SEARCH

SEARCH BY CITATION

Keywords:

  • actin cytoskeleton;
  • pathogenic bacterium;
  • post-translational modification;
  • Rho protein;
  • toxin;
  • virulence factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

Small GTPases of the Rho protein family are master regulators of the actin cytoskeleton and are targeted by potent virulence factors of several pathogenic bacteria. Their dysfunctional regulation can lead to severe human pathologies. Both host and bacterial factors can activate or inactivate Rho proteins by direct post-translational modifications: such as deamidation and transglutamination for activation, or ADP-ribosylation, glucosylation, adenylylation and phosphorylation for inactivation. We review and compare these unconventional ways in which both host cells and bacterial pathogens regulate Rho proteins.


Abbreviations used:
AMPylation

adenylylation

AT2R

angiotensin II type 2 receptor

AvrB

avirulence protein B

BACURD

BTB-containing adaptor for Cul3-mediated RhoA degradation

Cdc42

cell division cycle 42

CNF

cytotoxic necrotizing factor

DNT

dermonecrotic toxin

Doc

death on curing

EDIN

epidermal cell differentiation inhibitor

EMT

epithelial-to-mesenchymal transition

Fic

filamentation induced by cAMP

GAP

GTPase-activating protein

GDI

guanine-nucleotide-dissociation inhibitor

GEF

guanine-nucleotideexchange factor

HypE

Huntingtin yeast-interacting protein E

IbpA

immunoglobulin-binding protein A

LCT

large clostridial toxin

Par

cell polarity protein

PKA

cAMP-dependent kinase

PKG

cGMP-dependent kinase

Rnd

round

ROCK

p21-Rho kinase

SLK

Ste20-related kinase

Smurf

Smad ubiquitin regulatory factor

TGFR

transforming growth factor-β receptor

UPS

ubiquitin—proteasome system

VopS

Vibrio parahaemolyticus outer protein S

Yop

Yersinia outer protein

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

Rho proteins play essential roles in the regulation of virtually all actin-dependent processes (Jaffe and Hall, 2005; Heasman and Ridley, 2008). The primary sequences and functions of these proteins appear to be conserved in eukaryotes. Importantly, dysfunction of Rho-regulated signalling pathways is implicated in severe human diseases, such as cancer, mental retardation and immunological disorders (Ambruso et al., 2000; Sahai and Marshall, 2002; Nadif Kasri and Van Aelst, 2008).

Rho proteins also play a central role in the interactions between pathogenic bacteria and their hosts, in particular, by controlling essential aspects of innate and adaptive immune defences (Boquet and Lemichez, 2003; Bokoch, 2005). Not surprisingly, they are therefore directly targeted by numerous bacterial virulence factors (Chardin et al., 1989; Boquet and Lemichez, 2003; Aktories and Barbieri, 2005; Galan and Cossart, 2005). Two classes of bacterial virulence factors can be distinguished based on their mode of entry into the host-cell cytosol. The first, injected effectors, are proteins introduced by cell-bound bacteria through bacterial secretion apparatuses analogous to molecular syringes (Galan and Cossart, 2005). The second, exotoxins (also referred to as toxins), are secreted by bacteria into their environment to target distantly located host cells (Boquet and Lemichez, 2003; Aktories and Barbieri, 2005). These toxins bind to host-cell-surface receptors, enter cells by endocytosis and next translocate into the cytosol from endocytic compartments. Previous reviews have discussed how hijacking Rho proteins might benefit pathogenic bacteria by overwhelming host defences and facilitating bacterial invasion of host tissues (Boquet and Lemichez, 2003; Aktories and Barbieri, 2005; Bokoch, 2005; Galan and Cossart, 2005; Lemichez et al., 2010). This encompasses: (i) the paralysis of immune cells (i.e. inhibition of their migration and phagocytic properties), (ii) manipulation of host inflammatory responses, and (iii) breaching host-cell barriers by corrupting intercellular junctions and by triggering bacterial internalization into epithelial or endothelial cells for invasion.

Bacterial factors targeting Rho proteins are extremely diverse in terms of their structures and modes of action. Some virulence factors mimic Rho protein regulators (Galan and Cossart, 2005). Another group of virulence factors described in this review catalyse various types of direct post-translational modifications of Rho GTPases (Boquet and Lemichez, 2003; Aktories and Barbieri, 2005). The list of virulence factors and types of modifications of Rho proteins continue to grow. For example, Vibrio parahaemolyticus injected effector VP1686 [VopS (V. parahaemolyticus outer protein S)] and the virulence factor IbpA (immunoglobulin-binding protein A) of Histophilus somni were recently shown to catalyse AMPylation (adenylylation) of Rho proteins, unravelling a new type of post-translational modification of proteins potentially shared by host-cell factors (Worby et al., 2009; Yarbrough et al., 2009).

Decrypting the mode of action of bacterial virulence factors that target host Rho proteins directly has also provided new insights into the cellular regulation of these GTPases. Indeed, a growing number of studies point to the importance of various types of post-translational modifications of Rho proteins in physiology and pathophysiology. Our review aims to describe examples of host and microbial modifications of Rho proteins and draw parallels between their functions in physiology and during infection. We believe that comparisons of both systems should help to illuminate the complex regulation of these essential GTPases.

Rho GTPases

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

The family of Rho GTPases

Since the discovery of the Ras homologous protein, i.e. Rho protein, in Aplysia (Madaule and Axel, 1985), research on Rho proteins has established that these small GTPases are specialized in the regulation of the actin cytoskeleton in all eukaryotes (Jaffe and Hall, 2005; Heasman and Ridley, 2008). The human genome appears to encode approx. 20 Rho protein members, forming a distinct branch within the p21-Ras superfamily of small GTPases (Jaffe and Hall, 2005; Heasman and Ridley, 2008) (Figure 1). Much of the information regarding the regulation and function of this subfamily of proteins comes from studies on the GTPases RhoA, Rac1 and Cdc42 (cell division cycle 42). Most Rho proteins have the capacity to bind the guanine nucleotides GDP and GTP, as well as to hydrolyse GTP into GDP (Vetter and Wittinghofer, 2001). Several residues depicted in Figure 2, notably glutamine-63 of RhoA (glutamine-61 in Rac and Cdc42), a hot spot for post-translational modifications, are essential for hydrolysis of the γ-phosphate of GTP. Transition between both guanine-nucleotide-bound forms of Rho proteins produces conformational changes in the two flexible regions surrounding the γ-phosphate, referred to as the region switch I and II (Vetter and Wittinghofer, 2001) (Figure 2). Binding to GTP allows the switch I to bind to and activate downstream effector proteins.

image

Figure 1. Phylogenetic tree of Rho family members

Rho GTPases are classified into eight subfamillies. Amino-acid sequences of the 21 Rho GTPases were aligned using ClustalW program (http:www.ebi.ac.ukToolsclustalw2). The tree was generated by using the iTOL sofware (http:itol.embl.deindex.shtml).

Download figure to PowerPoint

image

Figure 2. GTPase essential residues and sites of post-translational modification

Sequence alignment of the primary sequences of RhoA, Rac1 and Cdc42. The switch I and II regions are highlighted in grey. Residues in bold correspond to the different G-boxes. The conserved C-terminal CAAX-box (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif is shown in italics. Known residues modified by post-translational modifications and the type of modification are indicated in red.

Download figure to PowerPoint

Function of Rho GTPases

Rho proteins play key roles as signalling molecules controlling the organization of the actin cytoskeleton downstream of cell-surface signalling receptors (Ridley and Hall, 1992; Ridley et al., 1992; Nobes and Hall, 1995). Rho GTPases control the polymerization and branching of actin filaments through their interplay with specific effector proteins at the interface of membranes (Jaffe and Hall, 2005; Le Clainche and Carlier, 2008; Heasman and Ridley, 2008). RhoA also controls the ability of myosin-II to bundle actin filaments into contractile actin cables. The control of actin cytoskeleton polymerization and organization allows Rho proteins to shape the morphology of cells, control the progression of cell division, co-ordinate the adhesion and cohesion of cells in endothelial and epithelial tissues, and drive cell migration and phagocytosis (Etienne-Manneville and Hall, 2002). Thus Rho proteins influence the organization and dynamic of cellular membranes and cohesion of cells in tissues.

By modulating the activity of various regulators of transcription Rho GTPases also control cell differentiation and survival. Regulation of transcription by Rho proteins can be direct as a consequence of the binding and activation of various signalling factors by Rho GTPases (Jaffe and Hall, 2005). Control of transcription can also be indirect as a consequence of both the production of various secondary messengers and the regulation of actin treadmilling (Posern and Treisman, 2006; Le Clainche and Carlier, 2008).

An apparently unique feature of Rho proteins, relative to other small GTPases, is their remarkable tendency to be targeted by bacterial virulence factors (Boquet and Lemichez, 2003; Aktories and Barbieri, 2005; Galan and Cossart, 2005). This has shed light on the function of Rho proteins as central elements of the hosts' innate and adaptive defences against pathogens (Boquet and Lemichez, 2003; Bokoch, 2005). For example, Rho proteins control the cohesion of the epithelial and endothelial layers, which form barriers that limit pathogen dissemination in tissues. Moreover Rho GTPases control cell death and thus epithelial cell renewal, a phenomenon also considered as an innate mechanism of defence aimed at restricting bacterial colonization of the epithelium. In addition, Rho proteins co-ordinate cellular defences by triggering the production of various chemokines in response to the perception of pathogens, and promote the migration of leucocytes to the site of infection (Arbibe et al., 2000). Some of these GTPases, particularly the Rac1 homologous protein Rac2 that is expressed in haematopoietic cells, control processes such as phagocytosis and the production of reactive oxygen species involved in micro-organism destruction (Diebold and Bokoch, 2005).

Regulation of Rho GTPases

Signals that emanate from cell-surface receptors are relayed to Rho GTPases by GEFs (guanine-nucleotide-exchange factors), which catalyse the release of GDP (Vetter and Wittinghofer, 2001). As the intrinsic kinetics of GDP release are slow, it needs to be stimulated by GEFs (Rossman et al., 2005) (Figure 3a). As for guanine-nucleotide exchange, the intrinsic GTPase activity of Rho proteins is slow. In order to terminate the signal, the hydrolysis of the γ-phosphate of GTP has to be catalysed by GAPs (GTPase-activating proteins) (Moon and Zheng, 2003) (Figure 3a).

image

Figure 3. Examples of post-translational modifications of Rho proteins

(a) Glucosylation or AMPylation of the switch I domain of Rho GTPases block their capacity to contact and activate effector proteins. (b) Proteolysis of the prenylated C-terminus of Rho proteins triggers their release into the cytosol. ADP-ribosylation and phosphorylation of RhoA trigger a tight association of RhoA with GDIs, the release of the complex from the membrane and trapping of RhoA into cytosol. (c) Various types of transglutamination of the conserved glutamine-63 of RhoA (glutamine-61 of Rac1 and Cdc42) abolish the intrinsic and GAP-stimulated GTPase activity of Rho GTPases, leading to their permanent activation. Activation of Rho proteins is followed by their ubiquitination and targeting to proteasomal degradation.

Download figure to PowerPoint

Rho proteins can cycle between the cytosol and membranes, their sites of action. Rho proteins are prenylated by geranylgeranylation on a conserved cysteine residue of the CAAX-box located at their C-termini for membrane anchorage (Figure 2). In addition, a stretch of positively charged amino acids, at the C-terminus of Rac1, probably drives its association to specific membrane locations (Yeung et al., 2006). The distribution of Rho proteins between membrane and the cytosol, and their activation by GEFs, are controlled by the family of GDIs (guanine-nucleotide-dissociation inhibitors) (Vetter and Wittinghofer, 2001; DerMardirossian and Bokoch, 2005). GDIs can hide the prenylated C-terminal part of Rho proteins and extract these proteins from the lipid environment of membranes (Figure 3a).

The GEF, GAP and GDI factors represent the core machinery controlling the spatio-temporal regulation of most Rho proteins. Nevertheless, recent advances have revealed that some Rho proteins have atypical biochemical properties (Aspenstrom et al., 2007; Heasman and Ridley, 2008). For example, the Rnd1 (Round 1), 2 and 3 proteins and RhoH have substitution of key amino-acid residues at positions glycine-14 and glutamine-63 of RhoA (Figure 2). These substitutions are used to generate dominant-positive mutants of RhoA, suggesting that Rnds and RhoH atypical GTPases are permanently bound to GTP and thus constitutively active (Chardin, 2006). The RhoU/WRCH1 (Wnt-responsive Cdc42 homologue) protein shows an increased property of nucleotide exchange, as compared with classic Rho GTPases (Aspenstrom et al., 2007). Finally, the abundance of RhoB, which is palmitoylated and permanently bound to endomembranes, is regulated at the levels of both transcriptional and protein degradation (Engel et al., 1998).

That atypical Rho proteins escape regulation by GEF, GAP or GDI factors highlights the importance of studying other modes of regulation of small GTPases.

Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

Direct inactivation of Rho proteins

Inactivation by blocking effector binding

Pathogens commonly inactivate Rho GTPases by blocking their binding to effectors through cross-linking bulky molecules to amino-acid residues located in the switch I region. Threonine-37 of RhoA (threonine-35 of Rac and Cdc42) is a hot spot for modifications by bacterial virulence factors, given its major role in contacting many Rho protein effectors (Figures 2 and 3a). This threonine residue is modified by a group of LCTs (large clostridial toxins), which trigger mono-glucosylation or N-acetyl-glucosaminylation of small GTPases (Just et al., 1995) (Table 1). Intoxication of cells by LCTs leads to disruption of the actin cytoskeleton, resulting in cell retraction, rounding and detachment. Interestingly, among the small GTPases tested for their sensitivity to these toxins, Rac1 (and potentially Rac2) was the only one modified by all toxin members (Table 1). This is probably related to the central role that these GTPases play in controlling inflammatory responses and the production of reactive oxygen species for pathogen destruction (Boquet and Lemichez, 2003; Bokoch, 2005).

Table 1.  Examples of post-translational modifications induced by bacterial virulence factors targeting Rho GTPasesResidues are given in single-letter code.
ModificationsVirulence factorsPathogensTargetsResiduesReferences
EndoproteolysisYopTYersinia enterocoliticaRhoA, Rac, Cdc42C-terminus (see Figure 2)Shao et al. (2003); Fueller and Schmidt (2008)
ADP-ribosylationC3 exoenzyme (C3bot)Clostridium botulinumRhoA>RhoB>RhoC (>>Rac1)N41 of RhoA (and equivalent)Chardin et al. (1989); Wilde et al. (2003)
 C3-like transferase (C3lim)Clostridium limosumRhoA>RhoB, RhoC Just et al. (1992b)
 C3-like transferase (C3cer)Bacillus cereusRhoA, RhoB>RhoC Just et al. (1992a); Wilde et al. (2003)
 EDIN-A, −B, −C (C3stau −1, −2, −3)Staphylococcus aureusRhoA>RhoB>RhoC (>>Rnd3 for EDIN-A) Yamaguchi et al. (2001, 2002); Wilde et al. (2003)
GlucosylationToxin A and BClostridium difficile (C. difficile VPI 10463)Rac, RhoA, Cdc42, not RhoDT37 of RhoA (and equivalent)Just et al. (1995); Jank et al. (2006)
 Toxin B-1470Clostridium difficile (C. difficile strain 1470)Rac, Cdc42, R-Ras, Ral, Rap, not RhoAT35 of Rac1 (and equivalent)Chaves-Olarte et al. (1999)
 Lethal toxinClostridium sordelliiRac, Cdc42, RhoG, Ras, Ral, Rap, not RhoAT35 of Rac1 (and equivalent)Popoff et al. (1996); Jank et al. (2006)
 Haemorrhagic toxinClostridium difficileRhoA, Rac, Cdc42T37 of RhoA (and equivalent)Genth et al. (1996)
N-acetyl-glucosaminylationAlpha-toxinClostridium novyiRhoA, Rac, Cdc42T37 of RhoA (and equivalent)Selzer et al. (1996)
AMPylationVopSVibrio parahaemolyticusRhoA, Rac, Cdc42T35 of Rac (and equivalent)Yarbrough et al. (2009)
 IbpAHistophilus somniRhoA, Rac, Cdc42Y34 of RhoA (and equivalent)Worby et al. (2009)
Transglutamination/deamidationDNTBordetella bronchisepticaRhoA, Rac, Cdc42Q63 of RhoAHoriguchi et al. (1997)
 CNF1Uropathogenic Escherichia coliRhoA, Rac, Cdc42, not RhoDQ63 of RhoA (and equivalent)Flatau et al. (1997); Schmidt et al. (1997); Jank et al. (2006)
 CNF2Enteropathogenic Escherichia coliRhoA, Rac, Cdc42 Stoll et al. (2009)
 CNF3Necrotoxigenic Escherichia coliRhoA, Rac, Cdc42 Orden et al. (2007)
 CNFYYersinia pseudotuberculosisRhoA>Rac, Cdc42 Hoffmann et al. (2004)

Exciting new findings have unravelled a new type of modification affecting residues of the switch I region (Figures 2 and 3a). The injected effector VopS of V. parahaemolyticus catalyses the covalent attachment of a molecule of AMP from ATP to the threonine-37 of RhoA (threonine-35 of Rac and Cdc42) (Yarbrough et al., 2009) (Table 1 and Figure 2). This covalent modification by AMPylation triggers an inactivation of Rho proteins and the ensuing destruction of the actin cytoskeleton (Yarbrough et al., 2009). The covalent attachment of AMP by a phosphodiester bond is predicted to be reversible (Kinch et al., 2009). This activity depends on a conserved histidine residue of the Fic (filamentation induced by cAMP) domain of VopS. The bacterial virulence factor IbpA of the respiratory pathogen H. somni also disrupts the actin cytoskeleton (Worby et al., 2009). It bears two Fic domains at its C-terminus. One of the two Fic domains (Fic2) of IbpA was recently shown to catalyse the AMPylation of RhoA, Rac1 and Cdc42 (Worby et al., 2009). Here, however, AMP is cross-linked to another residue of the switch I region: the conserved tyrosine residue at position 34 of RhoA (tyrosine-32 of Rac and Cdc42) (Table 1 and Figure 2). Much remains to be learnt about this newly discovered enzymatic reaction, which may be shared by eukaryotes. For example, the primary sequences of the Fic domains of IbpA are homologous with the Fic domain of the human HypE (Huntingtin yeast-interacting protein E) (Faber et al., 1998; Kinch et al., 2009). HypE, but not the mutant HypE (H295A), can adenylylate recombinant RhoA, Rac and Cdc42 and catalyses the modification of proteins of similar size in cell extracts (Worby et al., 2009) (Table 2). Nevertheless, expression of HypE in cells does not trigger a collapse of the actin cytoskeleton. This might be due to a tight regulation of its activity in cells. A Fic-like-domain-containing protein from Drosophila melanogaster shows auto-adenylylation activity in vitro (Kinch et al., 2009). Two other domains found in proteins, named Doc (death on curing), encoded by phage P1 of Escherichia coli, and the injected effector AvrB (avirulence protein B) of Pseudomonas syringae, display sequence and structural homologies with Fic domains respectively. Kinch et al. (2009) have proposed that these domains be unified with Fic domains to create a single superfamily of functional domains named Fido (for Fic, Doc and AvrB). It remains to be shown whether Doc and AvrB also confer AMPylation, and whether this activity is directed toward Rho GTPases.

Table 2.  Examples of post-translational modifications of Rho GTPasesResidues are given in single-letter code. CK1, casein kinase 1.
ModificationsEnzymesTargetResiduesReferences
AMPylationHypERhoA, Rac, Cdc42Y34 (RhoA)Worby et al. (2009)
TransglutaminationTransglutaminaseRhoAQ63Schmidt et al. (1998); Singh et al. (2001)
 Transglutaminase (serotonin)RhoA, Rac1, not Ras Walther et al. (2003); Dai et al. (2008)
PhosphorylationPKARhoAS188Lang et al. (1996)
 PKGRhoAS188Sauzeau et al. (2000); Rolli-Derkinderen et al. (2005)
 SLKRhoAS188Savoia et al. (2005)
 Src kinaseCdc42Y64Tu et al. (2003)
 AktRac1S71Kwon et al. (2000)
 ROCKRhoES11Riento et al. (2005)
 CK1 Ser/Thr kinaseRhoBS185Tillement et al. (2008)
UbiquitinationSmurf1RhoAK6, 7Wang et al. (2003); Ozdamar et al. (2005); Boyer et al. (2006a)
 Cul3/BACURDRhoA Chen et al. (2009)
  Rac1K147Doye et al. (2002); Lynch et al. (2006); Visvikis et al. (2008)
  Cdc42 Doye et al. (2002)
Inactivation by triggering the release of Rho proteins from the membrane

One way of inactivating Rho proteins is to remove these GTPases from the membrane. This can be achieved by cleaving the C-terminal part of Rho proteins containing the CAAX-box (Figures 2 and 3b). Indeed, Yersinia enterocolitica synthesizes the injected effector YopT (Yersinia outer protein T) (Shao et al., 2003). YopT is a cysteine protease with an endopeptidase activity that cleaves the C-terminal part of RhoA, Rac1 and Cdc42, thus severing their lipid anchor (Shao et al., 2003; Fueller and Schmidt, 2008) (Figure 2). This in turn triggers a release of the active form of Rac1 from membranes, resulting in its translocation to the nucleus (Wong and Isberg, 2005).

Another way to release Rho proteins from membranes is to corrupt their interaction with GDIs (Figure 3b). This strategy is shared by both cellular and bacterial factors and is best studied for the GTPase RhoA (Boquet and Lemichez, 2003; Aktories and Barbieri, 2005; Loirand et al., 2006). Various cellular kinases can phosphorylate RhoA, notably both the cAMP- and cGMP-dependent kinases, PKA and PKG respectively (Lang et al., 1996; Sauzeau et al., 2000, 2003; Loirand et al., 2006) (Table 2 and Figure 2). Phosphorylation of RhoA at serine-188 does not block its GTP-loading or its GTPase activity (Ellerbroek et al., 2003). Instead, both in vitro and in vivo studies show that this modification triggers a tight association of RhoA with GDIs and the extraction of RhoA from membranes (Lang et al., 1996; Sauzeau et al., 2000; Ellerbroek et al., 2003) (Figure 3b). Another study shows that, in vitro, the phosphorylation of RhoA blocks its association with ROCK (p21-Rho kinase), whereas it does not impair its association with rhotekin or mDIA1 (mammalian homologue of Drosophila Diaphanous protein 1) (Nusser et al., 2006). Phosphorylation of RhoA thus inactivates or eventually modulates the activity of RhoA. In contrast with regulation by GAPs, this may maintain a cytosolic pool of RhoA inaccessible to activation by GEFs, or, as suggested previously, a reservoir of GTP-loaded RhoA that can be mobilized in the absence of activation by GEFs (Loirand et al., 2006).

Several studies have also suggested a possible phosphorylation of other Rho proteins, such as Rac1 and Cdc42, at different positions (Table 2). As for RhoA, the phosphorylation of Cdc42 on tyrosine-64 by Src tyrosine kinase increases its association with GDIs (Tu et al., 2003). It has been suggested that the phosphorylation of serine-71 of Rac1 by the serine/threonine kinase Akt impairs the GTP-binding activity of Rac1 (Kwon et al., 2000). Phosphorylation might also contribute to regulation of atypical Rho GTPases, such as Rnd3 (RhoE), RhoH and RhoB (Riento et al., 2005; Gu et al., 2006; Tillement et al., 2008). Indeed, it has been shown by Riento et al. (2005) that the phosphorylation of serine-11 of Rnd3 by ROCKs in response to cell stimulation by PDGF (platelet-derived growth factor) stabilizes this GTPase and thus potentiates Rnd3 activity.

Phosphorylation of RhoA has been implicated in the regulation of vascular homoeostasis (Pacaud et al., 2005; Loirand et al., 2006). Studies involving the cardiovascular system implicate the phosphorylation of RhoA in smooth muscle cells as a key regulatory mechanism in blood vessels to prevent vascular dysfunction (Loirand et al., 2006). Indeed, the contraction of actomyosin cables driven by RhoA and its effector ROCK triggers vasoconstriction. Abnormal vasoconstrictions lead to vascular diseases, such as pulmonary hypertension (Pacaud et al., 2005). Consistently, expression of a phospho-resistant mutant of RhoA in smooth muscle of transgenic mice remodels the vascular system by causing perivascular fibrosis and coronary artery wall thickening (Sawada et al., 2009). Two important feedback regulations have been described. Vasodilatation can be induced by the NO/cGMP-dependent kinase PKG (Pacaud et al., 2005) and can be triggered by SLK (Ste20-related kinase) upon activation by the AT2R (angiotensin II type 2 receptor) pathway (Guilluy et al., 2008). Both the NO/PKG and AT2R/SLK pathways induced the phosphorylation of serine-188 of RhoA as a mechanism aimed at counteracting the effect of actin cable contraction mediated by RhoA in order to produce smooth muscle cell relaxation (Rolli-Derkinderen et al., 2005; Savoia et al., 2005).

To date, no bacterial virulence factors have been found to inactivate Rho proteins by phosphorylation. However, a parallel can be drawn with a group of virulence factors that inactivate RhoA by mono-ADP-ribosylation (Chardin et al., 1989; Wilde et al., 2003) (Table 1 and Figure 3b). In vitro experiments show that, although these ADP-ribosyltranferases can modify various Rho members, the C3 toxin of Clostridium botulinum and EDIN (epidermal cell differentiation inhibitor) of Staphylococcus aureus have a preference for RhoA (Wilde et al., 2003). ADP-ribosylation of asparagine-41 of RhoA in vitro does not block its binding to the PLD (phospholipase D) effector protein (Sekine et al., 1989; Genth et al., 2003). In contrast, several studies have established that in vivo ADP-ribosylation of RhoA induces its tight association with inhibitory factors (GDIs), leading to RhoA inactivation (Figure 3b) (Fujihara et al., 1997; Genth et al., 2003). Inhibition of RhoA by ADP-ribosyltransferases triggers various effects that depend on host cell types, including the paralysis of immune cell functions and the disruption of tight junctions in gut epithelial cell monolayers (Nusrat et al., 1995; Boquet and Lemichez, 2003). Rho GTPases, especially RhoA, regulate the formation of intercellular junctions and consequently endothelium permeability (Wojciak-Stothard and Ridley, 2003). A new cellular outcome of RhoA inhibition by ADP-ribosylation is observed when endothelial cells are intoxicated with either the ADP-ribosyltransferase C3 of C. botulinum or EDIN of S. aureus (Boyer et al., 2006b). Intoxication of various primary endothelial cell types with either of these toxins results in a dramatic loss of cellular actin cables (Boyer et al., 2006b). Intriguingly, this loss of actin cables in primary endothelial cells does not lead to cell retraction, as observed upon RhoA, Rac1 and Cdc42 inhibition by LCTs glucosylating toxins of Clostridium difficile. This instead leads to the formation of transcellular tunnels named macro-apertures (Boyer et al., 2006b). These tunnels form transiently, due to membrane-wave-driven closure. Formation and closure of macro-apertures by EDIN can be recapitulated by knocking down RhoA using RNAi (RNA interference). Interestingly, macro-apertures show some structural homologies with transmigratory cups that form in endothelial cells during the transcellular diapedesis of leucocytes (Carman and Springer, 2008). However, the mechanism by which leucocytes drive the formation of transcellular tunnels remains largely unknown. Endothelium intoxication by EDIN leads to a loss of barrier function (Boyer et al., 2006b; Rolando et al., 2009). These tunnels confer to S. aureus the ability to bind to the extracellular matrix beneath the endothelium (Boyer et al., 2006b; Rolando et al., 2009). This phenomenon may favour the bacterial colonization of the endothelium and potentially promote the dissemination of these pathogens in host tissues.

Direct activation of Rho proteins and their sensitization to ubiquitination

Permanent activation of Rho proteins

To date, all post-translational modifications described to activate Rho proteins target an essential glutamine residue of the switch II region (Figures 2 and 3c). The carboxamide nitrogen of this glutamine stabilizes the transition state during GTP hydrolysis and is thus essential to the intrinsic and GAP-stimulated GTPase activity of small GTPases. The first evidence that Rho proteins can be activated by post-translational modification came from studies carried out on CNF1 (cytotoxic necrotizing factor 1) toxin of uropathogenic E. coli (Flatau et al., 1997; Schmidt et al., 1997) (Table 1). In vitro, CNF1 catalyses the deamidation of glutamine-63 of RhoA (glutamine-61 of Rac1 and Cdc42) into a glutamic acid, reducing its intrinsic and GAP-stimulated GTPase activity (Figure 3c). This triggers a permanent activation of Rho proteins in host cells (Flatau et al., 1997; Schmidt et al., 1997; Lerm et al., 1999). DNT (dermonecrotic toxin) of Bordetella bronchiseptica deamidates and transglutaminates Rho proteins by catalysing the covalent cross-linking of biogenic amines and polyamines on to this conserved glutamine residue, also triggering an activation of Rho proteins (Masuda et al., 2000; Schmidt et al., 2001). These toxins belong to a family of virulence factors found in various clinical isolates of Gram-negative bacteria (Lemonnier et al., 2007) (Table 1).

Other examples of transglutamination of RhoA downstream of several physiological stimuli have now been reported (Table 2). For instance, activation of tissue transglutaminase-II by retinoic acid triggers the activation of RhoA/ROCK signalling (Singh et al., 2001). This leads to the formation of actin stress fibres and focal adhesions during cell differentiation (Singh et al., 2001). The vasoconstrictor 5-HT (5-hydroxytryptamine; serotonin) can also be cross-linked in vivo by transglutaminases to RhoA and Rac1 (Walther et al., 2003; Dai et al., 2008). The modification of RhoA by serotonylation triggers its activation and the release of α-granules in platelets (Walther et al., 2003). Serotonylation of RhoA was also proposed as a possible risk factor of pulmonary artery remodelling in hypertension (Guilluy et al., 2007).

Modification of Rho proteins by ubiquitination

Post-translational modifications leading to the permanent activation of RhoA, Rac1 and Cdc42 are counteracted by the depletion of activated-Rho proteins through the UPS (ubiquitin—proteasome system) (Figures 2 and 3c) (Doye et al., 2002). Ubiquitination involves the covalent attachment of ubiquitin, an 8-kDa polypeptide, to lysine residues on the target through a cascade of transfer reactions between ubiquitin-carrier proteins (Kerscher et al., 2006). Additional molecules of ubiquitin can subsequently be attached to one of the seven lysine residues of the previously cross-linked ubiquitin molecule, leading to the formation of various types of polyubiquitin chains. Permanent activation of Rho proteins by the CNF1 toxin was shown to be only transient, because of their cellular depletion (Doye et al., 2002; Munro et al., 2007). Depletion of Rho proteins is triggered by a classical lysine-48 polyubiquitination, a signal targeting proteins to the proteasome degradative machinery. Ubiquitin-mediated degradation of Rac1 is triggered by cell intoxication with DNT (Schmidt et al., 2001; Doye et al., 2006). Rac1 is also regulated by ubiquitin-mediated proteasomal degradation in cells stimulated by HGF (hepatocyte growth factor) (Lynch et al., 2006). Studies on the ubiquitination of Rac1 in response to their activation by CNF1 have shown that both the activation of Rac1 and its targeting to cellular membranes are required for Rac1 ubiquitination (Doye et al., 2006). Ubiquitin chains are primarily cross-linked to two lysine residues located at the N-terminus of RhoA and preferentially to lysine-147 of Rac1 (Ozdamar et al., 2005; Visvikis et al., 2008) (Figure 2). In this case, a cycle comprising Rho synthesis, permanent activation by the toxin and degradation was proposed to substitute for a classical GTPase-based cycle (Doye et al., 2002). The UPS-mediated control of Rho proteins drives both epithelial cell junction disassembly and epithelial cell motility within monolayers, also promoting phagocytosis by epithelial cells for bacterial internalization (Doye et al., 2002). Degradation of Rho proteins may also dampen the inflammatory responses resulting from Rho protein activation (Munro et al., 2004). These cellular phenomena driven by CNF1 are thus likely to accentuate the invasive capacities of uropathogenic E. coli producing this toxin.

Ubiquitination probably plays an important role in controlling the function of Rho proteins. The identification of Smurf1 (Smad ubiquitin regulatory factor 1) as the ubiquitin ligase of RhoA highlighted the importance of this regulation beyond infection, as it is also relevant during EMT (epithelial-to-mesenchymal transition) (Wang et al., 2003; Ozdamar et al., 2005). Indeed, Par6 (cell polarity protein 6) associates with TGFRs (transforming growth factor-β receptors) and is a substrate of TGFRII. Phosphorylation of Par6 then triggers its association with Smurf1 and the subsequent ubiquitin-mediated degradation of RhoA. This leads to the disruption of RhoA-regulated tight junctions of mammary gland epithelial cells, resulting in EMT (Ozdamar et al., 2005). Smurf1 is also the ubiquitin ligase responsible for CNF1-induced RhoA ubiquitination, indicating that it probably ubiquitinates the GTP-bound permanently activated form of RhoA (Boyer et al., 2006a). Smurf1 is an E3 ubiquitin ligase of the Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4)-like family containing a HECT (homologous with E6-associated protein C-terminus) catalytic domain (Chen and Matesic, 2007). Its knockdown triggers an increase of bone mass in mice (Yamashita et al., 2005). Smurf1 ubiquitinates several cellular targets. The contribution of the regulation of RhoA by ubiquitination to this phenotype thus remains to be defined. Of note, post-translational modifications of Rho proteins can interfere with each other. For instance, the phosphorylation of RhoA at serine-188 increases its interaction with GDIs and protects RhoA from Smurf1-induced ubiquitination (Rolli-Derkinderen et al., 2005). Finally, ubiquitination of Rho proteins occurs in response to their activation by serotonylation (Walther et al., 2003; Guilluy et al., 2007). Indeed, the transfer of serotonin on to Rho GTPases by transglutaminases potentiates the activation of RhoA, as well as its association with Smurf1 and ubiquitin-mediated proteasomal degradation (Guilluy et al., 2007). These findings have been complemented by studies on CNF1, which observed that ubiquitination of RhoA, Rac1 or Cdc42 is impaired in various cancer cell lines. These suggest: (i) a possible link between Rho protein ubiquitination and cancer, and (ii) that ubiquitination of Rho proteins is controlled by several pathways and potentially several ubiquitin ligases (Boyer et al., 2006a).

A recent work described that the GDP-loaded form of RhoA and its dominant-negative form are prone to ubiquitination by the SCF (Skp1-Cullin1-F-box protein)-like multimeric E3 ligase Cul3/BACURD (BTB-containing adaptor for Cul3-mediated RhoA degradation) (Table 2). RhoA-GDP ubiquitination by Cul3/BACURD, as by Smurf1, leads to its proteasomal degradation (Chen et al., 2009). In contrast with suppression of Smurf1, the knockdown of Cul3/BACURD increases cellular levels of RhoA. The Cul3/BACURD-dependent degradation of RhoA seems to play a role in cell migration by maintaining an appropriate cellular level of RhoA-GDP to be activated by GEFs (Chen et al., 2009). Both the Cul3/BACURD and Smurf1 ubiquitin-ligase systems probably affect different guanine-nucleotide-bound forms of RhoA.

Much remains to be learnt about the biochemical properties of ubiquitinated Rho proteins. Apart from its role in targeting substrates for proteasomal degradation, ubiquitination is now viewed as a molecular ‘bar code’ that directs the formation of multiprotein complexes. One may thus envisage that ubiquitination of activated Rho proteins might further increase their repertoire of biochemical properties.

Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

The study of the function of Rho proteins and their post-translational regulations from both a host and microbial perspective has shed light on the importance and mode of action of this class of small GTPases. Two apposed regulatory circuits appear to co-ordinate the activity of Rho proteins: (i) the canonical regulations of Rho GTPases by GEFs, GAPs and GDIs, and (ii) their regulation by direct post-translational modifications, as discussed in this review. The hierarchy and function of these two types of regulatory mechanisms remain to be clearly defined. Determining the various cellular actors that drive post-translational modifications of Rho proteins will help to reveal the site of these various modes of regulation of Rho proteins and their importance during bacterial infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

We thank L. Boyer, G. Gacon, M.R. Popoff and J. Bertoglio for fruitful discussions and comments on the review. We apologize to all those authors in the field whose papers we could not cite because of space limitations.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References

Work in our laboratory is supported by INSERM; a grant and fellowship to O.V. from the Agence Nationale de la Recherche [grant number ANR A05135AS]; and a grant and fellowship to M.P.M. from the Association pour la Recherche sur le Cancer [grant number ARC 4906].

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Rho GTPases
  5. Covalent modifications of Rho GTPases induced by cellular proteins and bacterial virulence factors
  6. Perspectives
  7. Acknowledgements
  8. Funding
  9. References