; Bhakoo et al., 1996; Ratnam and Kent, 1995). However, whether PLD and ChoK activation after transformation by ras takes place in a sequential or independent manner and the way it occurs, remains still unknown. In its active form, bound to GTP, Ras interacts with several downstream molecules eliciting dierent signal transduction pathways whose alterations may lead to tumorigenesis. The most extensively studied eectors to Ras are the Raf serine/threonine kinase-1, the Ral- GDS exchange factor, and the lipid kinase PI3K (Malumbres and Pellicer, 1999; Katz and McCormick, 1997; Herna ndez-Alcoceba et al., 2000; Crespo and Oncogene (2002) 21, 937 ± 946 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc *Correspondence: JC Lacal; E-mail: jclacal@iib.uam.es Received 23 August 2001; revised 19 October 2001; accepted 31 October 2001