RESUMO
The Wnt signaling pathways play pivotal roles in carcinogenesis. Modulation of the cell-surface abundance of Wnt receptors is emerging as an important mechanism for regulating sensitivity to Wnt ligands. Endocytosis and degradation of the Wnt receptors Frizzled (Fzd) and lipoprotein-related protein 6 (LRP6) are regulated by the E3 ubiquitin ligases zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), which are disrupted in cancer. In a genome-wide small interfering RNA screen, we identified the deubiquitylase ubiquitin-specific protease 6 (USP6) as a potent activator of Wnt signaling. USP6 enhances Wnt signaling by deubiquitylating Fzds, thereby increasing their cell-surface abundance. Chromosomal translocations in nodular fasciitis result in USP6 overexpression, leading to transcriptional activation of the Wnt/ß-catenin pathway. Inhibition of Wnt signaling using Dickkopf-1 (DKK1) or a Porcupine (PORCN) inhibitor significantly decreased the growth of USP6-driven xenograft tumors, indicating that Wnt signaling is a key target of USP6 during tumorigenesis. Our study defines an additional route to ectopic Wnt pathway activation in human disease, and identifies a potential approach to modulate Wnt signaling for therapeutic benefit.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores Frizzled/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Via de Sinalização Wnt , Animais , Proteínas de Ligação a DNA/genética , Receptores Frizzled/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Neoplasias Experimentais/genética , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Antígenos de Histocompatibilidade Menor , Modelos Biológicos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Pseudópodes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , TransfecçãoRESUMO
During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Baculoviridae , Western Blotting , Moléculas de Adesão Celular Neuronais/farmacologia , Células Cultivadas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Vetores Genéticos , Espectrometria de Massas , Camundongos , Proteínas Nucleares/isolamento & purificação , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteína Reelina , Serina Endopeptidases , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
BACKGROUND: The Rnd proteins Rnd1, Rnd2, and Rnd3 (RhoE) comprise a unique branch of Rho-family G-proteins that lack intrinsic GTPase activity and consequently remain constitutively "active." Prior studies have suggested that Rnd proteins play pivotal roles in cell regulation by counteracting the biological functions of the RhoA GTPase, but the molecular basis for this antagonism is unknown. Possible mechanisms by which Rnd proteins could function as RhoA antagonists include sequestration of RhoA effector molecules, inhibition of guanine nucleotide exchange factors, and activation of GTPase-activating proteins (GAPs) for RhoA. However, effector molecules of Rnd proteins with such properties have not been identified. RESULTS: Here we identify p190 RhoGAP (p190), the most abundant GAP for RhoA in cells, as an interactor with Rnd proteins and show that this interaction is mediated by a p190 region that is distinct from the GAP domain. Using Rnd3-RhoA chimeras and Rnd3 mutants defective in p190 binding, as well as p190-deficient cells, we demonstrate that the cellular effects of Rnd expression are mediated by p190. We moreover show that Rnd proteins increase the GAP activity of p190 toward GTP bound RhoA and, finally, demonstrate that expression of Rnd3 leads to reduced cellular levels of RhoA-GTP by a p190-dependent mechanism. CONCLUSIONS: Our results identify p190 RhoGAPs as effectors of Rnd proteins and demonstrate a novel mechanism by which Rnd proteins function as antagonists of RhoA.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Células 3T3 , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA , Expressão Gênica , Camundongos , Microscopia de Fluorescência , Testes de Precipitina , Proteínas Repressoras , Técnicas do Sistema de Duplo-Híbrido , Proteínas rho de Ligação ao GTP , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
SGEF (SH3-containing Guanine Nucleotide Exchange Factor) is a RhoGEF of unknown function. We found the SGEF protein to be expressed in many established cell lines and highly expressed in human liver tissue. SGEF stimulated the formation of large interconnected membrane ruffles across dorsal surfaces when expressed in fibroblasts. SGEF required its proline-rich amino-terminus to generate dorsal, but not lateral, membrane ruffles and a functional SH3 domain to colocalize with filamentous actin at sites of membrane protrusion. Full-length SGEF activated RhoG, but not Rac, when expressed in fibroblasts. Further, recombinant SGEF DH/PH protein exchanged nucleotide on RhoG, but not on Rac1 or Rac3, in vitro. Scanning electron microscopy of fibroblasts demonstrated that SGEF induced dorsal ruffles that were morphologically similar to those generated by constitutively active RhoG, but not constitutively active Rac1. Transient expression of SGEF stimulated fibroblast uptake of 10-kDa dextran, a marker of macropinocytosis. This required the full-length protein and a catalytically active DH domain. Finally, activated RhoG was found to be more effective than activated Rac, and comparable to SGEF, in its ability to trigger dextran uptake. Together, this work establishes SGEF as a RhoG exchange factor and provides evidence that both SGEF and RhoG regulate membrane dynamics in promotion of macropinocytosis.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Pinocitose/fisiologia , Actinas/análise , Actinas/metabolismo , Animais , Linhagem Celular , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/genética , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Pinocitose/genética , Mutação Puntual/genética , Proteínas rho de Ligação ao GTPRESUMO
An assay was developed that allows the precipitation of the active pools of Rho-GEFs, Rho-GAPs, or effectors from cell or tissue lysates. This assay can be used to identify GEFs, GAPs, and effectors involved in specific cellular pathways to determine their GTPase specificity and to monitor the temporal activation of GEFs and GAPs in response to upstream signals.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Células CHO , Cricetinae , Glutationa Transferase/genética , Humanos , Camundongos , Células NIH 3T3 , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/isolamento & purificaçãoRESUMO
BACKGROUND: Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. RESULTS: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. CONCLUSION: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.
Assuntos
Imunoprecipitação da Cromatina , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Intestinos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Reprodutibilidade dos Testes , Software , Células-Tronco/patologia , Via de Sinalização Wnt/genéticaRESUMO
The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/ß-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/ß-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of ß-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/ß-catenin signaling. Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a ß-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/ß-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/ß-catenin signaling to apoptosis in melanoma.
RESUMO
The multi-protein beta-catenin destruction complex tightly regulates beta-catenin protein levels by shuttling beta-catenin to the proteasome. Glycogen synthase kinase 3beta (GSK3beta), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark beta-catenin for ubiquitination and subsequent degradation. Because modulation of both beta-catenin and GSK3beta activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the beta-catenin/GSK3beta interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a beta-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3'oxime (BIO), a specific inhibitor of GSK3beta. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of beta-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear beta-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3beta and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFalpha, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of beta-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Lentivirus/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , beta Catenina/metabolismo , Anti-Inflamatórios/farmacologia , Linhagem Celular , Humanos , Indóis/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Metotrexato/farmacologia , Modelos Biológicos , Oximas/metabolismo , Fosforilação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Wnts are secreted ligands that activate several receptor-mediated signal transduction cascades. Homeostatic Wnt signaling through beta-catenin is required in adults, because either elevation or attenuation of beta-catenin function has been linked to diverse diseases. To contribute to the identification of both protein and pharmacological regulators of this pathway, we describe a combinatorial screen that merged data from a high-throughput screen of known bioactive compounds with an independent focused small interfering RNA screen. Each screen independently revealed Bruton's tyrosine kinase (BTK) as an inhibitor of Wnt-beta-catenin signaling. Loss of BTK function in human colorectal cancer cells, human B cells, zebrafish embryos, and cells derived from X-linked agammaglobulinemia patients with a mutant BTK gene resulted in elevated Wnt-beta-catenin signaling, confirming that BTK acts as a negative regulator of this pathway. From affinity purification-mass spectrometry and biochemical binding studies, we found that BTK directly interacts with a nuclear component of Wnt-beta-catenin signaling, CDC73. Further, we show that BTK increased the abundance of CDC73 in the absence of stimulation and that CDC73 acted as a repressor of beta-catenin-mediated transcription in human colorectal cancer cells and B cells.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Linhagem Celular , Cromatografia de Afinidade , Humanos , Espectrometria de Massas , Proteínas Tirosina Quinases/isolamento & purificaçãoRESUMO
The identification and characterization of previously unidentified signal transduction molecules has expanded our understanding of biological systems and facilitated the development of mechanism-based therapeutics. We present a highly validated small interfering RNA (siRNA) screen that functionally annotates the human genome for modulation of the Wnt/beta-catenin signal transduction pathway. Merging these functional data with an extensive Wnt/beta-catenin protein interaction network produces an integrated physical and functional map of the pathway. The power of this approach is illustrated by the positioning of siRNA screen hits into discrete physical complexes of proteins. Similarly, this approach allows one to filter discoveries made through protein-protein interaction screens for functional contribution to the phenotype of interest. Using this methodology, we characterized AGGF1 as a nuclear chromatin-associated protein that participates in beta-catenin-mediated transcription in human colon cancer cells.
Assuntos
Transativadores/metabolismo , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/fisiologia , Linhagem Celular Tumoral , Neoplasias do Colo , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.
Assuntos
Fibroblastos/citologia , Fibroblastos/enzimologia , Fibronectinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Transporte Proteico , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Fibras de Estresse/metabolismoRESUMO
Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.
Assuntos
Caderinas/fisiologia , Comunicação Celular/fisiologia , Junções Célula-Matriz/fisiologia , Integrinas/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Adesão Celular/fisiologia , Citoesqueleto/metabolismo , Humanos , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Cadherins are transmembrane receptors that mediate cell-cell adhesion in epithelial cells. A number of changes occur during cadherin-mediated junction formation, one of which is a rearrangement of the actin cytoskeleton. Key regulators of actin cytoskeletal dynamics in cells are the Rho family of GTPases. We have demonstrated in previous studies that cadherin signaling suppresses RhoA activity and activates Rac1. The signaling events downstream of cadherins that modulate the activity of Rho family proteins remain unknown. Here we have identified a pathway by which RhoA becomes inactivated by cadherins. To determine whether cadherins regulate RhoA through activation of a GTPase-activating protein (GAP) for RhoA, we used constitutively active RhoA to isolate activated GAPs. Using this assay, we have identified the RhoA-specific GAP, p190RhoGAP, downstream from engaged cadherins. We found that cadherin engagement induced tyrosine phosphorylation of p190RhoGAP and increased its binding to p120RasGAP. The increased precipitation of p190RhoGAP with 63LRhoA was blocked by addition of PP2 suggesting that Src family kinases are required downstream from cadherin signaling. The inhibition of RhoA activity by cadherins was antagonized by expression of a dominant negative p190RhoGAP. Taken together, these data demonstrate that p190RhoGAP activity is critical for RhoA inactivation by cadherins.
Assuntos
Caderinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células CHO , Adesão Celular , Cricetinae , Fibronectinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Genes Dominantes , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , Transfecção , Tirosina/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Clonagem Molecular , Adesões Focais/fisiologia , Biblioteca Gênica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Cinética , Leucemia Mieloide , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transfecção , Proteínas ras , Proteínas rho de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoCRESUMO
Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.
Assuntos
Astrócitos/metabolismo , Integrinas/química , Antígenos Thy-1/metabolismo , Proteína rhoA de Ligação ao GTP/química , Amidas/farmacologia , Animais , Adesão Celular , Linhagem Celular , Matriz Extracelular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Adesões Focais , Genes Dominantes , Integrinas/metabolismo , Ligantes , Microscopia de Fluorescência , Ligação Proteica , Piridinas/farmacologia , Ratos , Proteínas Recombinantes/química , Fatores de TempoRESUMO
New research suggests that, during tubulointerstitial fibrosis, alpha-smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-beta(1) (TGF-beta(1)) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-beta(1)-induced EMT in LLC-PK(1) cells and examined the role of the small GTPase Rho and its effector, Rho kinase, (ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-beta(1) treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, beta-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-beta(1) induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho-inhibiting C3 transferase and by dominant negative (DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-beta(1)-induced SMA promoter activation was potently abrogated by two Rho-inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN-ROK. Truncation analysis showed that the first CC(A/T)richGG (CArG B) serum response factor-binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-beta(1)-induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ROK pathway, whereas the transcriptional effects are partially ROK independent.
Assuntos
Actinas/metabolismo , Proteínas de Fase Aguda/fisiologia , Músculo Liso/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Animais , Diferenciação Celular/fisiologia , Citoesqueleto/fisiologia , Células Epiteliais/citologia , Células LLC-PK1 , Mesoderma/citologia , Suínos , Fator de Crescimento Transformador beta1RESUMO
Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.
Assuntos
Cadeias Leves de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Soluções Hipertônicas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Células LLC-PK1 , Pressão Osmótica , Fosforilação , Membro 2 da Família 12 de Carreador de Soluto , Suínos , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rhoRESUMO
Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively