Oligomerization of DH Domain Is Essential for Dbl-Induced Transformation

ABSTRACT The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homology (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, and the DH domain, together with the immediately adjacent pleckstrin homology (PH) domain, constitutes the minimum module bearing transforming function. In the present study, we demonstrate that the onco-Dbl protein exists in oligomeric form in vitro and in cells. The oligomerization is mostly homophilic in nature and is mediated by the DH domain. Mutagenesis studies mapped the region involved in oligomerization to the conserved region 2 of the DH domain, which is located at the opposite side of the Rho GTPase interacting surface. Residue His556 of this region, in particular, is important for this activity, since the H556A mutant retained the GEF catalytic capability and the binding activity toward Cdc42 and RhoA in vitro but was deficient in oligomer formation. Consequently, the Rho GTPase activating potential of the H556A mutant was significantly reduced in cells. The focus-forming and anchorage-independent growth activities of onco-Dbl were completely abolished by the His556-to-Ala mutation, whereas the abilities to stimulate cell growth, activate Jun N-terminal kinase, and cause actin cytoskeletal changes were retained by the mutant. The ability of onco-Dbl to oligomerize allowed multiple Rho GTPases to be recruited to the same signaling complex, and such an ability is defective in the H556A mutant. Taken together, these results suggest that oligomerization of onco-Dbl through the DH domain is essential for cellular transformation by providing the means to generate a signaling complex that further augments and/or coordinates its Rho GTPase activating potential.

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Autoinhibition Mechanism of Proto-Dbl

Molecular and Cellular Biology ◽

10.1128/mcb.21.5.1463-1474.2001 ◽

2001 ◽

Vol 21 (5) ◽

pp. 1463-1474 ◽

Cited By ~ 51

Author(s):

Feng Bi ◽

Balazs Debreceni ◽

Kejin Zhu ◽

Barbara Salani ◽

Alessandra Eva ◽

...

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Cell Transformation ◽

Intramolecular Interaction ◽

Nucleotide Exchange ◽

Ph Domain ◽

Intracellular Targeting ◽

Nucleotide Exchange Factor ◽

Transforming Activity ◽

Dh Domain

ABSTRACT The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family. Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues. The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function. Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences. Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential. Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain. Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity. The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain. Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.

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Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

10.1101/541219 ◽

2019 ◽

Author(s):

Nathalie R. Reinhard ◽

Sanne van der Niet ◽

Anna Chertkova ◽

Marten Postma ◽

Peter L. Hordijk ◽

...

Keyword(s):

Endothelial Cells ◽

Single Cell ◽

Rho Gtpases ◽

Rho Gtpase ◽

Actin Dynamics ◽

Pleckstrin Homology Domain ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

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ARHGEF18/p114RhoGEF coordinates PKA/CREB signaling and actomyosin remodeling to drive trophoblast cell-cell fusion during placenta morphogenesis

10.1101/2020.07.12.199141 ◽

2020 ◽

Author(s):

Robert Beal ◽

Ana Alonso-Carriazo Fernandez ◽

Dimitris K. Grammatopoulos ◽

Karl Matter ◽

Maria S. Balda

Keyword(s):

Gene Expression ◽

Cell Fusion ◽

Rho Gtpases ◽

Rho Gtpase ◽

Trophoblast Cell ◽

Cell Junctions ◽

Nucleotide Exchange ◽

Cell Cell

SUMMARYCoordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating PKA signalling, and is required for PKA-induced actomyosin remodelling, CREB-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signalling, gene expression and cell-cell fusion.

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The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity

Biochemical Journal ◽

10.1042/bj20061020 ◽

2006 ◽

Vol 400 (3) ◽

pp. 563-572 ◽

Cited By ~ 33

Author(s):

Mark A. Baumeister ◽

Kent L. Rossman ◽

John Sondek ◽

Mark A. Lemmon

Keyword(s):

Rho Gtpases ◽

Regulatory Role ◽

Pleckstrin Homology Domain ◽

Membrane Targeting ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Ph Domain ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Dh Domain

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain–phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-δ1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.

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Functional Analysis of the Caenorhabditis elegans UNC-73B PH Domain Demonstrates a Role in Activation of the Rac GTPase In Vitro and Axon Guidance In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.6823-6835.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 6823-6835 ◽

Cited By ~ 23

Author(s):

Terrance J. Kubiseski ◽

Joe Culotti ◽

Tony Pawson

Keyword(s):

Caenorhabditis Elegans ◽

Axon Guidance ◽

Catalytic Domain ◽

Nucleotide Exchange ◽

Ph Domain ◽

Rac Gtpase ◽

Ability To Act ◽

Dh Domain

ABSTRACT The Caenorhabditis elegans UNC-73B protein regulates axon guidance through its ability to act as a guanine nucleotide exchange factor (GEF) for the CeRAC/MIG-2 GTPases. Like other GEFs for Rho family GTPases, UNC-73B has a Dbl homology (DH) catalytic domain, followed by a C-terminal pleckstrin homology (PH) domain. We have explored whether the PH domain cooperates with the adjacent DH domain to promote UNC-73B GEF activity and axonal pathfinding. We show that the UNC-73B PH domain binds preferentially to monophosphorylated phosphatidylinositides in vitro. Replacement of residues Lys1420 and Arg1422 with Glu residues within the PH domain impaired this phospholipid binding but did not affect the in vitro catalytic activity of the DH domain. In contrast, a mutant UNC-73B protein with a Trp1502-to-Ala substitution in the PH domain still interacted with phosphorylated phosphatidylinositides but had lost its GEF activity. UNC-73B minigenes containing these mutations were microinjected into C. elegans and transferred to unc-73(e936) mutant worms. Unlike the wild-type protein, neither PH domain mutant was able to rescue the unc-73 axon guidance defect. These results suggest that the UNC-73B PH domain plays distinct roles in targeting and promoting GEF activity towards the Rac GTPase, both of which are important for the directed movements of motorneurons in vivo.

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Regulation of Rho GTPases by the Hematopoietic-Specific Guanine Nucleotide Exchange Factor Vav1 Is Critical for Hematopoietic Stem Cell Retention in the Endosteal Niche and Engraftment.

Blood ◽

10.1182/blood.v114.22.80.80 ◽

2009 ◽

Vol 114 (22) ◽

pp. 80-80

Author(s):

Abel Sanchez-Aguilera ◽

Yun-Jung Lee ◽

Cristina Lo Celso ◽

Kristina Brumme ◽

Charles P Lin ◽

...

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Nucleotide Exchange ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Guanine Nucleotide Exchange ◽

Endosteal Niche ◽

Gtpase Activation

Abstract Abstract 80 Background: Rho GTPases are molecular switches that regulate actin cytoskeleton dynamics, cell proliferation and survival. In hematopoietic stem cells and progenitors (HSC/P), several Rho GTPases (including Rac1, Rac2 and Cdc42) function as critical regulators of engraftment through the integration of diverse extracellular signals, such as those transmitted by growth factor, chemokine and adhesion receptors. In addition, Rac-deficient mice show significantly increased numbers of mobilized HSC/P. GTPase activation downstream of these and other receptors is mediated by a large family of guanine nucleotide exchange factors (GEF). Functional interactions between receptors, GEF and Rho GTPases are potentially complex and the crucial biochemical pathways regulating HSC activity have not been defined. Among the Rho/Rac GEFs, Vav1 shows hematopoietic-specific expression and has been previously implicated in immune cell processes, such as immunoreceptor signaling in lymphocytes and neutrophil migration. To further explore the mechanism of Rho GTPase regulation of HSC engraftment, we investigated the role of Vav1 GEF in Rho GTPase activation after ligation of multiple HSC receptors and the effect of genetic deletion of Vav1 on HSC homing, retention and engraftment in the hematopoietic microenvironment. Methods: GTPase activation (Rac, Cdc42, RhoA) was analyzed by in vitro pulldown assays. The HSC/P compartment of Vav1−/− mice was studied by flow cytometry, colony forming cell (CFC) assays, progenitor (CFC) homing, competitive and non-competitive repopulation assays. HSC localization in the endosteal niche was determined by intravital microscopy 1 h and 48 h after transplant. Results: At the biochemical level, Vav1−/− hematopoietic progenitors showed a dysfunctional Rho GTPase activation pattern, with increased baseline levels of GTP-bound Rac, Cdc42 and RhoA; however, in the absence of Vav1, these GTPases were unresponsive to stimulation by stem cell factor and SDF1α, critical proteins in HSC engraftment. In spite of this biochemical abnormality, Vav1−/− mice at baseline had nearly normal numbers of immunophenotypically defined HSC, myeloid and lymphoid progenitors in the bone marrow (BM), and normal hematopoietic progenitor content as defined by CFC, although reduced rather than increased circulating HSC/P. Vav1−/− HSC/P transplanted into irradiated recipients exhibited normal BM CFC homing efficiency (∼5%) and normal early endosteal localization of HSC in vivo (1 h after injection) as determined by intravital microscopy. Surprisingly-but in concordance with the normal BM homing of HSC/P in vivo- the loss of Vav1 did not affect hematopoietic progenitor chemotaxis or short-term adhesion to fibronectin in vitro. However, there was a significant decrease in the retention of HSC in the endosteal space at 48 h after transplant (Vav1−/− HSC numbers were reduced to 46%, relative to WT HSC) and this defect was associated with a profound loss of short- and long-term engraftment. In competitive repopulation assays, Vav1−/− cells virtually did not contribute to the graft (Table 1), whereas in a non-competitive setting, they either failed to rescue the recipient (60% survival vs 100% at 1 month, Vav1−/− vs WT) or showed significantly delayed hematopoietic reconstitution (Table 2). Conclusions: The hematopoietic-specific GEF Vav1 is essential for the appropriate microenvironment-induced Rho GTPase activation in HSC/P after transplant and is required for the retention of HSC/P in the BM endosteal niche and subsequent engraftment. Disclosures: No relevant conflicts of interest to declare.

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ARHGEF18/p114RhoGEF Coordinates PKA/CREB Signaling and Actomyosin Remodeling to Promote Trophoblast Cell-Cell Fusion During Placenta Morphogenesis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.658006 ◽

2021 ◽

Vol 9 ◽

Author(s):

Robert Beal ◽

Ana Alonso-Carriazo Fernandez ◽

Dimitris K. Grammatopoulos ◽

Karl Matter ◽

Maria S. Balda

Keyword(s):

Gene Expression ◽

Cell Fusion ◽

Rho Gtpases ◽

Rho Gtpase ◽

Trophoblast Cell ◽

Cell Junctions ◽

Nucleotide Exchange ◽

Cell Cell

Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signaling, gene expression and cell-cell fusion.

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A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions

Journal of Cell Science ◽

10.1242/jcs.115.14.2867 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2867-2879 ◽

Cited By ~ 4

Author(s):

Valérie Derrien ◽

Carole Couillault ◽

Michel Franco ◽

Stéphanie Martineau ◽

Philippe Montcourrier ◽

...

Keyword(s):

Amino Acid ◽

Coiled Coil ◽

Terminal Region ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Ph Domain ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Sec7 Domain

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

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Hormones Secretion and Rho GTPases in Neuroendocrine Tumors

Cancers ◽

10.3390/cancers12071859 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1859

Author(s):

Laura Streit ◽

Laurent Brunaud ◽

Nicolas Vitale ◽

Stéphane Ory ◽

Stéphane Gasman

Keyword(s):

Neuroendocrine Tumors ◽

Rho Gtpases ◽

Rho Gtpase ◽

Secretory Activity ◽

Guanine Nucleotide Exchange Factors ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Gtpase Activating Proteins

Neuroendocrine tumors (NETs) belong to a heterogeneous group of neoplasms arising from hormone secreting cells. These tumors are often associated with a dysfunction of their secretory activity. Neuroendocrine secretion occurs through calcium-regulated exocytosis, a process that is tightly controlled by Rho GTPases family members. In this review, we compiled the numerous mutations and modification of expression levels of Rho GTPases or their regulators (Rho guanine nucleotide-exchange factors and Rho GTPase-activating proteins) that have been identified in NETs. We discussed how they might regulate neuroendocrine secretion.

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Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly

The Journal of Cell Biology ◽

10.1083/jcb.201807106 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3397-3414 ◽

Cited By ~ 6

Author(s):

Jordan T. Silver ◽

Frederik Wirtz-Peitz ◽

Sérgio Simões ◽

Milena Pellikka ◽

Dong Yan ◽

...

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Adherens Junctions ◽

Nucleotide Exchange ◽

Temporal Regulation ◽

Apical Polarity ◽

Polarity Proteins ◽

Spatio Temporal ◽

Crumbs Complex

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity.

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