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.     

Lipid raft-associated GTPase signaling controls morphology and CD8+ T cell stimulatory capacity of human dendritic cells

Their eponymous morphology and unique ability to activate naive T cells are hallmark features of dendritic cells (DCs). Specific properties of the actin cytoskeleton may define both characteristics. In search for regulators that coordinate DC phenotype and function, we observed strongly increased expression of the actin-remodeling GTPases Cdc42 and Rac1 during DC development from human stem cells. Cdc42 and Rac1 are constitutively active in immature DCs, and their activity is further up-regulated by maturational stimuli such as LPS or CD40L. Activation of Rac1 is associated with its rapid recruitment into lipid rafts. Cdc42 is not recruited into rafts, but readily activated by raft-associated moieties. The functional interplay of rafts, GTPases, and cortical actin is further shown by GTPase activation and actin remodeling after pharmacological disruption of lipid rafts and by the loss of the actin-based DC morphology by transfection of dominant-negative Cdc42 and Rac1. Both Cdc42 and Rac1 also control the transport of essential immunostimulatory molecules to the DC surface. Transfection with dominant-negative GTPases led to reduced surface expression of MHC class I and CD86. Consecutively, DCs display a reduced stimulatory capacity for CD8(+) T cells, whereas MHC class II-dependent stimulation of CD4(+) T cells remains unperturbed. We conclude that Cdc42 and Rac1 signaling controls DC morphology and conditions DCs for efficient CD8(+) T cell stimulation.     

Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1. actin cytoskeleton; Cdc42; E-cadherin     

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     

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.     

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells

Cdc42 is a Rho-family GTPase that in yeast is important in establishing polarized bud growth. Here we show that Cdc42 is also essential in establishing and maintaining polarity in epithelial cells. Functional deletion of Cdc42 in Madin-Darby canine kidney (MDCK) cells results in the selective depolarization of basolateral membrane proteins; the polarity of apical proteins remains unaffected. This phenotype does not reflect major alterations in the actin cytoskeleton, but rather results from the selective inhibition of membrane traffic to the basolateral plasma membrane in both the endocytic and the secretory pathways. Thus, Cdc42 plays a critical part in epithelial-cell polarity, by, unexpectedly, regulating the fidelity of membrane transport.     

Actin pedestal formation by enteropathogenic Escherichia coli is regulated by IQGAP1, calcium, and calmodulin

During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca(2+)/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca(2+), and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca(2+) reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca(2+), and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.     

Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.     

Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell-cell dissociation during cell scattering

We have previously proposed that IQGAP1, an effector of Rac1 and Cdc42, negatively regulates cadherin-mediated cell-cell adhesion by interacting with beta-catenin and by causing the dissociation of alpha-catenin from cadherin-beta-catenin-alpha-catenin complexes and that activated Rac1 and Cdc42 positively regulate cadherin-mediated cell-cell adhesion by inhibiting the interaction of IQGAP1 with beta-catenin. However, it remains to be clarified in which physiological processes the Rac1-Cdc42-IQGAP1 system is involved. We here examined whether the Rac1-IQGAP1 system is involved in the cell-cell dissociation of Madin-Darby canine kidney II cells during 12-O-tetradecanoylphorbol-13-acetate (TPA)- or hepatocyte growth factor (HGF)-induced cell scattering. By using enhanced green fluorescent protein (EGFP)-tagged alpha-catenin, we found that EGFP-alpha-catenin decreased prior to cell-cell dissociation during cell scattering. We also found that the Rac1-GTP level decreased after stimulation with TPA and that the Rac1-IQGAP1 complexes decreased, while the IQGAP1-beta-catenin complexes increased during action of TPA. Constitutively active Rac1 and IQGAP1 carboxyl terminus, a putative dominant-negative mutant of IQGAP1, inhibited the disappearance of alpha-catenin from sites of cell-cell contact induced by TPA. Taken together, these results indicate that alpha-catenin is delocalized from cell-cell contact sites prior to cell-cell dissociation induced by TPA or HGF and suggest that the Rac1-IQGAP1 system is involved in cell-cell dissociation through alpha-catenin relocalization.     

Tumor necrosis factor-alpha promotes survival of opossum kidney cells via Cdc42-induced phospholipase C-gamma1 activation and actin filament redistribution

Although the renal proximal tubular epithelial cells are targeted in a variety of inflammatory diseases of the kidney, the signaling mechanism by which tumor necrosis factor (TNF)-alpha exerts its effects in these cells remains unclear. Here, we report that TNF-alpha elicits antiapoptotic effects in opossum kidney cells and that this response is mediated via actin redistribution through a novel signaling mechanism. More specifically, we show that TNF-alpha prevents apoptosis by inhibiting the activity of caspase-3 and this effect depends on actin polymerization state and nuclear factor-kappaB activity. We also demonstrate that the signaling cascade triggered by TNF-alpha is governed by the phosphatidylinositol-3 kinase, Cdc42/Rac1, and phospholipase (PLC)-gamma1. In this signaling cascade, Cdc42 was found to be selectively essential for PLC-gamma1 activation, whereas phosphatidylinositol-3,4,5-triphosphate alone is not sufficient to activate the phospholipase. Moreover, PLC-gamma1 was found to associate in vivo with the small GTPase(s). Interestingly, PLC-gamma1 was observed to associate with constitutively active (CA) Cdc42V12, but not with CA Rac1V12, whereas no interaction was detected with Cdc42(T17N). The inactive Cdc42(T17N) and the PLC-gamma1 inhibitor U73122 prevented actin redistribution and depolymerization, confirming that both signaling molecules are responsible for the reorganization of actin. Additionally, the actin filament stabilizer phallacidin potently blocked the nuclear translocation of nuclear factor-kappaB and its binding activity, resulting in abrogation of the TNF-alpha-induced inhibition of caspase-3. To conclude, our findings suggest that actin may play a pivotal role in the response of opossum kidney cells to TNF-alpha and implicate Cdc42 in directly regulating PLC-gamma1 activity.     

IQGAP1 regulates Salmonella invasion through interactions with actin, Rac1, and Cdc42

To infect host cells, Salmonella utilizes an intricate system to manipulate the actin cytoskeleton and promote bacterial uptake. Proteins injected into the host cell by Salmonella activate the Rho GTPases, Rac1 and Cdc42, to induce actin polymerization. Following uptake, a different set of proteins inactivates Rac1 and Cdc42, returning the cytoskeleton to normal. Although the signaling pathways allowing Salmonella to invade host cells are beginning to be understood, many of the contributing factors remain to be elucidated. IQGAP1 is a multidomain protein that influences numerous cellular functions, including modulation of Rac1/Cdc42 signaling and actin polymerization. Here, we report that IQGAP1 regulates Salmonella invasion. Through its interaction with actin, IQGAP1 co-localizes with Rac1, Cdc42, and actin at sites of bacterial uptake, whereas infection promotes the interaction of IQGAP1 with both Rac1 and Cdc42. Knockdown of IQGAP1 significantly reduces Salmonella invasion and abrogates activation of Cdc42 and Rac1 by Salmonella. Overexpression of IQGAP1 significantly increases the ability of Salmonella to enter host cells and required interaction with both actin and Cdc42/Rac1. Together, these data identify IQGAP1 as a novel regulator of Salmonella invasion.     

Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells

The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro . We have examined the pathway(s) by which epithelial cells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internalization occurs by an actin-dependent Toxin B-inhibited pathway which becomes downregulated as epithelial cells become polarized, suggesting that one or more of the Rho family GTPases is involved in bacterial internalization. Here, we demonstrate that activation of the Rho family GTPases by cytotoxic necrotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examination of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rac1 (Rac1V12) or Cdc42 (Cdc42V12), is sufficient to increase uptake of PA103 pscJ . This relative increase persists when bacterial infection is established at the basolateral surface of polarized cells, suggesting that the effect of RhoAV14 is not simply due to its known ability to disrupt tight junction integrity in polarized cells. RhoAV14-mediated stimulation of bacterial uptake is actin dependent as it is abrogated by exposure to latrunculin A. We also find that endogenous Rho GTP levels in epithelial cells are increased by infection with an internalized strain of P. aeruginosa; conversely, a poorly internalized isogenic strain expressing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fusion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103 pscJ internalization in MDCK or HeLa cells. Models consistent with these data are presented.     

Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets

BACKGROUND: Sheets of cells move together as a unit during wound healing and embryonic tissue movements, such as those occurring during gastrulation and neurulation. We have used epithelial wound closure as a model system for such movements and examined the mechanisms of closure and the importance of the Rho family of Ras-related small GTPases in this process. RESULTS: Wounds induced in Madin-Darby canine kidney (MDCK) epithelial cell monolayers close by Rac- and phosphoinositide-dependent cell crawling, with formation of lamellipodia at the wound margin, and not by contraction of a perimarginal actomyosin purse-string. Although Rho-dependent actin bundles usually form at the margin, neither Rho activity nor formation of these structures is required for wound closure to occur at a normal rate. Cdc42 activity is also not required for closure. Inhibition of Rho or Cdc42 results, however, in statistically significant decreases in the regularity of wound closure, as determined by the ratio of wound margin perimeter over the remaining denuded area at different times. The Rac-dependent force generation for closure is distributed over several rows of cells from the wound margin, as inhibition of motility in the first row of cells alone does not inhibit closure and can be compensated for by generation of motile force in cells behind the margin. Furthermore, we observed high levels of Rac-dependent actin assembly in the first few rows of cells from the wound margin. CONCLUSIONS: Wounds in MDCK cell sheets do not close by purse-string contraction but by a crawling behavior involving Rac, phosphoinositides and active movement of multiple rows of cells. This finding suggests a new distributed mode of signaling and movement that, nevertheless, resembles individual cell motility. Although Rho and Cdc42 activities are not required for closure, they have a role in determining the regularity of closure.     

PTP mu-dependent growth cone rearrangement is regulated by Cdc42

PTP mu is expressed in the developing nervous system and promotes growth and guidance of chick retinal ganglion cells. Using a newly developed growth cone rearrangement assay, we examined whether the small G-proteins were involved in PTP mu-dependent signaling. The stimulation of retinal cultures with purified PTP mu resulted in a striking morphological change in the growth cone, which becomes dominated by filopodia within 5 min of addition. This rearrangement in response to PTP mu stimulation was mediated by homophilic binding. We perturbed GTPase signaling using Toxin B, which inhibits Cdc42, Rac, and Rho, as well as the toxin Exoenzyme C3 that inhibits Rho. The PTP mu-induced growth cone rearrangement was blocked by Toxin B, but not by Exoenzyme C3. This result suggests that either Cdc42 or Rac are required but not Rho. To determine which GTPase was involved in PTP mu signaling, we utilized dominant-negative mutants of Cdc42 and Rac. Dominant-negative Cdc42 blocked PTP mu-induced rearrangement, while wild-type Cdc42 and dominant-negative Rac did not. Together, these results suggest a molecular signaling cascade beginning with PTP mu homophilic binding at the plasma membrane and the activation of Cdc42, which acts on the actin cytoskeleton to result in rearrangement of the growth cone.     

Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.     

Glucose-stimulated Cdc42 signaling is essential for the second phase of insulin secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.     

SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell.

The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1. Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allow S. typhimurium to specifically activate different sets of RhoGTPase signaling cascades.     

Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells

Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, in this study we demonstrate that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. We have confirmed a previous report that Akt is activated by expression of Rac and Cdc42 and also observed colocalization of endogenous phosphorylated Akt with Rac and Cdc42 at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Finally, we found that dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells.