Rho-specific Guanine Nucleotide Exchange Factor Research Articles

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

A novel function for Cyclin A2: Control of cell invasion via RhoA signaling

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.

Polarity-regulating Kinase Partitioning-defective 1b (PAR1b) Phosphorylates Guanine Nucleotide Exchange Factor H1 (GEF-H1) to Regulate RhoA-dependent Actin Cytoskeletal Reorganization

Partitioning-defective 1b (PAR1b), also known as microtubule affinity-regulating kinase 2 (MARK2), is a member of evolutionally conserved PAR1/MARK serine/threonine kinase family, which plays a key role in the establishment and maintenance of cell polarity at least partly by phosphorylating microtubule-associated proteins (MAPs) that regulate microtubule stability. PAR1b has also been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system. Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). This observation suggests a functional link between PAR1b and GEF-H1. Here we show that PAR1b induces phosphorylation of GEF-H1 on serine 885 and serine 959. We also show that PAR1b-induced serine 885/serine 959 phosphorylation inhibits RhoA-specific GEF activity of GEF-H1. As a consequence, GEF-H1 phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b not only regulates microtubule stability through phosphorylation of MAPs but also influences actin stress fiber formation by inducing GEF-H1 phosphorylation. The dual function of PAR1b in the microtubule-based cytoskeletal system and the actin-based cytoskeletal system in the coordinated regulation of cell polarity, cell morphology, and cell movement.

Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-β and miR-24: role in epithelial-to-mesenchymal transition

In the present study we analyzed the regulation of the two isoforms of the RhoA-specific guanine nucleotide exchange factor Net1 by transforming growth factor-β (TGF-β) in keratinocytes. We report that short-term TGF-β treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-β-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-β. Interestingly, long-term TGF-β treatment resulted in Net1 mRNA downregulation and Net1A protein degradation by the proteasome. Furthermore, we identified the microRNA miR-24 as a novel post-transcriptional regulator of Net1A expression. Silencing of Net1A resulted in disruption of E-cadherin- and zonula occludens-1 (ZO-1)-mediated junctions, as well as expression of the transcriptional repressor of E-cadherin, Slug and the mesenchymal markers N-cadherin, plasminogen activator inhibitor-1 (PAI-1) and fibronectin, indicating that late TGF-β-induced downregulation of Net1A is involved in epithelial-to-mesenchymal transition (EMT). Finally, miR-24 was found to be implicated in the regulation of the EMT program in response to TGF-β and was shown to be directly involved in the TGF-β-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short- and long-term TGF-β-mediated regulation of EMT.

Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and β-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, β-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.

Mechanistic Insights into Specificity, Activity, and Regulatory Elements of the Regulator of G-protein Signaling (RGS)-containing Rho-specific Guanine Nucleotide Exchange Factors (GEFs) p115, PDZ-RhoGEF (PRG), and Leukemia-associated RhoGEF (LARG)

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 10(7)-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.

A-Kinase Anchoring Protein (AKAP)-Lbc Anchors a PKN-based Signaling Complex Involved in α1-Adrenergic Receptor-induced p38 Activation

The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.

The Small GTPase RhoA Localizes to the Nucleus and Is Activated by Net1 and DNA Damage Signals

BackgroundRho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells.Principal FindingsUsing an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus.Conclusions/SignificanceThese results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.

The Application of Hybrid Methods to Study Complex, Multi-Domain Protein - RhoA-Specific Guanidine Nucleotide Exchange Factor - PDZ-RhoGEF

As the focus of structural biology shifts towards studies of increasingly complex, multi-domain proteins and their complexes, the use of hybrid methodologies is becoming a necessity. Here, we present our findings regarding the mechanism of regulation of a multi-domain, RhoA-specific guanine nucleotide exchange factor, PDZ-RhoGEF (PRG). PRG belongs to a small family of RGS GEFs and is an important component of the ‘RhoA cycle’.Activity of RhoA is tightly regulated by a sophisticated interplay between three proteins - guanine nucleotide exchange factor (RhoGEF), GTPase-activating protein (RhoGAP), and guanine nucleotide dissociation inhibitor (RhoGDI). GEFs catalyze the exchange of a nucleotide on RhoA, and consequently cause RhoA activation. Active RhoA transmits signal to its effectors (e.g. Rho-dependent kinase (ROCK)), and consequently triggers a number of physiological responses, including reorganization of actin cytoskeleton, neurite retraction and neuronal morphogenesis, and Ca2+ - sensitization of smooth muscle.The classical paradigm for regulation of multidomain GEFs states that these proteins are autoinhibited by the supramodular arrangement of their domains, which may interfere with the binding of upstream and downstream binding partners, and that a combination of inter-protein interactions initiates a conformational change, leading to the relief of autoinhibition. Using a broad array of biophysical and biochemical techniques, including SAXS, EPR, DLS, CD and others, we are investigating the structure of several multidomain constructs of PRG and we present current data suggesting a functional role for interdomain linkers.

HGAL, a germinal center specific protein, decreases lymphoma cell motility by modulation of the RhoA signaling pathway

AbstractHGAL is a germinal center (GC)–specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL enhances activation of RhoA and its down-stream effectors by a novel mechanism – direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL and cHL.

Mechanotransduction by GEF-H1 as a novel mechanism of ventilator-induced vascular endothelial permeability

Pathological lung overdistention associated with mechanical ventilation at high tidal volumes (ventilator-induced lung injury; VILI) compromises endothelial cell (EC) barrier leading to development of pulmonary edema and increased morbidity and mortality. We have previously shown involvement of microtubule (MT)-associated Rho-specific guanine nucleotide exchange factor GEF-H1 in the agonist-induced regulation of EC permeability. Using an in vitro model of human pulmonary EC exposed to VILI-relevant magnitude of cyclic stretch (18% CS) we tested a hypothesis that CS-induced alterations in MT dynamics contribute to the activation of Rho-dependent signaling via GEF-H1 and mediate early EC response to pathological mechanical stretch. Acute CS (30 min) induced disassembly of MT network, cell reorientation, and activation of Rho pathway, which was prevented by MT stabilizer taxol. siRNA-based GEF-H1 knockdown suppressed CS-induced disassembly of MT network, abolished Rho signaling, and attenuated CS-induced stress fiber formation and EC realignment compared with nonspecific RNA controls. Depletion of GEF-H1 in the murine two-hit model of VILI attenuated vascular leak induced by lung ventilation at high tidal volume and thrombin-derived peptide TRAP6. These data show for the first time the critical involvement of microtubules and microtubule-associated GEF-H1 in lung vascular endothelial barrier dysfunction induced by pathological mechanical strain.

Dynamic regulation of LARG in blastopore closure and archenteron formation during Xenopus laevis gastrulation

Rho GTPases have important roles in regulating cell migration and are activated by Rho-specific guanine nucleotide exchange factors (RhoGEFs). However, the role of leukemia-associated RhoGEF (LARG), responding to G12/13 family, has not been studied in vertebrate development. Here, the in vivo biochemical function of LARG was examined during early embryonic development in African frog Xenopus laevis. Gain-of-function study was performed by injecting the RNA of full-length xLARG to 2 cell-stage embryos. The ectopic expression of this protein resulted in the defect of blastopore closure during early embryogenesis. Expression of the dominant-negative form caused the defect in cell movement and following archenteron formation during late gastrulation, which is represented by the blister formation in the ventral side of the embryos. The phenotype was rescued by co-expressing the mutant with Rho or wild type xLARG, confirming the specificity of the dominant-negative activity of xLARG mutant. In this study, I showed for the first time that the spatiotemporal expression of xLARG is very dynamic and specifically regulated in early Xenopus embryonic development and xLARG may mediate Gα13 signal to activate Rho to exert its function in gastrulation movement and archenteron formation. My results implicate that the dynamic regulation of maternal and zygotic xLARG expression and its biochemical activity is necessary for proper gastrulation.

HGAL, a Lymphoma Prognostic Biomarker, Regulates Lymphocyte and Lymphoma Cell Motility by Modulation of RhoA Signaling Pathway.

Abstract 316Identification of genes whose expression correlates with clinical outcome is of paramount importance for designing upfront patient-tailored treatments that will potentially improve the outcome of lymphoma patients. Furthermore, new molecular prognostic biomarkers may lead to the discovery of novel pathophysiological mechanisms underlying lymphoma pathogenesis, potentially highlighting unique biological processes regulated by these proteins in lymphoma cells and in their normal cellular counterparts. HGAL is a novel germinal center (GC)-specific gene whose expression predicts survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Recently, we have demonstrated that HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and preventing lymphoma dissemination; however, the molecular mechanism of HGAL effects on cell motility is unknown. Herein, we demonstrated that HGAL serves as a physiological regulator of the RhoA signaling pathway. HGAL over-expression or siRNA-mediated knock-down leads to increased or decreased levels of GTP-bound RhoA, respectively, but does not affect levels of GTP-bound CDC42 and RAC1. HGAL-induced activation of RhoA results in inhibition of lymphocyte and lymphoma cell motility and chemotaxis by activation of its downstream effector ROCK leading to: a) phosphorylation of myosin regulatory light chain (MRLC) that regulates actin-activated Mg-ATPase activity of myosin II; b) phosphorylation of myosin phosphatase (myosin PPTase) subunit MYPT1 inhibiting myosin light chain dephosphorylation; and c) phosphorylation of cofilin leading to cytoskeletal reorganization, as reflected by increased formation of focal adhesions and stress fibers. Opposite effects on RhoA downstream effectors are observed upon knock-down of HGAL expression. Furthermore, HGAL over-expression or knock-down modulate additional functions of RhoA, including transcriptional activation by serum response factor and RhoA transforming potential, as assessed by foci formation in NIH 3T3 cells. Co-immunoprecipitation experiments demonstrated that the effect of HGAL on RhoA is indirect and is mediated by RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate. Moreover, we delineate the structural domains of HGAL and PDZ-RhoGEF that mediate the interaction between these proteins. Over-expression of HGAL mutants lacking the PDZ binding domain, mediating the interaction between HGAL and these RhoGEFs, fails to induce activation of RhoA and does not inhibit lymphoma cell motility and chemotaxis. Similarly, knock-downs of PDZ-RhoGEF and/or LARG reverse the inhibitory effects of HGAL on lymphoma cell motility and chemotaxis. These observations reveal a novel molecular mechanism underlying the inhibitory effects of HGAL on the motility of normal GC lymphocyte and GC-derived lymphoma cells. Since HGAL is specifically expressed only during the GC stage of B lymphocyte differentiation, these findings highlight the uniqueness and complexity of cell processes occurring during the GC reaction. They further elucidate the pathophysiologic mechanism underlying the favorable outcome of DLBCL and cHL patients whose tumors express high levels of HGAL protein. Disclosures:No relevant conflicts of interest to declare.

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130(Cas) on Tyr-410 and an increase in the abundance of the Crk.p130(Cas) complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130(Cas) and disrupts the Crk.p130(Cas) complex. We further determined that suppression of expression of Cdc42, Ack1, p130(Cas), or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk.p130(Cas) complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.

On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF

BackgroundThe Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Gα12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).ResultsHere we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition.ConclusionOur results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.

Lfc and Tctex-1 regulate the genesis of neurons from cortical precursor cells

The mechanisms that regulate symmetric, proliferative divisions versus asymmetric, neurogenic divisions of mammalian neural precursors are still not well understood. We found that Lfc (Arhgef2), a Rho-specific guanine nucleotide exchange factor that interacts with spindle microtubules, and its negative regulator Tctex-1 (Dynlt1) determine the genesis of neurons from precursors in the embryonic murine cortex. Specifically, genetic knockdown of Arhgef2 in cortical precursors either in culture or in vivo inhibited neurogenesis and maintained cells as cycling radial precursors. Conversely, genetic knockdown of Dynlt1 in radial precursors promoted neurogenesis and depleted cycling cortical precursors. Coincident silencing of these two genes indicated that Tctex-1 normally inhibits the genesis of neurons from radial precursors by antagonizing the proneurogenic actions of Lfc. Moreover, Lfc and Tctex-1 were required to determine the orientation of mitotic precursor cell divisions in vivo. Thus, Lfc and Tctex-1 interact to regulate cortical neurogenesis, potentially by regulating mitotic spindle orientation.

A Role for Gab1/SHP2 in Thrombin Activation of PAK1

To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.

The RhoA-specific guanine nucleotide exchange factor p63RhoGEF binds to activated Gα 16 and inhibits the canonical phospholipase Cβ pathway

Heterotrimeric G proteins regulate diverse physiological processes by modulating the activities of intracellular effectors. Members of the Gα q family link G protein-coupled receptor activation to phospholipase Cβ (PLCβ) activity and intracellular calcium signaling cascades. However, they differ markedly in biochemical properties as well as tissue distribution. Recent findings have shown that some of the cellular activities of Gα q family members are independent of PLCβ activation. A guanine nucleotide exchange factor, p63RhoGEF, has been shown to interact with Gα q proteins and thus provides linkage to RhoA activation. However, it is not known if p63RhoGEF can associate with other Gα q family members such as Gα 16. In the present study, we employed co-immunoprecipitation studies in HEK293 cells to demonstrate that p63RhoGEF can form a stable complex with the constitutively active mutant of Gα 16 (Gα 16QL). Interestingly, overexpression of p63RhoGEF inhibited Gα 16QL-induced IP 3 production in a concentration-dependent manner. The binding of PLCβ 2 to Gα 16QL could be displaced by p63RhoGEF. Similarly, p63RhoGEF inhibited the binding of tetratricopeptide repeat 1 to Gα 16QL, leading to a suppression of Gα 16QL-induced Ras activation. In the presence of p63RhoGEF, Gα 16QL-induced STAT3 phosphorylation was significantly reduced and Gα 16QL-mediated SRE transcriptional activation was attenuated. Taken together, these results suggest that p63RhoGEF binds to activated Gα 16 and inhibits its signaling pathways.