Abstract
Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.
SUPPLEMENTAL MATERIAL
We thank Brigitte Denker for excellent technical assistance, Lothar Gröbe and Maria Höxter for FACS, Tadaomi Takenawa for kindly providing WAVE2 knockout cells, Gustav Bernroider for help with statistics, and Juergen Wehland for providing Listeria strains and for helpful discussions.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 621 to K.R. and SPP1150 to T.E.B.S. and K.R.), Associazione Italiana Ricerca sul Cancro (AIRC) (to G.S.), and Human Science Frontier Program (RGP0072/2003-C to G.S.). T.B. was supported by a Georg-Christoph-Lichtenberg fellowship of Lower Saxony.