Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization

Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells.     

Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering

The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.     

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.     

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.     

Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-beta-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-beta-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-beta-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil.     

Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta

We describe here a signal transduction pathway controlling the establishment of mammalian cell polarity. Scratching a confluent monolayer of primary rat astrocytes leads to polarization of cells at the leading edge. The microtubule organizing center, the microtubule cytoskeleton, and the Golgi reorganize to face the new free space, and directed cell protrusion and migration specifically occur perpendicularly to the scratch. We show here that the interaction of integrins with extracellular matrix at the newly formed cell front leads to the activation and polarized recruitment of Cdc42, which in turn recruits and activates a cytoplasmic mPar6/PKCzeta complex. Localized PKCzeta activity, acting through the microtubule motor protein dynein, is required for all aspects of induced polarity in these cells.     

The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.     

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.     

Lipid-dependent recruitment of neuronal Src to lipid rafts in the brain

Although most Src family tyrosine kinases are modified by palmitoylation as well as myristoylation, Src itself is only myristoylated. Dual acylation is important for attachment to liquid-ordered microdomains or lipid rafts. Accordingly, Src is excluded from lipid rafts in fibroblasts. Evidence of partial genetic redundancy between Src and Fyn for brain-specific targets suggests that these two kinases may occupy overlapping subcellular locations. Neuronal Src (NSrc), an alternative isoform of Src with a 6-amino acid insert in the Src homology 3 domain, is highly expressed in neurons. We investigated whether this structural difference in NSrc allows it to associate with lipid rafts. We found that perinatal mouse brains express predominantly NSrc, which is partly (10-20%) in a lipid raft fraction from brain but not fibroblasts. The association of Src with brain lipid rafts does not depend on the NSrc insert but depends on the amino-terminal myristoylation signal. A crude lipid fraction from brain promotes NSrc entry into rafts in vitro. Moreover, lipid raft-localized NSrc is more catalytically active than NSrc from the soluble fraction, possibly because raft localization alters access to other tyrosine kinases and phosphatases. These findings suggest that NSrc may be involved in signaling from lipid rafts in mouse brain.     

CD28 engagement promotes actin polymerization through the activation of the small Rho GTPase Cdc42 in human T cells

Engagement of the costimulatory molecule CD28 is an important step in the optimal activation of T cells. Nevertheless, the specific role of CD28 in the formation of the immunological synapse and cytoskeletal changes that occur upon TCR/CD3 complex engagement is still poorly understood. Using Ab-coated surfaces, we show that CD28 engagement in the absence of any other signal induced the formation of cytoplasmic elongations enriched in filamentous actin (F-actin), in this work called filopodia or microspikes. Such structures were specific for engagement of CD28 on mAb-coated surfaces because they could not be observed in surfaces coated with either poly(L-lysine) or anti-CD3 mAb. The signaling pathway coupling CD28 to cytoskeletal rearrangements required Src-related kinase activity and promoted Vav phosphorylation and Cdc42 activation independently of the zeta-chain-associated kinase (ZAP-70). CD28-induced filopodia required Cdc42 GTPase activity, but not the related Rho GTPase Rac1. Moreover, Cdc42 colocalized to areas of increased F-actin. Our results support a specific role for the activation of the small Rho GTPase Cdc42 in the actin reorganization mediated by CD28 in human T cells.     

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation.     

Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas

We previously showed that activation of the small GTPase Cdc42 promotes breast cell migration on a collagen matrix. Here we further define the signaling pathways that drive this response and show that Cdc42-mediated migration relies on the adaptor molecule p130(Cas). Activated Cdc42 enhanced p130(Cas) phosphorylation and its binding to Crk. Cdc42-driven migration and p130(Cas) phosphorylation were dependent on the Cdc42 effector Ack1 (activated Cdc42-associated kinase). Ack1 formed a signaling complex that also included Cdc42, p130(Cas), and Crk, formation of which was regulated by collagen stimulation. The interaction between Ack1 and p130(Cas) occurred through their respective SH3 domains, while the substrate domain of p130(Cas) was the major site of Ack1-dependent phosphorylation. Signaling through this complex is functionally relevant, because treatment with either p130(Cas) or Ack1 siRNA blocked Cdc42-induced migration. These results suggest that Cdc42 exerts its effects on cell migration in part through its effector Ack1, which regulates p130(Cas) signaling.     

Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration

BACKGROUND: Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal cells. Polarization is essential for migration of a variety of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact with betaPIX, a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42. Cdc42 controls cell polarity from yeast to mammals during asymmetric cell division and epithelial cell polarization, as well as during cell migration. Cdc42 is, in particular, required for polarization and orientation of astrocytes in a scratch-induced polarized migration assay. Using this assay, we characterized Scrib function during polarized cell migration. RESULTS: Depletion of Scrib by siRNA or expression of dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation, cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with betaPIX at the front edge of polarizing astrocytes. Perturbation of Scrib localization or of Scrib-betaPIX interaction inhibits betaPIX polarized recruitment. We further show that betaPIX is required for astrocyte polarization and that both the Scrib-binding motif and the GEF activity of betaPIX are essential for its function. Scrib and betaPIX control Cdc42 activation and localization during astrocyte polarization. Thereby, Scrib regulates Cdc42-dependent APC and Dlg1 recruitment to the leading edge to promote cell orientation. CONCLUSION: We conclude that Scrib plays a key role in the establishment of cell polarity during migration. By interacting with betaPIX, Scrib controls localization and activation of the small GTPase Cdc42 and regulates Cdc42-dependent polarization pathways.     

Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

Nectins, Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, initiate cell-cell adhesion by their trans interactions and recruit cadherins to cooperatively form adherens junctions (AJs). In addition, the trans interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which increases the velocity of the formation of AJs. We examined here how nectins induce the activation of Cdc42 in MDCK epithelial cells and L fibroblasts. Nectins recruited and activated c-Src at the nectin-based cell-cell adhesion sites. FRG, a GDP/GTP exchange factor specific for Cdc42, was then recruited there, tyrosine phosphorylated by c-Src, and activated, causing an increase in the GTP-bound active form of Cdc42. Inhibition of the nectin-induced activation of c-Src suppressed the nectin-induced activation of FRG and Cdc42. Inhibition of the nectin-induced activation of FRG or depletion of FRG by RNA interference suppressed the nectin-induced activation of Cdc42. These results indicate that nectins induce the activation of Cdc42 through c-Src and FRG locally at the nectin-based cell-cell adhesion sites.     

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.     

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.     

Selective inhibition of T cell activation via CD147 through novel modulation of lipid rafts

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.     

Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

Activated protein C (APC) is a serine protease that regulates thrombin (IIa) production through inactivation of blood coagulation factors Va and VIIIa. APC also has non-hemostatic functions related to inflammation, proliferation, and apoptosis through various mechanisms. Using two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, we investigated the role of APC in cell chemotaxis and invasion. Treatment of cells with increasing APC concentrations (1-50 microg/ml) increased invasion and chemotaxis in a concentration-dependent manner. Only the active form of APC increased invasion and chemotaxis of the MDA-MB-231 cells when compared to 3 inactive APC derivatives. Using a modified "checkerboard" analysis, APC was shown to only affect migration when plated with the cells; therefore, APC is not a chemoattractant. Blocking antibodies to endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) attenuated the effects of APC on chemotaxis in the MDA-MB-231 cells. Finally, treatment of the MDA-MB-231 cells with the proliferation inhibitor, Na butyrate, showed that APC did not increase migration by increasing cell number. Therefore, APC increases invasion and chemotaxis of cells by binding to the cell surface and activating specific signaling pathways through EPCR and PAR-1.     

EGFL6 promotes endothelial cell migration and angiogenesis through the activation of extracellular signal-regulated kinase

Angiogenesis is required for bone development, growth, and repair. It is influenced by the local bone environment that involves cross-talks between endothelial cells and adjacent bone cells. However, data regarding factors that directly contribute to angiogenesis by bone cells remain poorly understood. Here, we report that EGFL6, a member of the epidermal growth factor (EGF) repeat superfamily proteins, induces angiogenesis by a paracrine mechanism in which EGFL6 is expressed in osteoblastic-like cells but promotes migration and angiogenesis of endothelial cells. Co-immunoprecipitation assays revealed that EGFL6 is secreted in culture medium as a homodimer protein. Using scratch wound healing and transwell assays, we found that conditioned medium containing EGFL6 potentiates SVEC (a simian virus 40-transformed mouse microvascular endothelial cell line) endothelial cell migration. In addition, EGFL6 promotes the endothelial cell tube-like structure formation in Matrigel assays and angiogenesis in a chick embryo chorioallantoic membrane. Furthermore, we show that EGFL6 recombinant protein induces phosphorylation of ERK in SVEC endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Together, these results demonstrate, for the first time, that osteoblastic-like cells express EGFL6 that is capable of promoting endothelial cell migration and angiogenesis via ERK activation. Thus, the EGLF6 mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts and might offer an important new target for the potential treatment of bone diseases, including osteonecrosis, osteoporosis, and fracture healing.