Rho-specific Guanine Nucleotide Exchange Factor Research Articles

Gef10—the third member of a Rho-specific guanine nucleotide exchange factor subfamily with unusual protein architecture

According to cDNA sequence homologies, Gef10 is related to the Rho-specific guanine nucleotide exchange factors GrinchGEF and p164-RhoGEF. Like these GEFs, Gef10 exhibits only weak homology to known pleckstrin homology domains, but contains a putative WD40-like domain. As detected by RT-PCR, Gef10 is transcribed in at least two splice variants in different human tissues. Although the Gef10 sequence contains two putative transmembrane segments, recombinantly expressed Gef10 displays a cytosolic localisation. As detected by guanine nucleotide exchange activity assay, precipitation assay of GTP-bound Rho proteins and serum response element dependent gene transcription Gef10 activates RhoA-C, but not Rac1 or Cdc42. In the reporter gene assay, Gef10 preferentially activated RhoB. When expressed in NIH3T3 cells, Gef10 induced actin stress fibre, but not lamellipodia or filopodia formation. We conclude that Gef10 is the third member of a Rho-specific GEF family with unusual protein architecture.

Transformation by the Rho-specific Guanine Nucleotide Exchange Factor Dbs Requires ROCK I-mediated Phosphorylation of Myosin Light Chain

Dbs was identified in a cDNA-based expression screen for sequences that can cause malignant growth when expressed in murine fibroblasts. In previous studies we have shown that Dbs is a Rho-specific guanine nucleotide exchange factor that can activate RhoA and/or Cdc42 in a cell-specific manner. In this current study we have used a combination of genetic and pharmacological approaches to examine the relative contributions of RhoA x PRK and RhoA x ROCK signaling to Dbs transformation. Our analysis indicates that ROCK is activated in Dbs-transformed cells and that Dbs transformation is dependent upon ROCK I activity. In contrast, there appears to be no requirement for PRK activation in Dbs transformation. Dbs transformation is also associated with increased phosphorylation of myosin light chain and stress fiber formation, both of which occur in a ROCK-dependent manner. Suppression of myosin light chain expression by small interfering RNAs impairs Dbs focus formation, thus establishing a direct link between actinomyosin contraction and Rho-specific guanine nucleotide exchange factor transformation.

Hyaluronan-CD44 Interaction with Leukemia-associated RhoGEF and Epidermal Growth Factor Receptor Promotes Rho/Ras Co-activation, Phospholipase Cϵ-Ca2+ Signaling, and Cytoskeleton Modification in Head and Neck Squamous Cell Carcinoma Cells

In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.

Angiotensin II Up-Regulates the Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (RhoGEF), a Regulator of G Protein Signaling Domain-Containing RhoGEF, in Vascular Smooth Muscle Cells

In vascular smooth muscle, stimulation of heterotrimeric G protein-coupled receptors (GPCRs) by various contractile agonists activates intracellular signaling molecules to result in an increase in cytosolic Ca2+ and the subsequent phosphorylation of myosin light chain (MLC) by Ca2+/calmodulin-dependent MLC kinase. In addition, a portion of agonist-induced contraction is partially mediated by the Ca2+-independent activation of the small G protein RhoA and a downstream target, Rho-kinase. The activation of RhoA is controlled by several regulatory proteins, including guanine nucleotide exchange factors (GEFs). GEFs activate RhoA by promoting the release of GDP and then facilitating the binding of GTP. There are three Rho-specific GEFs (RhoGEFs) in vascular smooth muscle that contain a binding domain [regulator of G protein signaling (RGS) domain] capable of linking GPCRs to RhoA activation: PDZ-RhoGEF, leukemia-associated RhoGEF (LARG), and p115RhoGEF. We hypothesized that RGS domain-containing RhoGEFs, especially LARG, participate in linking GPCR to RhoA activation in vascular smooth muscle. We observed that angiotensin II up-regulates LARG via the AT1 receptor, and this up-regulation is signaled via the phosphatidylinositol 3-kinase pathway. Furthermore, angiotensin II treatment caused a small, but significant, increase in the component of contractile responses sensitive to Rho-kinase antagonism. These observations support the hypothesis that RhoGEFs, particularly LARG, participate in linking GPCR to RhoA activation in vascular smooth muscle.

Rac1 and Cdc42 regulate hyphal growth and cytokinesis in the dimorphic fungus Ustilago maydis

Small GTP-binding proteins of the highly conserved Rho family act as molecular switches regulating cell signalling, cytoskeletal organization and vesicle trafficking in eukaryotic cells. Here we show that in the dimorphic plant pathogenic fungus Ustilago maydis deletion of either cdc42 or rac1 results in loss of virulence but does not interfere with viability. Cells deleted for cdc42 display a cell separation defect during budding. We have previously shown that the Rho-specific guanine nucleotide exchange factor (GEF) Don1 is required for cell separation in U. maydis. Expression of constitutive active Cdc42 rescues the phenotype of don1 mutant cells indicating that Don1 triggers cell separation by activating Cdc42. Deletion of rac1 affects cellular morphology and interferes with hyphal growth, whereas overexpression of wild-type Rac1 induces filament formation in haploid cells. This indicates that Rac1 is both necessary and sufficient for the dimorphic switch from budding to hyphal growth. Cdc42 and Rac1 share at least one common essential function because depletion of both Rac1 and Cdc42 is lethal. Expression of constitutively active Rac1(Q61L) is lethal and results in swollen cells with a large vacuole. The morphological phenotype, but not lethality is suppressed in cla4 mutant cells suggesting that the PAK family kinase Cla4 acts as a downstream effector of Rac1.

Modulation of Muscle Regeneration, Myogenesis, and Adipogenesis by the Rho Family Guanine Nucleotide Exchange Factor GEFT

Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.

A Novel Mechanism of G Protein-dependent Phosphorylation of Vasodilator-stimulated Phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.

GrinchGEF—A novel Rho-specific guanine nucleotide exchange factor

RhoGTPases, which are activated by specific guanine nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a new RhoGEF (GrinchGEF) containing the typical Dbl homology domain, a putative WD40-like domain, and two predicted transmembrane helices. In contrast to most other RhoGEFs, it exhibits no sequence similarities to known pleckstrin homology domains. GrinchGEF mRNA was highly abundant in skeletal muscle and pancreas. Despite the predicted transmembrane domains, subcellular localization studies revealed a cytosolic distribution. In vitro, GrinchGEF induced the GDP/GTP exchange at RhoA, but not at Rac1 or Cdc42. In intact cells, GrinchGEF induced specifically Rho activation and enhanced RhoA–C-specific downstream effects.

The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

Selective Uncoupling of Gα12 from Rho-mediated Signaling

The heterotrimeric G protein G12 has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated α-subunit of G12 has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G12-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G12 activation, we produced a series of substitution mutants in the regions of Gα12 predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Gα12 that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Gα12-mediated signals. This mutant of Gα12 fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Gα12 retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger β-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Gα12 that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G12 proteins mediate diverse biological responses.

Physical and Functional Interactions of the Lysophosphatidic Acid Receptors with PDZ Domain-containing Rho Guanine Nucleotide Exchange Factors (RhoGEFs)

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRs) including LPA1, LPA2, and LPA3. LPA-induced cytoskeletal changes are mediated by Rho family small GTPases, such as RhoA, Rac1, and Cdc42. One of these small GTPases, RhoA, may be activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors (RhoGEFs) under LPA stimulation although the detailed mechanisms are poorly understood. Here, we show that the C terminus of LPA1 and LPA2 but not LPA3 interact with the PDZ domains of PDZ domain-containing RhoGEFs, PDZ-RhoGEF, and LARG, which are comprised of PDZ, RGS, Dbl homology (DH), and pleckstrin homology (PH) domains. In LPA1- and LPA2-transfected HEK293 cells, LPA-induced RhoA activation was observed although the C terminus of LPA1 and LPA2 mutants, which failed to interact with the PDZ domains, did not cause LPA-induced RhoA activation. Furthermore, overexpression of the PDZ domains of PDZ domain-containing RhoGEFs served as dominant negative mutants for LPA-induced RhoA activation. Taken together, these results indicate that formation of the LPA receptor/PDZ domain-containing RhoGEF complex plays a pivotal role in LPA-induced RhoA activation.

Plexins: axon guidance and signal transduction.

Axon guidance represents a key stage in the formation of neuronal network. Axons are guided by a variety of guidance factors, such as semaphorins, ephrins and netrin. Plexins function as receptors for the repulsive axonal guidance molecules semaphorins. Intracellular domains of plexins are responsible for initiating cellular signal transduction inducing axon repulsion. Recent advances have revealed molecular mechanisms for plexin-mediated cytoskeletal reorganization, leading to repulsive responses, and small GTPases play important roles in this signaling. Plexin-B1 activates Rho through Rho-specific guanine nucleotide exchange factors, leading to neurite retraction. Plexin-B1 possesses an intrinsic GTPase-activating protein activity for R-Ras and induces growth cone collapse through R-Ras inactivation. In this review we survey current understanding of the signaling mechanisms of plexins.

PAK1 Negatively Regulates the Activity of the Rho Exchange Factor NET1

Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.

Association of CNK1 with Rho Guanine Nucleotide Exchange Factors Controls Signaling Specificity Downstream of Rho

Rho is a small GTPase that controls signal transduction pathways in response to a large number of extracellular stimuli. With over 15 potential Rho target proteins identified to date, however, it is not clear how distinct signaling outputs can be generated downstream of a particular stimulus. Several of the known Rho targets are structurally reminiscent of scaffold proteins, which are generally thought to play an important role in controlling signaling specificity. Here, we show that the Rho target CNK1 is a scaffold protein that interacts with Net1 or p115RhoGEF, two Rho-specific guanine nucleotide exchange factors (GEFs), as well with MLK2 and MKK7, two of the kinase components in the JNK MAP kinase cascade. CNK1 acts cooperatively with the two GEFs to activate JNK MAP kinase, but not other Rho-mediated pathways. In HeLa cells, serum or sphingosine-1-phosphate stimulate Rho-dependent activation of the JNK MAP kinase cascade, and this requires endogenous CNK1. We conclude that CNK1 couples a subset of Rho exchange factors to activation of the JNK MAP kinase pathway and that signaling specificity is achieved through complexes containing both upstream activators and downstream targets of Rho.

The Guanine Nucleotide Exchange Factor p63RhoGEF, a Specific Link between Gq/11-coupled Receptor Signaling and RhoA

The monomeric GTPase RhoA, which is a key regulator of numerous cellular processes, is activated by a variety of G protein-coupled receptors, through either G12 or G(q) family proteins. Here we report that p63RhoGEF, a recently identified RhoA-specific guanine nucleotide exchange factor, enhances the Rho-dependent gene transcription induced by agonist-stimulated G(q/11)-coupled receptors (M3-cholinoceptor, histamine H1 receptor) or GTPase-deficient mutants of G alpha(q) and G alpha11. We further demonstrate that active G alpha(q) or G alpha11, but not G alpha12 or G alpha13, strongly enhances p63RhoGEF-induced RhoA activation by direct protein-protein interaction with p63RhoGEF at its C-terminal half. Moreover, the activation of p63RhoGEF by G alpha(q/11) occurs independently of and in competition to the activation of the canonical G alpha(q/11) effector phospholipase C beta. Therefore, our results elucidate a new signaling pathway by which G alpha(q/11)-coupled receptors specifically induce Rho signaling through a direct interaction of activated G alpha(q/11) subunits with p63RhoGEF.

The Sec14 Homology Domain Regulates the Cellular Distribution and Transforming Activity of the Rho-specific Guanine Nucleotide Exchange Factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose overexpression cause deregulated growth in murine fibroblasts. Dbs contains multiple recognizable motifs including a centrally located Rho-specific guanine nucleotide exchange factor domain, a COOH-terminal Src homology 3 domain, two spectrin-like repeats, and a recently identified NH(2)-terminal Sec14 homology domain. The transforming potential of Dbs is substantially activated by the removal of inhibitory sequences that lie outside of the core catalytic sequences, and in this current study we mapped this inhibition to the Sec14 domain. Surprisingly removal of the NH(2) terminus did not alter the catalytic activity of Dbs in vivo but rather altered its subcellular distribution. Whereas full-length Dbs was distributed primarily in a perinuclear structure that coincides with a marker for the Golgi apparatus, removal of the Sec14 domain was associated with translocation of Dbs to the cell periphery where it accumulated within membrane ruffles and lamellipodia. However, translocation of Dbs and the concomitant changes in the actin cytoskeleton were not sufficient to fully activate Dbs transformation. The Sec14 domain also forms intramolecular contacts with the pleckstrin homology domain, and these contacts must also be relieved to achieve full transforming activity. Collectively these observations suggest that the Sec14 domain regulates Dbs transformation through at least two distinct mechanisms, neither of which appears to directly influence the in vivo exchange activity of the protein.

The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1.

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.

P63RhoGEF and GEFT are Rho-specific guanine nucleotide exchange factors encoded by the same gene.

Activation of Rho GTPases, which play pivotal roles in diverse cellular functions, is catalysed by specific guanine nucleotide exchange factors (GEFs). We and others (Souchet et al. (2002)) independently cloned a human cDNA encoding a 580 aa protein (p63RhoGEF), which contains a tandem of Dbl homology and pleckstrin homology domains typical for RhoGEFs. In accordance with Souchet et al., recombinant p63RhoGEF interacted with and catalysed GDP/GTP exchange at RhoA, but not Rac1 or Cdc42. Recently, an N-terminally truncated form of p63RhoGEF, termed GEFT, was described as a Rac/Cdc42-specific GEF (Guo et al. 2003). As judged by RT-PCR with specific primers, we were able to detect mRNA variants encoding p63RhoGEF and GEFT within several tissues and cell lines. Apparently, they co-exist within one cell and are derived from the same gene. When expressed in human embryonic kidney cells, both p63RhoGEF and GEFT caused activation of RhoA, but not Rac1 or Cdc42, and induced serum response factor-mediated gene transcription, which was fully blunted by the Rho-inactivating C3 transferase. In line with these data, expression of either p63RhoGEF or GEFT in J82 human bladder carcinoma cells induced the formation of actin stress fibres. We therefore conclude that p63RhoGEF and GEFT are apparently isoforms derived from the same gene and that GEFT, similar to p63RhoGEF, activates RhoA in several cell types.

Phosphorylation-independent membrane relocalization of ezrin following association with Dbl in vivo.

Ezrin, a widespread protein involved in cell migration, morphogenesis and cell adhesion, belongs to a large family of proteins known as ERM (ezrin, radixin, moesin). These three closely related proteins are thought to function as linkers between plasma membrane and actin cytoskeleton and their function is regulated by the small GTP-binding protein Rho. It has been previously shown that the active form of radixin can bind in vitro to Dbl, a Rho-specific guanine nucleotide exchange factor, although an in vivo interaction has not yet been demonstrated. In this paper, we attempted to investigate whether ezrin can also associate with Dbl. We show here that Dbl protein can effectively bind both in vitro and in vivo to the N-terminal region (amino acids 1-531) of a constitutively active mutant of ezrin and with the full-length molecule. We found that this binding is mediated by the Dbl pleckstrin homology domain, responsible for the proper subcellular localization of the Dbl protein. Moreover, we show that Dbl induces localization to the plasma membrane of both the active deletion mutant and the full-length ezrin proteins. Finally, we show that the relocalization of ezrin is independent of Dbl GEF activity. These results indicate that Dbl could induce translocation of ezrin to the plasma membrane through a mechanism that does not require ezrin C-terminus phosphorylation by Rho-associated kinases.