Bacterial toxins and the Rho GTP-binding protein: what microbes teach us about cell regulation

 Cell Death and Differentiation (1998) 5, 720 ± 728 1998 Stockton Press All rights reserved 13509047/98 $12.00 http://www.stockton-press.co.uk/cdd Review Bacterial toxins and the Rho GTP-binding protein: what microbes teach us about cell regulation Carla Fiorentini1,3, Michel Gauthier2, Gianfranco Donelli1 and Patrice Boquet2 1 2 3 Department of Ultrastructures, Istituto Superiore di SanitaÁ, Viale Regina Elena 299, 00161 Rome, Italy INSERM U452 Faculte de MeÂdecine, avenue de Valombrose, 06107, Nice Cedex 2, France corresponding author: C. Fiorentini, tel: +39-6-49903006; fax: +39-6-49387140; e-mail: ® Received 22.12.97; revised 31.3.98; accepted 13.5.98 Edited by R.A. Knight Abstract In the present review activities of two bacterial toxins, Clostridium botulinum exoenzyme C3 and Escherichia coli CNF1, both acting on the GTP-binding protein Rho are analyzed. Proteins belonging to the Rho family regulate the actin cytoskeleton and act as molecular switches in a number of signal transduction pathways. C3 and CNF1 have opposite effects on Rho thus representing useful tools for studies on cell division, cell differentiation and apoptosis. Keywords: Rho; C3; CNF1; cell regulation Abbreviations: CNF, cytotoxic necrotizing factor; GEF, guanine exchange factor; GAP, GTPase activating protein; LPA, lysophospatidic acid; ROK, Rho kinase; PIP2, phosphatidyl-inositol-4,5phosphate; PI4-P, phosphatidyl inositol 4 phosphate; ECM, extracellular matrix; SRE, serum responsive element; SRF, serum response factor Introduction Almost 10 years separate the discovery of a toxin which exerts an inhibitory activity on the p21 Rho small GTPase: the Clostridium botulinum exoenzyme C3 (Aktories et al, 1987; Rubin et al, 1988; Chardin et al, 1989) from that of a toxin which activates the same GTP-binding protein: the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli (Flatau et al, 1997; Schmidt et al, 1997). Exoenzyme C3 and CNF1 are now major tools for laboratories working on the actin cytoskeleton and signal transduction. Aims of the present review are (i) to analyze the activities of these two bacterial toxins having opposite effects on the GTP-binding protein Rho; and (ii) to examine how these toxins can provide clues for explaining roles played by Rho in cell division, cell differentiation or apoptosis. The GTP-binding protein Rho: a target for bacterial toxins Rho protein (Madaule and Axel, 1985), discovered shortly after Ras (Chang et al, 1982), and YPT1 (a Rab-like GTPase from Saccharomyces cerevisiae) (Gallwitz et al, 1983), belong to a family of regulatory molecules now grouped under the name of `Ras superfamily'. This superfamily encompasses three main groups of proteins: Ras, Rho and Rab which differ according to their sequence homology and their function. A certain number of other GTP-binding molecules (Arf and Ran) which harbour similarities with Ras are now linked to this superfamily of proteins (Zerial and Huber, 1995). The Ras group (Ras, Rap and Ral) is implicated in signal transduction of mitogenic signals, the Rho, Rac and Cdc42 subgroup regulates the F-actin cytoskeleton and the Rab subfamily (Rab 1 to Rab 30) controls intracellular traffic (Downward, 1990). Small GTP-binding proteins are under an active form, and are thus able to trigger a cascade of signalling events when they are associated with GTP. Linked to GDP, they are in the resting state. An activated small GTP-binding protein becomes inactive by hydrolyzing GTP into GDP. GTP hydrolysis by small GTP-binding proteins alone is normally exceedingly slow. In association with a protein named GTPase activating protein (GAP), GTP hydrolysis is very rapid. Activation of small GTP-binding proteins is due to the removal of their bound GDP. The protein which performs this task is the guanine exchange factor (GEF). The simple removal of GDP from the small GTP-binding protein allows binding of GTP since there is a large excess in cells of GTP over GDP. Two polypeptide domains change their conformation in small GTP-binding proteins when the molecule is associated either with GTP or GDP. These polypeptides are called switches (Milburn et al, 1990). Switch 1 (residues 30 to 39 in Ras, 32 to 41 in Rho) corresponds to the Ras polypeptide contacting its downstream effector (in the case of Ras it is Raf) whereas switch 2 (residues 60 to 76 in Ras, 62 to 78 in Rho) is implicated in the GTP to GDP hydrolysis. Rho is mostly localized in the cytosol, associated with a molecule (guanine dissociation inhibitor GDI) which maintains its conformation in the inactive form (linked to GDP) (Fukumoto et al, 1990). When a growth factor (derived from a lipid, lysophosphatidic acid (LPA)) binds to its receptor (belonging to a family of receptors acting through heterotrimeric G proteins), it activates Rho via the Rho exchange factor at the level of the membrane. This mechanism and its precise localization are still poorly elucidated. Activated-Rho has two main targets for the regulation of the actin cytoskeleton: a serine-threonine kinase named Rho kinase (ROK) (Matsui et al, 1996; Ishizaki et al, 1996) and a kinase inducing, by phosphorylation (on position 5 of the inositol ring), the formation of CNF1 and C3 activities on Rho C Fiorentini et al 721 phosphatidyl-inositol-4,5-phosphate (PIP2) from phosphatidyl inositol 4 phosphate (PI4-P) (Chong et al, 1994; Ren et al, 1996). By regulating these two kinases, Rho might control the actin cytoskeleton by three mechanisms: (i) by acting on Rho kinase, it will provoke the bundling of actin filaments by directly (Amano et al, 1997) or indirectly (via phosphorylation of the myosin light chain phosphatase resulting in the inhibition of this enzyme) (Kimura et al, 1996) phosphorylating the myosin type 2 light chain allowing these molecules to associate with actin filaments and thereby provoking contractility (Fujihara et al, 1997); (ii) by locally raising the PIP2 concentration, Rho activates molecules bridging actin filaments and cell membraneassociated proteins such as vinculin (Gilmore and Burridge, 1996) ezrin, moesin or radixin (ERM group) (Hirao et al, 1996) and (iii) probably by provoking a PIP2 dependent actin polymerization (as described for Rac; Hartwig et al, 1995) by uncapping actin filament barbed ends (where addition of new actin subunits occurs). By these three mechanisms, Rho will allow extension of the cell surface (also called cell spreading). According to Cramer and Mitchison (1995), cell spreading results from actin polymerization at the cell periphery but also from the association of actin and myosin which induces cell contractility. Rho also induces, by a mechanism implicating ezrin (a protein belonging to the ERM group), the formation of focal adhesion contacts (Mackay et al, 1997). Focal contacts are structures by which cells are anchored to the extracellular matrix (ECM) via integrins. As we will see below, anchoring to ECMs through integrins is an indispensabl (...truncated)