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Vol. 18, Issue 12, 4859-4871, December 2007

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Diacylglycerol Kinase- Mediates Hepatocyte Growth Factor-induced Epithelial Cell Scatter by Regulating Rac Activation and Membrane Ruffling

Federica Chianale^*,, Santina Cutrupi^*,,,, Elena Rainero^*, Gianluca Baldanzi^*,||, Paolo E. Porporato^*, Sara Traini^*, Nicoletta Filigheddu^¶, Viola F. Gnocchi^*, Massimo M. Santoro^#, Ornella Parolini^||, Wim J. van Blitterswijk^@, Fabiola Sinigaglia^*, and Andrea Graziani^*

Departments of *Medical Sciences, ^¶Clinical and Experimental Medicine, and ^#Scienze dell'Ambiente e della Vita, University of Piemonte Orientale "A. Avogadro," 28100 Novara, Italy; Department of Animal and Human Biology and Center for Complex System in Molecular Biology and Medicine – SysBioM, University of Torino, 10123 Torino, Italy; ^||Centro Ricerche "E. Menni," Ospedale Poliambulanza, 25124 Brescia, Italy; and ^@The Netherlands Cancer Institute, Amsterdam, 1066 CX Amsterdam, The Netherlands

Submitted February 28, 2007; Revised August 9, 2007; Accepted September 17, 2007
Monitoring Editor: J. Silvio Gutkind

Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgk in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgk is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgk, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgk, obtained either pharmacologically by R59949 [GenBank] treatment, or by expression of Dgk dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgk, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgk, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.

These authors contributed equally to this work.

Address correspondence to: Andrea Graziani (andrea.graziani{at}med.unipmn.it).