The first CH domain of affixin activates Cdc42 and Rac1 through alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor

Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of alphaPIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of alphaPIX, alphaPIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation.     

Cytotoxic necrotizing factor 1 of Escherichia coli stimulates Rho/Rho-kinase-dependent myosin light-chain phosphorylation without inactivating myosin light-chain phosphatase in endothelial cells

Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin of Escherichia coli that constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.     

Proteasomal degradation of cytotoxic necrotizing factor 1-activated rac

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli has been shown to activate members of the Rho family by deamidation of glutamine 63. This amino acid is essential for hydrolysis of GTP, and any substitution results in a constitutively active Rho. Activation of Rho induces the formation of stress fibers, filopodia, and membrane ruffles due to activation of RhoA, Cdc42, and Rac, respectively. Here we show that the level of endogenous Rac decreased in CNF1-treated HEK293 and HeLa cells. The amount of mRNA remained unaffected, leaving the possibility that Rac is subject to proteolytic degradation. Treatment of cells with lactacystin, an inhibitor of the 26S proteasome, protected Rac from degradation. We have previously shown that CNF1 activates the c-Jun N-terminal kinase (JNK) only transiently in HeLa cells (M. Lerm, J. Selzer, A. Hoffmeyer, U. R. Rapp, K. Aktories, and G. Schmidt, Infect. Immun. 67:496-503, 1998). Here we show that CNF1-induced JNK activation is stabilized in the presence of lactacystin. The data indicate that Rac is degraded by a proteasome-dependent pathway in CNF1-treated cells.     

Escherichia coli Cytotoxic Necrotizing Factor 1 Effaces Microvilli and Decreases Transmigration of Polymorphonuclear Leukocytes in Intestinal T84 Epithelial Cell Monolayers

Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho. Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A. Nusrat, M. Giry, J. R. Turner, S. P. Colgan, C. A. Parkos, E. Lemichez, P. Boquet, and J. L. Madara, Proc. Natl. Acad. Sci. USA 92:10629–10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration. Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin. However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction. CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs. Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.     

Rho activation regulates CXCL12 chemokine stimulated actin rearrangement and restitution in model intestinal epithelia

Chemokines are critical regulatory factors that direct migration, proliferation and maturation of receptor expressing target cells within gut mucosa. The aim of the present study was to define the cellular mechanisms whereby engagement of the essential chemokine CXCL12 to CXCR4 regulates restitutive epithelial cell migration. Non-transformed IEC-6 cells or polarized T84 epithelial monolayers were wounded and F-actin accumulation assessed using fluorescence microscopy and flow cytometry. Immunoblot analysis, pull-down assays, fluorescence microscopy and wound healing assays defined activation of Rho, Rho-kinase (ROCK), and myosin light chain (MLC) and the role for those Rho effectors in CXCL12-regulated epithelial restitution. CXCL12 increased RhoGTP and F-actin localization to the leading edge of wounded IEC-6 and T84 monolayers. CXCL12 congruently stimulated an increase in active MLC that was inhibited by blockade of ROCK and myosin light chain kinase and regulated epithelial migration. Our data in model intestinal epithelia suggest CXCR4 and CXCL12 may function as an autocrine and paracrine mucosal signaling network regulating the competency of the epithelial barrier to withstand injury and mediate repair following damage.     

Focal adhesion kinase regulates laminin-induced oligodendroglial process outgrowth

In the central nervous system (CNS), myelination of axons occurs when oligodendrocyte progenitor cells undergo terminal differentiation, and initiate process formation and axonal ensheathment. Although Fyn, a member of the Src-family kinases (SFKs), plays an important role in this differentiation process, the substrates of Fyn in oligodendrocytes are largely unknown. Using mass spectrometric analysis, we identified focal adhesion kinase (FAK) as a tyrosine-phosphorylated protein in the rat-derived CG4 oligodendrocyte cell line. Tyrosine phosphorylation of FAK was enhanced during differentiation of CG4 cells in a Fyn-dependent manner. In addition, phosphorylation of FAK was stimulated by laminin, one of the ligands for integrin. Knockdown of FAK expression in CG4 cells suppressed process outgrowth on laminin. Rac1 and Cdc42 activities, which are required for oligodendrocyte process formation, were down-regulated in FAK-knockdown cells. Expression of wild-type (WT) FAK in FAK-knockdown CG4 cells restored outgrowth of processes, but the Y397F mutant lacking the autophosphorylation site did not. These results suggest that FAK/Fyn-mediated activation of Rac1 and Cdc42 is critical for laminin-induced outgrowth of oligodendrocyte processes.     

IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells

The process of injury and repair involves spreading, migration and cell proliferation. The functions of Rho GTPases and their effector IQGAP1 are poor known in this process of airway epithelium. In the present study, we employed a widely used in vitro model by scratching a monolayer of BECs. We found that scratching induced decreasing of the GTP-bound Rac1 and Cdc42, but increasing the amounts of IQGAP1 at different time points. Next, we confirmed that IQGAP1 interacted with the constitutively active Rac1 (Rac1^V12) and Cdc42 (Cdc42^V12) rather than the dominant negative Rac1 (Rac1^N17) and Cdc42 (Cdc42^N17). Over-expressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was defective to interact with Rho GTPases, induced translocation of β-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and increased the expression levels of its target genes of c-myc and cyclin D1. Likewise, the amounts of c-myc and cyclin D1 increased after scratching. Our results suggested that IQGAP1 mediated cell proliferation through activating Tcf in a manner independent of Rac1 and Cdc42 in wound repair of BECs.     

TNF-stimulated MAP kinase activation mediated by a Rho family GTPase signaling pathway

The biological response to tumor necrosis factor (TNF) involves activation of MAP kinases. Here we report a mechanism of MAP kinase activation by TNF that is mediated by the Rho GTPase family members Rac/Cdc42. This signaling pathway requires Src-dependent activation of the guanosine nucleotide exchange factor Vav, activation of Rac/Cdc42, and the engagement of the Rac/Cdc42 interaction site (CRIB motif) on mixed-lineage protein kinases (MLKs). We show that this pathway is essential for full MAP kinase activation during the response to TNF. Moreover, this MLK pathway contributes to inflammation in vivo.     

Disruption of epithelial barrier integrity by Salmonella enterica serovar typhimurium requires geranylgeranylated proteins

Epithelial cells that line the human intestinal mucosa constitute the initial sites of host invasion by bacterial pathogens. A number of bacteria, such as Salmonella and Yersinia spp., have been shown to disrupt the integrity of the epithelial barrier, although little is known about the mechanisms underlying that effect. We found that polarized MDCK-1 epithelial cells infected with invasive Salmonella enterica serovar Typhimurium SL1344 exhibited marked changes in F-actin organization, an increase in the paracellular flux of dextran, and a rapid decrease in transepithelial electrical resistance (TER). In contrast, infection with an isogenic noninvasive mutant (hilA) increased the TER in these cells. Pretreating MDCK-1 cells with the inhibitors for tyrosine kinase (genistein) or phosphatidylinositol 3-kinase (wortmannin) did not affect invasion and subsequent perturbation of the epithelial barrier by serovar Typhimurium. Instead, the geranylgeranyltransferase 1 inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, clearly reversed the capacity of serovar Typhimurium to disrupt the epithelial barrier. The substrates for GGTI-298 include Rho family GTPases, as indicated by inhibiting prenylation of Rac1 and Cdc42. Infection with wild-type serovar Typhimurium increased the level of activated Rac1 and Cdc42 and caused these proteins to accumulate apically in MDCK-1 cells. This Salmonella-induced accumulation of Rac1 and Cdc42 and alteration of the junction-associated proteins ZO-1, occludin, and E-cadherin in MDCK-1 cells were markedly inhibited by GGTI-298. These results suggest that activation of geranylgeranylated proteins, including Rac1 and Cdc42, is critical for disruption of barrier integrity by serovar Typhimurium in polarized MDCK-1 cells.     

Phospholipase C gamma negatively regulates Rac/Cdc42 activation in antigen-stimulated mast cells

The Rho GTPases Rac and Cdc42 play a central role in the regulation of secretory and cytoskeletal responses in antigen-stimulated mast cells. In this study, we examine the kinetics and mechanism of Rac and Cdc42 activation in the rat basophilic leukemia RBL-2H3 cells. The activation kinetics of both Rac and Cdc42 show a biphasic profile, consisting of an early transient peak at 1 min and a late sustained activation phase at 20-40 min. The inhibition of phospholipase C (PLC)gamma causes a twofold increase in Rac and Cdc42 activation that coincides with a dramatic production of atypical filopodia-like structures. Inhibition of protein kinase C using bisindolylmaleimide mimics the effect of PLCgamma inhibition on Rac activation, but not on Cdc42 activation. In contrast, depletion of intracellular calcium leads to a complete inhibition of the early activation peak of both Rac and Cdc42, without significant effects on the late sustained activation. These data suggest that PLCgamma is involved in a negative feedback loop that leads to the inhibition of Rac and Cdc42. They also suggest that the presence of intracellular calcium is a prerequisite for both Rac and Cdc42 activation.     

Differential regulation of human lung epithelial and endothelial barrier function by thrombin

Lung epithelial and endothelial barrier dysfunction is critical to the physiologic derangement observed in acute lung injury, but remains poorly understood. We utilized human alveolar epithelial (A549) and endothelial cells (EC) to study cytoskeletal remodeling, myosin light chain (MLC) phosphorylation and barrier regulation evoked by the edemagenic agent, thrombin. Thrombin-challenged human EC monolayers demonstrated increased MLC phosphorylation, actin stress fiber formation and loss of barrier integrity reflected by decreased transmonolayer electrical resistance (TER). In contrast, thrombin produced prominent circumferential localization of actin fibers, increased MLC phosphorylation and increased TER across epithelial monolayers, consistent with barrier protection. Reductions in MLC phosphorylation induced by cell pretreatment with pharmacological inhibitors of MLC kinase (ML-7) and Rho kinase (Y-27632) significantly attenuated thrombin-mediated TER changes and MLC phosphorylation in both lung cell types. Thrombin-produced, time-dependent activation of Rho GTPase in both epithelial and EC, whereas Rac GTPase activation was observed only in A549 cells. Molecular inhibition of Rac activity by adenoviral transfer of dominant-negative Rac mutant abolished thrombin-induced TER increases in alveolar epithelial cells. Finally, A549 cells, but not endothelium, demonstrated increased levels of tight junction proteins (ZO-1 and occludin) after thrombin at the cell-cell interface areas linked to thrombin-elicited barrier protection. These results demonstrate differential pulmonary endothelial and alveolar epithelial barrier regulation via unique actomyosin remodeling and cytoskeletal interactions with tight junction complexes, which confer selective barrier responses to edemagenic stimuli.     

Cdc42 and Rac small G proteins activated by trans-interactions of nectins are involved in activation of c-Jun N-terminal kinase,but not in association of nectins and cadherin to form adherens junctions,in fibroblasts

BACKGROUND: Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules which associate with cadherins to form adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin-1 and -3 are members of the nectin family which most strongly trans-interact, causing cell-cell adhesion. The trans-interaction between nectin-1 and -3 induces the activation of both Cdc42 and Rac small G proteins in epithelial cells. We studied the roles of Cdc42 and Rac activated in this way in L fibroblasts stably expressing both nectin-1 and E-cadherin (nectin-1-EL cells). RESULTS: The trans-interaction between nectin-1 and -3 induced the activation of Cdc42 and Rac in nectin-1-EL cells. Cdc42, and presumably Rac, activated in this way, induced the activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). Cdc42 or Rac was not essential for the association of nectin-1 and E-cadherin to form AJs. Reorganization of the actin cytoskeleton was not required for the association of nectin-1 and E-cadherin. CONCLUSION: These results indicate that Cdc42 and Rac activated by the trans-interaction of nectins selectively induce the activation of JNK, but are not essential for the association of nectins and cadherin to form AJs in fibroblasts.     

RhoA activation promotes transendothelial migration of monocytes via ROCK

Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells.     

Importance of spatial activation of Cdc42 and rac small G proteins by frabin for microspike formation in MDCK cells

BACKGROUND: Frabin is an actin filament (F-actin)-binding protein that shows GDP/GTP exchange activity for Cdc42 small G protein (Cdc42). Frabin furthermore induces indirect activation of Rac small G protein (Rac) in intact cells. We have recently shown that in nonepithelial cells, frabin induces the formation of both filopodia- and lamellipodia-like processes through the activation of Cdc42 and Rac, respectively. In epithelial cells such as MDCK cells, Cdc42 and Rac regulate cell-cell adherens junctions (AJs) via the accumulation of F-actin and E-cadherin, although neither Cdc42 nor Rac induces the formation of filopodia or lamellipodia. In this study, we have examined the effects of frabin on the reorganization of the actin cytoskeleton in MDCK cells. RESULTS: Frabin induces the formation of microspikes at the basal area of the lateral membranes through the activation of Cdc42 and Rac in MDCK cells, although a dominant active mutant of Cdc42 or Rac alone, or both, did not induce the formation of microspikes. Furthermore, frabin weakly increased the accumulation of F-actin and E-cadherin at cell-cell AJs and the formation of stress fibres through the activation of Cdc42 and Rac, under conditions where the dominant active mutant of Cdc42 or Rac markedly showed these effects. The Cdc42- and Rac-induced formation of stress fibres was dependent on the activation of Rho small G protein. CONCLUSION: These results indicate that the frabin-dependent spatial activation of Cdc42 and Rac is important for the formation of microspikes.     

Deamidation of Cdc42 and Rac by Escherichia coli Cytotoxic Necrotizing Factor 1: Activation of c-Jun N-Terminal Kinase in HeLa Cells

Recently, Escherichia coli cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G. Schmidt, P. Sehr, M. Wilm, J. Selzer, M. Mann, and K. Aktories, Nature 387:725–729, 1997; G. Flatau, E. Lemichez, M. Gauthier, P. Chardin, S. Paris, C. Fiorentini, and P. Boquet, Nature 387:729–733, 1997). Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells. Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles. CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase. Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin. The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1. Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2′(3′)-O-(N-methylanthraniloyl)GDP. The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1.     

Cytotoxic necrotizing factor type 1 production by uropathogenic Escherichia coli modulates polymorphonuclear leukocyte function

Many strains of uropathogenic Escherichia coli (UPEC) produce cytotoxic necrotizing factor type 1 (CNF1), a toxin that constitutively activates the Rho GTPases RhoA, Rac1, and Cdc42. We previously showed that CNF1 contributes to the virulence of UPEC in a mouse model of ascending urinary tract infection and a rat model of acute prostatitis and that a striking feature of the histopathology of the mouse bladders and rat prostates infected with CNF1-positive strains is an elevation in levels of polymorphonuclear leukocytes (PMNs). We also found that CNF1 synthesis leads to prolonged survival of UPEC in association with human neutrophils. Here, we tested the hypothesis that CNF1 production by UPEC diminishes the antimicrobial capacity of mouse PMNs by affecting phagocyte function through targeting Rho family GTPases that are critical to phagocytosis and the generation of reactive oxygen species. We found that, as with human neutrophils, CNF1 synthesis provided a survival advantage to UPEC incubated with mouse PMNs. We also observed that CNF1-positive UPEC down-regulated phagocytosis, altered the distribution of the complement receptor CR3 (CD11b/CD18), enhanced the intracellular respiratory burst, and increased levels of Rac2 activation in PMNs. From these results, we conclude that modulation of PMN function by CNF1 facilitates UPEC survival during the acute inflammatory response.     

In Vivo Modifications of Small GTPase Rac and Cdc42 by Bordetella Dermonecrotic Toxin

Bordetella dermonecrotic toxin (DNT) is known to activate the small GTPase Rho through deamidation or polyamination. In this study, we examined whether Rac and Cdc42, the two other members of the Rho family, serve as intracellular targets for the toxin. Immunoprecipitation and immunoblot assays revealed that DNT deamidated or polyaminated intracellular Rac and Cdc42. After the modifications, both Rac and Cdc42 lost their GTP-hydrolyzing, but not GTP-binding, activities. The interactions of the modified Rac and Cdc42 with their respective effectors were strictly dependent on GTP. MC3T3-E1 cells treated with DNT at high concentrations demonstrated extensive formations of lamellipodia and filopodia, which indicate the intracellular activation of Rac and Cdc42, respectively.     

An essential function for the calcium-promoted Ras inactivator in Fcgamma receptor-mediated phagocytosis

Fc receptor (FcR)-mediated phagocytosis requires activation of the Rho GTPases Cdc42 and Rac1, but how they are recruited to the FcR is unknown. Here we show that the calcium-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, functions as an adaptor for Cdc42 and Rac1 during FcR-mediated phagocytosis. CAPRI-deficient macrophages had impaired FcgammaR-mediated phagocytosis and oxidative burst, as well as defective activation of Cdc42 and Rac1. CAPRI interacted constitutively with both Cdc42 and Rac1 and translocated to phagocytic cups during FcgammaR-mediated phagocytosis. CAPRI-deficient mice had an impaired innate immune response to bacterial infection. These results suggest that CAPRI provides a link between FcgammaR and Cdc42 and Rac1 and is essential for innate immune responses.     

Rho-activating Escherichia coli cytotoxic necrotizing factor 1: macropinocytosis of apoptotic bodies in human epithelial cells

Some pathogenic Escherichia coli strains produce a protein toxin, named cytotoxic necrotizing factor 1 (CNF1), which permanently activates proteins belonging to the Rho family. In epithelial cells, the consequence of this activation is the rearrangement of the actin cytoskeleton and the promotion of an intense and generalized ruffling activity. This leads, in turn, to the induction of a phagocytic-like behavior called macropinocytosis that, in the case of CNF1, depends on the coordinate activation of Rho, Rac and Cdc42. Following internalization, the ingested material is discharged into Rab-7 and Lamp-1-positive acidic vesicles where it probably undergoes degradation. By exerting this activity, CNF1-activated epithelial cells might support the scavenging activity of macrophages during bacterial overgrowth.