Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Rac1, RhoA, and Cdc42 participate in HeLa cell invasion by group B streptococcus

The group B streptococcus (GBS) is an important human pathogen with the ability to cause invasive disease. To do so, the bacteria must invade host cells. It has been well documented that GBS are able to invade a variety of nonphagocytic host cell types, and this process is thought to involve a number of pathogen-host cell interactions. While some of the molecular aspects of the GBS-host cell invasion process have been characterized, many events still remain unclear. The objective of this investigation was to evaluate the role of the Rho-family GTPases Rac, Rho, and Cdc42 in GBS invasion into epithelial cells. The epithelial cell invasion process was modeled using HeLa 229 cell culture. Treatment of HeLa cells with 10 microM compactin, a pan-GTPase inhibitor, abolished GBS internalization, suggesting that GTPases are involved in the GBS invasion process. The addition of Toxin B or exoenzyme C3 to HeLa cells before GBS infection reduced invasion by 50%, further suggesting that the Rho-family GTPases are involved in GBS entry. Examining invasion of GBS into HeLa cells with altered genetic backgrounds was used to confirm these findings; GBS invasion into HeLa cells transiently transfected with dominant negative Rac1, Cdc42, or RhoA reduced invasion by 75%, 51%, and 42%, respectively. Results of this study suggest that the Rho-family GTPases are required for efficient invasion of HeLa cells by GBS.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.     

Regulation of calcium signalling by the small GTP-binding proteins Ras and Rac1

In order to investigate a possible interaction of the small GTP-binding proteins Ras and Rac1 with Ca²⁺-mediated signalling cascades the effects of dominant negative mutants of Ras and Rac1 on Ca²⁺ signalling have been studied after stimulation of either the EGFR or the nerve growth factor receptor (TRK). Expression of dominant negative Ras blocks the release of Ca²⁺ from internal stores after activation of EGFR whereas the calcium signal elicited by the activated TRK receptor is unaffected. The sensitivity to dominant negative Ras is determined by the structure of the PLCγ-binding sites of the corresponding receptors. Exchange of the PLCγ-binding domain of the EGFR by the PLCγ-binding site of TRK renders the EGFR-induced calcium signal insensitive to the expression of dominant negative Ras. Substitution of the PLCγ-binding site of TRK by the PLCγ-binding region of EGFR renders TRK sensitive to dominant negative Ras. The inhibition of Ca²⁺ release by dominant negative Ras is accompanied by a reduction in PLCγ binding to the EGFR and a concomitant decrease of EGF-induced inositol-1,3,5-trisphosphate (InsP₃) formation. The depression of PLCγ binding to EGFR is explained by a competition of PLCγ with other SH2-domain containing proteins for the same low affinity binding regions of the EGFR. This conclusion is supported by the observation that microinjection of several SH2-domain containing proteins including Ras-GP, lipase-free fragment of PLCγ or Janus kinase binding protein (JAB), reduces the association of PLCγ to the EGFR, not, however, to TRK. In contrast to dominant negative Ras which does not affect the Ca²⁺ transient induced by the activation of the TRK receptor, a dominant negative mutant of Rac significantly depresses the Ca²⁺ signals induced by EGFR as well as by TRK. The different behavior of Rac and Ras supports the notion that the two small GTP-binding proteins act through separate pathways. It is demonstrated that dominant negative Rac significantly reduces the formation of phosphatidylinositol-4,5-bisphosphate (PIP₂), the substrate of PLCγ. This effect is not observed after expression of dominant negative Ras. In summary, the data provide further evidence for a cross-talk between small GTP-binding proteins and Ca²⁺ signalling in which both G-proteins interfere with the formation of InsP₃ although by different mechanisms.     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

Immunodetection of Rho-like plant proteins with Rac1 and Cdc42Hs antibodies

A few small GTP-binding proteins of the Ras superfamily have been identified in plants, including members of the Rho family: the proteins belonging to this group are known in mammalian and yeast cells to be involved in the control of polarity, cell morphogenesis and movement by regulating cytoskeleton organization. An investigation into where some Rho-like proteins are located in plant cells was made. The antibodies used, anti-Cdc42Hs and anti-Rac 1, were raised against conserved characteristic sequences of Cdc42Hs and Rac1 mammalian proteins respectively. In fixed cells, Cdc42Hs antibody recognized epitopes generally co-localized with microtubules which may be implicated in the establishment of cell polarity, whereas the proteins recognized by Rac1 antibody seemed to be associated with organelle membranes. The same anti-bodies were used in Western blots of proteins from tobacco BY-2 and lucerne A2 suspension cells: Cdc42Hs antibody recognized three bands whereas Rac1 antibody revealed only one band of 18 kDa Mr. A [³⁵S]GTP overlay revealed four bands of the same Mr as those recognized in Western blots by Cdc42Hs and Rac1 antibodies.Key words: Rho G-proteins, Cdc42, Rac1, Immunofluorescence, plant cells.      

Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins

The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.     

Regulation of anoikis by Cdc42 and Rac1

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.     

RhoG signals in parallel with Rac1 and Cdc42

RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo. First, we determined that RhoG was regulated by guanine nucleotide exchange factors that also activate Rac and/or Cdc42. Vav2 (which activates RhoA, Rac1, and Cdc42) and to a lesser degree Dbs (which activates RhoA and Cdc42) activated RhoG in vitro. Thus, RhoG may be activated concurrently with Rac1 and Cdc42. Second, some effectors of Rac/Cdc42 (IQGAP2, MLK-3, PLD1), but not others (e.g. PAKs, POSH, WASP, Par-6, IRSp53), interacted with RhoG in a GTP-dependent manner. Third, consistent with this differential interaction with effectors, activated RhoG stimulated some (JNK and Akt) but not other (SRF and NF-kappaB) downstream signaling targets of activated Rac1 and Cdc42. Finally, transient transduction of a tat-tagged Rac1(17N) dominant-negative fusion protein inhibited the induction of lamellipodia by the Rac-specific activator, Tiam1, but not by activated RhoG. Together, these data argue that RhoG function is mediated by signals independent of Rac1 and Cdc42 activation and instead by direct utilization of a subset of common effectors.     

RhoB links PDGF signaling to cell migration by coordinating activation and localization of Cdc42 and Rac

The small GTPase RhoB regulates endocytic trafficking of receptor tyrosine kinases (RTKs) and the non-receptor kinases Src and Akt. While receptor-mediated endocytosis is critical for signaling processes driving cell migration, mechanisms that coordinate endocytosis with the propagation of migratory signals remain relatively poorly understood. In this study, we show that RhoB is essential for activation and trafficking of the key migratory effectors Cdc42 and Rac in mediating the ability of platelet-derived growth factor (PDGF) to stimulate cell movement. Stimulation of the PDGF receptor-β on primary vascular smooth muscle cells (VSMCs) results in RhoB-dependent trafficking of endosome-bound Cdc42 from the perinuclear region to the cell periphery, where the RhoGEF Vav2 and Rac are also recruited to drive formation of circular dorsal and peripheral ruffles necessary for cell migration. Our findings identify a novel RhoB-dependent endosomal trafficking pathway that integrates RTK endocytosis with Cdc42/Rac localization and cell movement.     

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

Cdc42, Rac1, and their effector IQGAP1 as molecular switches for cadherin-mediated cell-cell adhesion.

Cell-cell adhesion is a dynamic process in various cellular and developmental situations. Cadherins, well-known Ca(2+)-dependent adhesion molecules, are thought to play a major role in the regulation of cell-cell adhesion. However, the molecular mechanism underlying the rearrangement of cadherin-mediated cell-cell adhesion is largely unknown. Cdc42 and Rac1, belonging to the Rho small GTPase family, have recently been shown to be involved in the regulation of cell-cell adhesion. In addition, IQGAP1, an effector for Cdc42 and Rac1, has been shown to regulate the cadherin function through interaction with beta-catenin, a molecule associated with cadherin. In this review, we will summarize the mode of action of Cdc42 and Rac1 as well as IQGAP1 as molecular switches for the cadherin function, and then discuss physiological processes in which the Cdc42/Rac1/IQGAP1 system may be involved.     

Nitric oxide causes macrophage migration via the HIF-1-stimulated small GTPases Cdc42 and Rac1

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1α^−/− mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS^−/− mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO–HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.     

Role of the small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni

Host cell invasion of the food-borne pathogen Campylobacter jejuni is one of the primary reasons of tissue damage in humans but molecular mechanisms are widely unclear. Here, we show that C. jejuni triggers membrane ruffling in the eukaryotic cell followed by invasion in a very specific manner first with its tip followed by the flagellar end. To pinpoint important signalling events involved in the C. jejuni invasion process, we examined the role of small Rho family GTPases. Using specific GTPase-modifying toxins, inhibitors and GTPase expression constructs we show that Rac1 and Cdc42, but not RhoA, are involved in C. jejuni invasion. In agreement with these observations, we found that internalization of C. jejuni is accompanied by a time-dependent activation of both Rac1 and Cdc42. Finally, we show that the activation of these GTPases involves different host cell kinases and the bacterial fibronectin-binding protein CadF. Thus, CadF is a bifunctional protein which triggers bacterial binding to host cells as well as signalling leading to GTPase activation. Collectively, our results suggest that C. jejuni invade host target cells by a unique mechanism and the activation of the Rho GTPase members Rac1 and Cdc42 plays a crucial role in this entry process.