RESUMO
Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, we find GLs 1 and 2 act as "decoys" that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Transdução de Sinais , Fuso Acromático/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Ciclo Celular , Polaridade Celular , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gßγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.
Assuntos
Artrite Reumatoide/genética , Quimiotaxia de Leucócito , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neutrófilos/metabolismo , Artrite Reumatoide/imunologia , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Interleucina-8/metabolismo , Leucopoese , Leucotrieno B4/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Polimorfismo de Nucleotídeo Único , Tretinoína/metabolismoRESUMO
Dental pulp stem cells (DPSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulating differentiation of DPSCs may be a useful tool for regenerative medicine and cell-based therapy in oral diseases. Multisignaling pathways are involved in osteogenic differentiation of DPSCs. Recent studies show that cAMP/PKA/CREB signaling could stimulate the expression of genes such as bone morphogenic proteins 2 (BMP2), inhibitor of DNA binding 2 (ID2), bone sialoprotein, osteocalcin, and type XXIV collagen, which have been implicated in osteogenesis and bone formation. Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, plays an important role in regulating the phosphorylation of cyclic AMP response element-binding protein (p-CREB). However, the involvement of AGS3 in osteogenic differentiation of DPSCs has not been explored. Our data indicated that increased expression of AGS3 would inhibit osteogenic differentiation of DPSCs exposed to inflammatory cytokine tumor necrosis factor α (TNF-α) via cAMP/PKA/CREB signaling. The negative role of AGS3 in osteogenic differentiation was further confirmed by knocking down and over expression of AGS3. Our findings may provide clinical implications for osteoporosis.
Assuntos
Polpa Dentária/citologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Células-Tronco Multipotentes/citologia , Osteogênese/fisiologia , Fator de Necrose Tumoral alfa , Adulto , Idoso , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Pessoa de Meia-Idade , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/fisiologia , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Polaridade Celular , Saccharomyces cerevisiae/citologiaRESUMO
Proteins containing the G protein regulator (GPR) domain bind the major neural G protein Gα(o) in vitro. However, the biological functions of GPR proteins in neurons remain undefined, and based on the in vitro activities of GPR proteins it is unclear whether these proteins activate or inhibit G protein signaling in vivo. We found that the conserved GPR domain protein AGS-3 activates Gα(o) signaling in vivo to allow Caenorhabditis elegans to alter several behaviors after food deprivation, apparently so that the animals can more effectively seek food. AGS-3 undergoes a progressive change in its biochemical fractionation upon food deprivation, suggesting that effects of food deprivation are mediated by modifying this protein. We analyzed one C. elegans food-regulated behavior in depth; AGS-3 activates Gα(o) in the ASH chemosensory neurons to allow food-deprived animals to delay response to the aversive stimulus octanol. Genetic epistasis experiments show the following: (1) AGS-3 and the guanine nucleotide exchange factor RIC-8 act in ASH in a mutually dependent fashion to activate Gα(o); (2) this activation requires interaction of the GPR domains of AGS-3 with Gα(o); and (3) Gα(o)-GTP is ultimately the signaling molecule that acts in ASH to delay octanol response. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gα(o) signaling in vivo.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Privação de Alimentos/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas Nucleares/fisiologia , Ligação Proteica/genética , Transdução de Sinais/genéticaRESUMO
Despite over two decades of research, the mechanism of Rab targeting to specific intracellular membranes is still not completely understood. Present evidence suggests that the original hypothesis that the message for targeting resides solely in the hypervariable C-terminus is incorrect, and a second mechanism involving a GDF [GDI (guanine-nucleotide-dissociation inhibitor) displacement factor] to disrupt stable Rab-GDI complexes has only been shown to apply in one case, despite the need for targeting over 60 human Rab proteins. Evidence for the involvement of Rab-effector interactions has only been presented for a few cases or in a very specific context. There is mounting evidence that GEFs (guanine-nucleotide-exchange factors) are essential for membrane targeting, although contributions from additional factors are likely to be of importance, at least in specific cases.
Assuntos
Membranas Intracelulares/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Modelos Biológicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas rab de Ligação ao GTP/químicaRESUMO
Rab GTPases are master regulators of membrane traffic. By binding to distinct sets of effector proteins, Rabs catalyse the formation of function-specifying membrane microdomains. They are delivered to membranes by a protein named GDI (guanine-nucleotide-dissociation inhibitor) and are stabilized there after nucleotide exchange by effector binding. In the present mini-review, I discuss what we know about how Rab GTPases are delivered to the correct membrane-bound compartments and how Rab GTPase cascades order Rabs within the secretory and endocytic pathways. Finally, I describe how Rab cascades may establish the distinct compartments of the Golgi complex to permit ordered processing, sorting and secretion of secretory cargoes.
Assuntos
Complexo de Golgi/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Complexo de Golgi/ultraestrutura , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Ligação Proteica , Transporte ProteicoRESUMO
Rab proteins constitute the largest family within the Ras superfamily of small GTPases (>60 in mammals) and are essential regulators of transport between intracellular organelles. Key to this activity is their targeting to specific compartments within the cell. However, although great strides have been made over the last 25 years in assigning functions to individual Rabs and identifying their downstream effectors, the mechanism(s) regulating their targeting to specific subcellular membranes remains less well understood. In the present paper, we review the evidence supporting the proposed mechanisms of Rab targeting and highlight insights into this process provided by studies of Rab27a.
Assuntos
Melanossomas/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Membranas Intracelulares/enzimologia , Melanócitos/enzimologia , Transporte Proteico , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab27 de Ligação ao GTPRESUMO
Asymmetric division of Drosophila neuroblasts (NBs) and the Caenorhabditis elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. We show here that the Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pl cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13Fsingle and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Gai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry.
Assuntos
Divisão Celular/fisiologia , Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Modelos Biológicos , Neurônios Aferentes/fisiologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura , Fator de Crescimento Transformador beta/fisiologiaRESUMO
The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4-7 and 6-11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor alpha (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Príons/genética , Príons/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Expressão Gênica/fisiologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Humanos , Camundongos , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Príons/antagonistas & inibidores , Príons/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteômica , RNA Interferente Pequeno/farmacologia , Via Secretória/efeitos dos fármacos , Via Secretória/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal ß-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fusão de Membrana , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Vacúolos/fisiologia , Citoplasma/metabolismo , Citosol/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.
Assuntos
Neoplasias do Colo/secundário , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neoplasias da Próstata/secundário , Idoso , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Corantes Fluorescentes , Géis , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Hidrólise , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Análise em Microsséries , Pessoa de Meia-Idade , Tripsina/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Insulina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Glucose/farmacologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Espectrometria de Massas/métodos , Camundongos , Mutação de Sentido Incorreto , Fosforilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells.
Assuntos
Gangliosídeo G(M3)/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Melanoma Experimental/patologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Camundongos , Antígenos de Histocompatibilidade Menor , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/fisiologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Ovarian cancer is a gynecological malignancy with the highest mortality. Chemoresistance is an important subject for the treatment of ovarian cancer, because obtaining significant drug resistance to the first line chemotherapy, paclitaxel, causes major therapeutic obstacles. It is essential to improve the survival rate of ovarian cancer patients by mining the biomarkers indicating the drug resistance and prognosis, and by further understanding underlying mechanisms of drug resistance. In the present study, we established paclitaxel-resistant subline (SKpac) from human epithelial ovarian cancer cell line, SKOV3, and performed comparative analysis of whole proteomes between paclitaxel-resistant SKpac sublines and paclitaxel-sensitive parental SKOV3 cells to identify differentially expressed proteins and useful biomarkers indicating chemoresistance. Proteins related to chemoresistant process were identified by two-dimensional gel electrophoresis (2DE) with mass spectrometry (MALDI-TOF and LC-MS/MS). Eighteen spots were differentially expressed and were identified in SKpac chemoresistant cells compared to SKOV3. The expressions of ALDH 1A1, annexin A1, hnRNP A2, and GDI 2 proteins were validated by Western blot, which was consistent with proteomic analysis. Among the selected proteins, downregulation of hnRNP A2 and GDI 2 was found to be the most significant finding in SKpac cells and chemoresistant ovarian cancer tissues. Our results suggest that hnRNP A2 and GDI 2 may represent potential biomarkers of the paclitaxel-resistant ovarian cancers for tailored cancer therapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Dissociação do Nucleotídeo Guanina/análise , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/análise , Neoplasias Ovarianas/fisiopatologia , Paclitaxel/farmacologia , Linhagem Celular Tumoral , Feminino , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Proteômica/métodosRESUMO
Despite the recent advances in the diagnosis of bladder cancer, recurrence after surgical intervention for muscle invasive disease is still problematic as nearly half of the patients harbor occult distant metastases and this, in turn, is associated with poor 5-year survival rate. We have recently identified Rho family GDP dissociation inhibitor 2 (RhoGDI2) protein as functional metastasis suppressor and a prognostic marker in patients after cystectomy. In identifying the mechanisms underlying metastasis suppression by RhoGDI2, we found this protein to be associated with the c-Src kinase in human tumors, where the expression of both is diminished as a function of stage. Interestingly, c-Src bound to and phosphorylated RhoGDI2 resulting in enhanced metastasis suppressive potency. In this review, we will discuss the established roles of c-Src and RhoGDI2 in bladder cancer and speculate on their therapeutic relevance.
Assuntos
Carcinoma de Células de Transição/secundário , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Neoplasias da Bexiga Urinária/patologia , Sequência de Aminoácidos , Proteína Tirosina Quinase CSK , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/cirurgia , Sequência Conservada , Cistectomia , Receptores ErbB/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Fosforilação , Prognóstico , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/cirurgia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico , Quinases da Família src/fisiologiaRESUMO
The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPase function. GDIs control the access of Rho GTPases to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. We discuss here our current understanding of how Rho GTPase-GDI complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. We propose that phosphorylation mediated by diverse kinases might provide a means of controlling and coordinating Rho GTPase activation.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Ativadores de Enzimas/farmacologia , Fatores de Troca do Nucleotídeo Guanina/farmacologia , Humanos , Lipídeos/farmacologia , Fosforilação , Proteínas/fisiologia , Proteínas Supressoras de Tumor , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/fisiologia , Inibidor gama de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Insulin stimulates GLUT4 (glucose transporter 4) translocation in adipocytes and muscles. An emerging picture is that Rab10 could bridge the gap between the insulin signalling cascade and GLUT4 translocation in adipocytes. In the present study, two potential effectors of Rab10, GDI (guanine-nucleotide-dissociation inhibitor)-1 and GDI-2, are characterized in respect to their roles in insulin-stimulated GLUT4 translocation. It is shown that both GDI-1 and GDI-2 exhibit similar distribution to GLUT4 and Rab10 at the TGN (trans-Golgi network) and periphery structures. Meanwhile, GDI-1 clearly interacts with Rab10 with higher affinity, as shown by both immunoprecipitation and in vivo FRET (fluorescence resonance energy transfer). In addition, the participation of GDIs in GLUT4 translocation is illustrated when overexpression of either GDI inhibits insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Taken together, we propose that GDI-1 is preferentially involved in insulin-stimulated GLUT4 translocation through facilitating Rab10 recycling.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Insulina/farmacologia , Proteínas rab de Ligação ao GTP/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Linhagem Celular , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Transporte ProteicoRESUMO
BACKGROUND/AIMS: The acquisition of resistance to 5-FU is one of the most prominent obstacles to successful chemotherapy, and the mechanisms underlying the resistance are not fully understood. The aim of this study is to identify novel mediators of 5-FU resistance in colon cancer cells. METHODOLOGY: LoVo colon cancer cells were induced to 5-FU resistance in vitro. The global protein profiles between LoVo and its 5-FU resistant derivative cell line LoVo/5-FU were analyzed by two dimensional gel electrophoresis-based comparative proteomics. The identified proteins expression was confirmed by Western blot analysis. The cytotoxicity of 5-FU was measured in LoVo/5-FU after knockdown of RhoGDI2 (one of the identified protien). RESULTS: Three differentially expressed proteins were identified. RhoGDI2 and CapG were upregulated, whereas proapoptotic protein Maspin was down-regulated in LoVo/5-FU and validated by Western blotting. Furthermore, knockdown of RhoGDI2 expression by transfection with the RhoGDI2-specific siRNA significantly reduced the resistance to 5-FU in LoVo/5-FU (p < 0.05). CONCLUSIONS: These novel data suggest that these differentially expressed proteins may contribute to the development of 5-FU resistance in colon cancer cells.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Fluoruracila/farmacologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Inibidores de Dissociação do Nucleotídeo Guanina/antagonistas & inibidores , Humanos , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.