AnatomyOrganismsDiseasesChemicals and DrugsAnalytical, Diagnostic and Therapeutic Techniques and EquipmentPhenomena and ProcessesInformation Science
Guanine Nucleotide Exchange FactorsRho Guanine Nucleotide Exchange Factorsras Guanine Nucleotide Exchange FactorsGuanine NucleotidesProto-Oncogene Proteins c-vavGuanosine Diphosphateras-GRF1cdc42 GTP-Binding ProteinGuanosine Triphosphaterac1 GTP-Binding Proteinrho GTP-Binding ProteinsADP-Ribosylation Factorsrac GTP-Binding Proteinsrap1 GTP-Binding ProteinsrhoA GTP-Binding ProteinGuanine Nucleotide-Releasing Factor 2GTPase-Activating ProteinsSOS1 ProteinGTP-Binding ProteinsADP-Ribosylation Factor 1GTP PhosphohydrolasesProtein Structure, TertiaryMolecular Sequence Dataral Guanine Nucleotide Exchange FactorSon of Sevenless ProteinsProtein BindingAmino Acid SequenceSignal TransductionEukaryotic Initiation Factor-2Brab GTP-Binding Proteinsras ProteinsGuanineral GTP-Binding Proteinsrap GTP-Binding ProteinsCOS CellsProteinsGuanine Nucleotide Dissociation InhibitorsNucleotidesrab5 GTP-Binding ProteinsSequence Homology, Amino AcidGTP-Binding Protein alpha Subunits, G12-G13Coat Protein Complex IAdaptor Proteins, Signal TransducingMonomeric GTP-Binding ProteinsEnzyme ActivationMutationCell LineCell Cycle ProteinsGuanosine 5'-O-(3-Thiotriphosphate)p21-Activated KinasesTwo-Hybrid System TechniquesRecombinant Fusion ProteinsProtein TransportEukaryotic Initiation Factor-2ran GTP-Binding ProteinHSP110 Heat-Shock Proteins3T3 CellsProto-Oncogene Proteins p21(ras)TransfectionActinsProto-Oncogene Proteinsrab1 GTP-Binding ProteinsPhosphorylationSaccharomyces cerevisiae ProteinsHeLa CellsBrefeldin ANIH 3T3 CellsCell Membranerho-Specific Guanine Nucleotide Dissociation InhibitorsBinding SitesCarrier ProteinsCytoskeletonOncogene Proteinssrc Homology DomainsSaccharomyces cerevisiaeCloning, MolecularCell MovementA Kinase Anchor ProteinsKineticsModels, BiologicalStress FibersFungal Proteinscdc42 GTP-Binding Protein, Saccharomyces cerevisiaeGolgi ApparatusGRB2 Adaptor ProteinRecombinant ProteinsBase Sequenceras GTPase-Activating ProteinsMicroscopy, FluorescencePhosphoproteinsCercopithecus aethiopsSequence AlignmentProtein-Serine-Threonine KinasesGTP-Binding Protein alpha SubunitsModels, MolecularVesicular Transport ProteinsCells, CulturedrhoB GTP-Binding ProteinMembrane ProteinsHeterotrimeric GTP-Binding Proteins

Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42. (73/3221)

Guanine nucleotide exchange factors of the Dbl family regulate the actin cytoskeleton through activation of Rho-like GTPases. At present the Dbl family consists of more than thirty members; many have not been phenotypically or biochemically characterized. Guanine nucleotide exchange factors universally feature a Dbl homology domain followed by a pleckstrin homology domain. Employing data base screening we identified a recently cloned cDNA, KIAA0424, showing substantial sequence homology with Rac activators such as Tiam1, Sos, Vav, and PIX within the catalytic domain. This cDNA appears to be the human homologue of the Ascidian protein Posterior End Mark-2 (PEM-2). We refer to this exchanger as hPEM-2. hPEM-2 encodes a protein of 70 kDa and features an N-terminal src homology 3 domain, followed by tandem Dbl homology and pleckstrin homology domains. The gene is highly expressed in brain and is localized on the human X-chromosome. Employing biochemical activity assays for Rho-like GTPases we found that hPEM-2 specifically activates Cdc42 and not Rac or RhoA. Ectopic expression of hPEM-2 in NIH3T3 fibroblasts revealed a Cdc42 phenotype featuring filopodia formation, followed by cortical actin polymerization and cell rounding. hPEM-2 represents an exchange factor, which may have a role in the regulation of a number of cellular processes through Cdc42.  (+info)

Activation of the PAK-related kinase by human immunodeficiency virus type 1 Nef in primary human peripheral blood lymphocytes and macrophages leads to phosphorylation of a PIX-p95 complex. (74/3221)

Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication in both primary T lymphocytes and monocyte-derived macrophages. This enhancement phenotype has been linked to the ability of Nef to modulate the activity of cellular kinases. We find that despite the reported high-affinity interaction between Nef and the Src kinase Hck in vitro, a Nef-Hck interaction in the context of HIV-1-infected primary macrophages is not detectable. However, Nef binding and activation of the PAK-related kinase and phosphorylation of its substrate could be readily detected in both infected primary T lymphocytes and macrophages. Furthermore, we show that this substrate is a complex composed of the recently characterized PAK interacting partner PIX (PAK-interacting guanine nucleotide exchange factor) and its tightly associated p95 protein. PAK and PIX-p95 appear to be differentially activated and phosphorylated depending on the intracellular environment in which nef is expressed. These results identify the PIX-p95 complex as a novel effector of Nef in primary cells and suggest that the regulation of the PAK signaling pathway may differ in T cells and macrophages.  (+info)

Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins. (75/3221)

Human DOCK180, which was originally identified as a major protein bound to the Crk oncogene product, is an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. After DOCK180, at least three putative human proteins that manifest high amino acid sequence similarity to DOCK180 have been registered in the GenBank/EMBL database. We have designated one of them, KIAA0209, as DOCK2 and characterize here. DOCK2 mRNA was expressed mostly in peripheral blood cells, followed by slight expression in the spleen and thymus, whereas DOCK180 was expressed in all tissues tested except in peripheral blood cells. Immunostaining of human cadaver tissues revealed that the expression of DOCK2 was limited to the lymphocytes and macrophages of various organs. DOCK2 bound to and activated Rac1, as did DOCK180; however, DOCK2 did not bind to CrkII, which transduces signals at focal adhesions. Thus, DOCK180 and DOCK2 are regulators of Rac and function in adherent and non-adherent cells, respectively.  (+info)

Activation of Ras by phorbol esters in cardiac myocytes. Role of guanine nucleotide exchange factors. (76/3221)

The relationship between protein kinase C (PKC) activation and Ras function was investigated in cardiac cells. Ras function was required for ERK activation by phorbol esters in cardiac myocytes, but not in cardiac fibroblasts. Accordingly, treatment with phorbol esters resulted in GTP loading of Ras in cardiac myocytes, but not fibroblasts. Ras activation by phorbol esters was abolished by a PKC specific inhibitor, but was insensitive to tyrosine kinase inhibitors. Ras activation was mediated by stimulation of guanine nucleotide exchange. These results suggest the existence of a novel pathway for Ras activation, specific to cardiac myocytes, with implications for myocardial hypertrophy.  (+info)

DelGEF, an RCC1-related protein encoded by a gene on chromosome 11p14 critical for two forms of hereditary deafness. (77/3221)

We have cloned a human cDNA, DELGEF (deafness locus associated putative guanine nucleotide exchange factor), derived from a 225 kb genomic sequence of chromosome 11p14, critical for the Usher 1C syndrome and for DFNB18, a locus for non-syndromic sensorineural deafness. The amino acid sequence of the protein hDelGEF1 is homologous to the nucleotide exchange factor RCCI for the small GTPase Ran. hDelGEF2 is derived from the same DELGEF gene by alternative splicing. In addition, we have identified a murine homologue, mDelGEF. The ubiquitously expressed soluble protein hDelGEF1 is found both in the cyytoplasm and in the nucleus. Overexpressed hDelGEF2 colocalizes with mitochondria.  (+info)

Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons. (78/3221)

c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells.  (+info)

Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis. (79/3221)

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.  (+info)

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. (80/3221)

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.  (+info)