RESUMO
The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.
Assuntos
Guanosina Trifosfato/química , Mutação de Sentido Incorreto , Proteínas rac de Ligação ao GTP/química , Substituição de Aminoácidos , Ativação Enzimática , Guanosina Trifosfato/genética , Guanosina Trifosfato/imunologia , Humanos , Hidrólise , NADPH Oxidase 2/química , NADPH Oxidase 2/genética , NADPH Oxidase 2/imunologia , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Quinases Ativadas por p21/química , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/imunologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologia , Proteína RAC2 de Ligação ao GTPRESUMO
Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Adesão Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas dos Microfilamentos/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Micetozoários , Fagocitose , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Alinhamento de Sequência , Relação Estrutura-Atividade , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genéticaRESUMO
The P-Rex family are Dbl-type guanine-nucleotide exchange factors for Rac family small G proteins. They are distinguished from other Rac-GEFs through their synergistic mode of activation by the lipid second messenger phosphatidyl inositol (3,4,5) trisphosphate and the Gßγ subunits of heterotrimeric G proteins, thus acting as coincidence detectors for phosphoinositide 3-kinase and G protein coupled receptor signaling. Work in genetically-modified mice has shown that P-Rex1 has physiological importance in the inflammatory response and the migration of melanoblasts during development, whereas P-Rex2 controls the dendrite morphology of cerebellar Purkinje neurons as well as glucose homeostasis in liver and adipose tissue. Deregulation of P-Rex1 and P-Rex2 expression occurs in many types of cancer, and P-Rex2 is frequently mutated in melanoma. Both GEFs promote tumor growth or metastasis. This review critically evaluates the P-Rex literature and tools available and highlights exciting recent developments and open questions.
Assuntos
Diabetes Mellitus/metabolismo , Neoplasias/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Humanos , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Sistemas do Segundo Mensageiro , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genéticaRESUMO
While numerous studies support regulation of Ras GTPases by reactive oxygen and nitrogen species, the Rho subfamily has received considerably less attention. Over the last few years, increasing evidence is emerging that supports the redox sensitivity of Rho GTPases. Moreover, as Rho GTPases regulate the cellular redox state by controlling enzymes that generate and convert reactive oxygen and nitrogen species, redox feedback loops likely exist. Here, we provide an overview of cellular oxidants, Rho GTPases, and their inter-dependence.
Assuntos
Proteínas rho de Ligação ao GTP/metabolismo , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Oxirredução , Processamento de Proteína Pós-Traducional , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Sistemas do Segundo Mensageiro , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b 558 (a heterodimer of Nox2 and p22(phox)) and 4 cytosolic components: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67(phox) playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67(phox); (5) Induction of a conformational change in p67(phox), promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general.
Assuntos
NADPH Oxidases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/química , Fagócitos/citologia , Fagócitos/enzimologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac de Ligação ao GTP/químicaRESUMO
Rho GTPases are a class of evolutionarily conserved proteins comprising 20 members, which are predominantly known for their role in regulating the actin cytoskeleton. They are primarily regulated by binding of GTP/GDP, which is again controlled by regulators like GEFs, GAPs, and RhoGDIs. Rho GTPases are thus far well known for their role in the regulation of actin cytoskeleton and migration. Here we present an overview on the role of Rho GTPases in regulating cell shape and plasticity of cell migration. Finally, we discuss the emerging roles of ubiquitination and sumoylation in regulating Rho GTPases and cell migration.
Assuntos
Proteínas rho de Ligação ao GTP/metabolismo , Movimento Celular , Forma Celular , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Sumoilação , Ubiquitinação , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/classificaçãoRESUMO
Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of ß2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, ß2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for ß2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region.
Assuntos
Diglicerídeos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Domínios Proteicos Ricos em Prolina , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Fator de Crescimento Epidérmico/farmacologia , Proteínas Ativadoras de GTPase/química , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-Atividade , Vanadatos/farmacologia , Proteínas rac de Ligação ao GTP/química , Domínios de Homologia de srcRESUMO
The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocina CXCL12/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Quimiotaxia , Ativação Enzimática , Feminino , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/química , Humanos , Dados de Sequência Molecular , Invasividade Neoplásica , Proteínas rac de Ligação ao GTP/químicaRESUMO
We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac2 and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Experiments using GST fusion proteins or GST-free counterparts, purified proteins revealed that the PX domain is sufficient to exert GEF activity similar to full-length PLD2. The PLD2-GEF catalytic site is formed by a hydrophobic pocket of residues Phe-107, Phe-129, Leu-166, and Leu-173, all of which are in the PX domain. A nearby Arg-172 is also important in the overall activity. PX mutants altering any of those five amino acids fail to have GEF activity but still bind to Rac2, while their lipase activity was mostly unaffected. In addition to the PX domain, a region in the pleckstrin homology domain (Ile-306-Ala-310) aids in the PX-mediated GEF activity by providing a docking site to hold Rac2 in place during catalysis. We conclude that PLD2 is a unique GEF, with the PX being the major catalytic domain for its GEF activity, whereas the pleckstrin homology domain assists in the PX-mediated activity. The physiological relevance of this novel GEF in cell biology is demonstrated here in chemotaxis and phagocytosis of leukocytes, as the specific PX and PH mutants abolished cell function. Thus, this study reveals for the first time the catalytic site that forms the basis for the mechanism behind the GEF activity of PLD2.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfolipase D/química , Animais , Células COS , Domínio Catalítico , Movimento Celular , Quimiotaxia , Chlorocebus aethiops , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/química , Humanos , Leucócitos/metabolismo , Lipase/química , Macrófagos/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fagocitose , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Proteínas rac de Ligação ao GTP/química , Proteína RAC2 de Ligação ao GTPRESUMO
Phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for defense against microbes. Rac2 is a low molecular weight GTP-binding protein that has been implicated in the regulation of phagocyte NADPH oxidase. Here we report that Cys(157) of Rac2 is a target of S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by thioltransferase in the presence of GSH. S-glutathionylated Rac2 enhanced the binding of GTP, presumably due to structural alterations. These results elucidate the redox regulation of cysteine in Rac2 and a possible mechanism for regulating NADPH oxidase activation.
Assuntos
Cisteína/metabolismo , Glutationa/metabolismo , Guanosina Trifosfato/metabolismo , NADP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Cisteína/química , Cisteína/genética , Ditiotreitol/química , Ativação Enzimática , Glutationa/química , Guanosina Trifosfato/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genética , Proteína RAC2 de Ligação ao GTPRESUMO
Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.
Assuntos
Transformação Celular Neoplásica/patologia , Receptores ErbB/metabolismo , Glioblastoma/enzimologia , Glioblastoma/patologia , Fosfotirosina/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Humanos , Dados de Sequência Molecular , Fosforilação , Prognóstico , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas rac de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Estrogen receptor alpha (ERα) is a ligand-dependent nuclear receptor that is important in breast cancer genesis, behavior and response to hormone-based therapies. A T7 phage display screen against full-length human ERα, coupled with genome-wide exon arrays, was used to identify RAC3 as a putative ERα co-regulator. RAC3 is a Rho family small GTPase that is associated with cytoskeletal rearrangement. We demonstrate a novel role for nuclear RAC3 as an ERα transcriptional activator, with prognostic implications for metastatic disease. Through in vitro and cell-based studies, RAC3 was shown to exist in a GTP-bound state and act as a ligand specific ERα co-activator of E2-induced transcription. Overexpression of RAC3 induced pro-growth and pro-migratory genes that resulted in increased migration of ERα-positive breast cancer cells. Chemical inhibition and genetic knockdown of RAC3 antagonized E2-induced cell proliferation, cell migration and ERα mediated gene expression, indicating that RAC3 is necessary for full ERα transcriptional activity. In agreement with the molecular and cellular data, RAC3 overexpression in ERα-positive breast cancers correlated with a significant decrease in recurrence free survival and a significant increase in the odds ratio of metastasis. In conclusion, RAC3 is a novel ERα co-activator that promotes cell migration and has prognostic value for ERα-positive breast cancer metastasis. RAC3 may also be a useful therapeutic target for ERα-positive breast cancers.
Assuntos
Movimento Celular , Receptor alfa de Estrogênio/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Prognóstico , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/deficiência , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Polarized cell expansion plays an important role in plant morphogenesis. Tip growth is a dramatic form of this process, which is widely used as a model to study its regulation by RAC/ROP GTPase signalling. During the dominant haploid phase of its life cycle, the moss Physcomitrella patens contains different types of cells that expand by tip growth. Physcomitrella is a highly attractive experimental system because its genome has been sequenced, and transgene integration by homologous recombination occurs in this plant at frequencies allowing effective gene targeting. Furthermore, together with the vascular spikemoss Selaginella moellendorffii, whose genome has also been sequenced, the non-vascular moss Physcomitrella provides an evolutionary link between green algae and angiosperms. BLAST searches established that the Physcomitrella and Selaginella genomes encode not only putative RAC/ROP GTPases, but also homologues of all known regulators of polarized RAC/ROP signalling, as well as of key effectors acting in signalling cascades downstream of RAC/ROP activity. Nucleotide sequence relationships within seven different families of Physcomitrella, Selaginella, Arabidopsis thaliana and Nicotiana tabacum (tobacco) genes with distinct functions in RAC/ROP signalling were characterized based on extensive maximum likelihood and Neighbor-Joining analyses. The results of these analyses are interpreted in the light of current knowledge concerning expression patterns and molecular functions of RAC/ROP signalling proteins in angiosperms. A key aim of this study is to facilitate the use of Physcomitrella as a model to investigate the molecular control of tip growth in plants.
Assuntos
Bryopsida/crescimento & desenvolvimento , Meristema/crescimento & desenvolvimento , Modelos Biológicos , Proteínas de Plantas/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Bryopsida/citologia , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Meristema/citologia , Dados de Sequência Molecular , Família Multigênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Análise de Sequência de DNA , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180.
Assuntos
Modelos Moleculares , Proteínas rac de Ligação ao GTP/química , Animais , Sítios de Ligação , Proteínas do Citoesqueleto , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Família Multigênica/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In addition to their pleiotropic functions under physiological conditions, transcription factors STAT3 and STAT5 also have oncogenic activities, but how activated STATs are transported to the nucleus has not been fully understood. Here we show that an MgcRacGAP mutant lacking its nuclear localizing signal (NLS) blocks nuclear translocation of p-STATs both in vitro and in vivo. Unlike wild-type MgcRacGAP, this mutant did not promote complex formation of phosphorylated STATs (p-STATs) with importin alpha in the presence of GTP-bound Rac1, suggesting that MgcRacGAP functions as an NLS-containing nuclear chaperone. We also demonstrate that mutants of STATs lacking the MgcRacGAP binding site (the strand betab) are hardly tyrosine phosphorylated after cytokine stimulation. Intriguingly, mutants harboring small deletions in the C'-adjacent region (betab-betac loop region) of the strand betab became constitutively active with the enhanced binding to MgcRacGAP. The molecular basis of this phenomenon will be discussed, based on the computer-assisted tertiary structure models of STAT3. Thus, MgcRacGAP functions as both a critical mediator of STAT's tyrosine phosphorylation and an NLS-containing nuclear chaperone of p-STATs.
Assuntos
Núcleo Celular/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Chaperonas Moleculares/metabolismo , Sinais de Localização Nuclear/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Galinhas , Guanosina Trifosfato/metabolismo , Humanos , Chaperonas Moleculares/química , Proteínas Mutantes/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Fatores de Transcrição STAT/química , Saccharomyces cerevisiae , Deleção de Sequência , Tetraciclina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , alfa Carioferinas/metabolismo , Proteínas rac de Ligação ao GTP/químicaRESUMO
The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine 5'-monophosphate (AMP). The resulting AMPylation prevented the interaction of Rho GTPases with downstream effectors, thereby inhibiting actin assembly in the infected cell. Eukaryotic proteins were also directly modified with AMP, potentially expanding the repertoire of posttranslational modifications for molecular signaling.
Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Vibrio parahaemolyticus/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Forma Celular , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Treonina/química , Treonina/metabolismo , Vibrio parahaemolyticus/patogenicidade , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/químicaRESUMO
DOCK4, a member of DOCK180 family proteins, was originally identified as a product of a gene deleted during tumor progression. Although its tumor suppression properties have been reported, the regulation mechanism of this protein has not been fully elucidated. DOCK4 shares two conserved domains called as DHR-1 and DHR-2 domain as other members including DOCK180. Although DHR-1 in DOCK180 is reported to bind to PIP(3), whether that of DOCK4 exhibits similar function has yet not been examined. In a search for novel PIP(3) binding proteins by the PIP(3) analog beads binding assay, we found that DOCK4 and its novel splicing variant, whose exon1 and exon52 are different from the known one, bind to PIP(3). Binding assay using deletion mutants of DOCK4 revealed that the binding region falls into the DHR-1 domain. These results raise the possibility that DOCK4 may be regulated by PIP(3) to exert its function.
Assuntos
Processamento Alternativo , Proteínas Ativadoras de GTPase/química , Regulação da Expressão Gênica , Mutação , Proteínas rac de Ligação ao GTP/química , Proteínas de Transporte , Linhagem Celular , Clonagem Molecular , Éxons , Humanos , Microscopia Confocal , Modelos Biológicos , Ligação Proteica , Isoformas de Proteínas , TransfecçãoRESUMO
Tumor necrosis factor (TNF) is an important cytokine in immunity and inflammation and induces many cellular responses, including apoptosis and necrosis. TNF signaling enables the generation of superoxide in phagocytic and vascular cells through the activation of the NADPH oxidase Nox2/gp91. Here we show that TNF also activates the Nox1 NADPH oxidase in mouse fibroblasts when cells undergo necrosis. TNF treatment induces the formation of a signaling complex containing TRADD, RIP1, Nox1, and the small GTPase Rac1. TNF-treated RIP1-deficient fibroblasts fail to form such a complex, indicating that RIP1 is essential for Nox1 recruitment. Moreover, the prevention of TNF-induced superoxide generation with dominant-negative mutants of TRADD or Rac1, as well as knockdown of Nox1 using siRNA, inhibits necrosis. Thus our study suggests that activation of Nox1 through forming a complex with TNF signaling components plays a key role in TNF-induced necrotic cell death.
Assuntos
Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , NADH NADPH Oxirredutases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antioxidantes/farmacologia , Hidroxianisol Butilado/farmacologia , Linhagem Celular , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Complexos Multiproteicos , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Necrose , Neuropeptídeos/química , Neuropeptídeos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Superóxidos/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Necrosis is a caspase-independent cell death process involving the generation of reactive oxygen species (ROS). In a recent issue of Molecular Cell, Kim et al. (2007) reported on a novel TNF receptor 1 necrotic signaling complex inducing TRADD- and RIP1-dependent recruitment and activation of the ROS-generating Nox1 NADPH oxidase complex.
Assuntos
NADPH Oxidases/metabolismo , Necrose/metabolismo , Transdução de Sinais , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antioxidantes/farmacologia , Hidroxianisol Butilado/farmacologia , Linhagem Celular , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
IQGAP1 is a scaffolding protein that binds to a diverse array of signaling and structural molecules that are often associated with cell polarization and adhesion. Through interaction with its target proteins, IQGAP1 participates in multiple cellular functions, including Ca2+-calmodulin signaling, definition of cytoskeletal architecture, regulation of Cdc42 and Rac1 dependent cytoskeletal changes, and control of E-cadherin mediated intercellular adhesion. These analysis have been largely restricted to cells of epithelial and fibroblast origin. The present studies were initiated to examine the role of IQGAP1 in cellular interactions involving the lymphoid cells. A mass spectrometric based analysis of IQGAP1 containing complexes isolated from the human NK-like cell line, YTS, identified several known and new potential IQGAP1 interaction partners including receptor of activated C kinase 1 (RACK1) and the small GTPase, Rac2. Immunofluorescence analysis of YTS cells indicated that a minor component of IQGAP1 was localized at the cell membrane with the remainder diffusely distributed through out the cytoplasm. However, at sites of cellular contact, there was a marked accumulation of IQGAP1. Staining for RACK1 and Rac2 revealed that both of these proteins accumulated these contact sites. Antibody-based studies suggested that a subset of RACK1 was associated in an IQGAP1-containing complex, which prevented recognition of RACK1 by monoclonal antibody. These results suggest that RACK1, Rac2, and IQGAP1 are components of complexes involved in NK cell homotypic adhesion.