Hindawi Publishing Corporation Journal of Signal Transduction Volume 2012, Article ID 570183, 10 pages doi:10.1155/2012/570183 Research Article Increased Cell-Matrix Adhesion upon Constitutive Activation of Rho Proteins by Cytotoxic Necrotizing Factors from E. Coli and Y. Pseudotuberculosis Martin May, 1 Tanja Kolbe, 1 Tianbang Wang, 1 Gudula Schmidt, 2 and Harald Genth 1 1 Institute for Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 2 Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, 79104 Freiburg, Germany Correspondence should be addressed to Harald Genth, genth.harald@mh-hannover.de Received 28 March 2012; Accepted 3 June 2012 Academic Editor: Kris DeMali Copyright © 2012 Martin May et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cytotoxic necrotizing factors (CNFs) encompass a class of autotransporter toxins produced by uropathogenic E. coli (CNF1) or Y. pseudotuberculosis (CNFy). CNF toxins deamidate and thereby constitutively activate RhoA, Rac1, and Cdc42. In this study, the effects of CNF1 on cell-matrix adhesion are analysed using functional cell-adhesion assays. CNF1 strongly increased cell-matrix binding of suspended Hela cells and decreased the susceptibly of cells to trypsin-induced cell detachment. Increased cell-matrix binding was also observed upon treatment of Hela cells with isomeric CNFy, that specifically deamidates RhoA. Increased cell- matrix binding thus appears to depend on RhoA deamidation. In contrast, increased cell spreading was specifically observed upon CNF1 treatment, suggesting that it rather depended on Rac1/Cdc42 deamidation. Increased cell-matrix adhesion is further presented to result in reduced cell migration of adherent cells. In contrast, migration of suspended cells was not affected upon treatment with CNF1 or CNFy. CNF1 and CNFy thus reduced cell migration specifically under the condition of pre-established cell-matrix adhesion. 1. Introduction Cell-matrix adhesion involves several processes including integrin binding, cell spreading, and flattening against the substrate. Cultured cells, that spread out on ligand coated surfaces, rearrange their cytoskeleton and begin to move. Integrins thereby cluster together in “focal complexes” at the leading edge. These focal complexes grow into mature focal contacts, also called focal adhesions (FAs) [1]. Focal adhe- sions contain over 100 different proteins, including integrins, adapter proteins, and intracellular signaling proteins. Clus- tered integrins anchor actin filaments to the cell membrane and link them with the extracellular matrix (ECM) through adapter proteins such as talin and vinculin. The adapter protein paxillin links integrins to signaling proteins, forming a scaffold for Src kinases, the focal adhesion kinase (FAK), or the p21-activated kinase (PAK) [2–5]. The turnover of FAs in moving cells is driven by small GTPases of the Rho subfamily. FA formation and disassembly at the leading edge is driven by Rac1 and the localized suppression of Rho activity. Disassembly of FAs at the cell rear requires RhoA activity [6]. The activity of Rho proteins is regulated by the GTPase cycle. Rho proteins are active in the GTP-bound state and inactive in the GDP-bound state. In their active conformation Rho proteins interact with effector proteins to transmit downstream signaling. The cycling between these states is governed by guanine nucleotide exchange factors (GEF) and GTPase activating proteins (GAP), which catalyse the exchange of GDP to GTP or stimulate the intrinsic GTP hydrolase, respectively. A critical amino acid for GAP-induced as well as for intrinsic GTPase activity is Gln-63 in RhoA (Gln-61 in Rac1 and Cdc42). Gln-63/61 is deamidated by cytotoxic necrotizing factors (CNF), a class of autotransporter toxins produced by uropathogenic E. coli (CNF1-3) or Y. pseudotuberculosis (CNFy) [7, 8]. Deamidation results in inhibition of GAP- induced as well as of intrinsic GTPase activity, resulting in so called “constitutively active” Rho proteins. CNF1-induced