RESUMO
A common benchmark in the brain tissue mechanics literature is that the properties of acute brain slices should be measured within 8 h of the experimental animal being sacrificed. The core assumption is that-since there is no substantial protein degradation during this time-there will be no change to elastic modulus. This assumption overlooks the possibility of other effects (such as osmotic swelling) that may influence the mechanical properties of the tissue. To achieve consistent and accurate analysis of brain mechanics, it is important to account for or mitigate these effects. Using atomic force microscopy (AFM), tissue hydration and volume measurements, we find that acute brain slices in oxygenated artificial cerebrospinal fluid (aCSF) with a standard osmolarity of 300 mOsm/l experience rapid swelling, softening, and increases in hydration within the first 2 hours after slicing. Reductions in elastic modulus can be partly mitigated by addition of chondroitinase ABC enzyme (CHABC). Increasing aCSF osmolarity to 400 mOsm/l does not prevent softening but may hasten equilibration of samples to a point where measurements of relative elastic modulus are consistent across experiments.
Assuntos
Encéfalo , Módulo de Elasticidade , Encéfalo/metabolismo , Microscopia de Força Atômica , Água/metabolismo , Fatores de Tempo , Feminino , Animais , Camundongos , Concentração OsmolarRESUMO
The concept of slippery lubricant-infused surfaces has shown promising potential in antifouling for controlling detrimental biofilm growth. In this study, nontoxic silicone oil was either impregnated into porous surface nanostructures, referred to as liquid-infused surfaces (LIS), or diffused into a polydimethylsiloxane (PDMS) matrix, referred to as a swollen PDMS (S-PDMS), making two kinds of slippery surfaces. The slippery lubricant layers have extremely low contact angle hysteresis, and both slippery surfaces showed superior antiwetting performances with droplets bouncing off or rolling transiently after impacting the surfaces. We further demonstrated that water droplets can remove dust from the slippery surfaces, thus showing a "cleaning effect". Moreover, "coffee-ring" effects were inhibited on these slippery surfaces after droplet evaporation, and deposits could be easily removed. The clinically biofilm-forming species P. aeruginosa (as a model system) was used to further evaluate the antifouling potential of the slippery surfaces. The dried biofilm stains could still be easily removed from the slippery surfaces. Additionally, both slippery surfaces prevented around 90% of bacterial biofilm growth after 6 days compared to the unmodified control PDMS surfaces. This investigation also extended across another clinical pathogen, S. epidermidis, and showed similar results. The antiwetting and antifouling analysis in this study will facilitate the development of more efficient slippery platforms for controlling biofouling.
RESUMO
Phospholipase D (PLD) is distributed widely in nature, being present in various isoforms in bacteria, protozoa, fungi, plants, and animals. It catalyzes the hydrolysis of phospholipids, primarily phosphatidylcholine (PC), into phosphatidic acid (PA) and the head group, choline. It also catalyzes a transphosphatidylation reaction in which water is replaced by a primary alcohol to yield a phosphatidyl alcohol. This reaction is exclusive to PLD and is employed as a specific assay for the enzyme in in vivo systems. When the purified enzyme is assayed in vitro, the release of choline from PC can be utilized. This chapter describes production of a recombinant mammalian isozyme of PLD (PLD1) in baculovirus-infected insect cells and its purification. It also provides details of the assay procedure in the presence and absence of regulatory proteins in vitro. The assay of the enzyme in cells in vivo is also documented using labeling of endogenous PC by incubating the cells with (3)H-labeled fatty acid. Details of the assay utilizing the transphosphatidylation reaction are presented. In this, 1-butanol is employed as the primary alcohol and [(3)H]phosphatidylbutanol is isolated by thin-layer chromatography of lipid extracts from the cells. A variation of this assay is described using deuterated 1-butanol (1-butanol-d(10)) and detection of the synthesized deuterated phosphatidylbutanol species by mass spectrometry. Convenient alternative assays for PLD and diacylglycerol (DAG) lipase activity based on fluorescence are also described. Many of the materials for these assays are available commercially, with the exception of the fluorescently labeled DAG substrate, which can be synthesized enzymatically in a simple one-step procedure.
Assuntos
Fosfolipase D/metabolismo , Animais , Cromatografia em Gel/métodos , Ativação Enzimática , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Marcação por Isótopo/métodos , Cinética , Mamíferos , Fosfolipase D/genética , Fosfolipase D/isolamento & purificação , Proteína Quinase C/metabolismo , Radioisótopos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Especificidade por SubstratoRESUMO
NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.
Assuntos
Apoptose/genética , Regulação para Baixo , Retículo Endoplasmático/metabolismo , NF-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspase 8 , Caspases/análise , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Endorribonucleases , Ativação Enzimática/efeitos dos fármacos , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cinética , Células L , Luciferases/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/genética , Testes de Precipitina , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 2 Associado a Receptor de TNF/genética , Tapsigargina/farmacologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Tunicamicina/farmacologiaRESUMO
In neuronal cells, current evidence suggests that G(13)alpha and RhoA play significant roles in LPA-mediated neurite retraction; however, the contribution of other G-proteins to this process is less well-understood. We provide evidence that LPA activation of G(13), G(q) and G(i) occurs rapidly in neuroblastoma cells, but that stimulation of RhoA is transient whereas the activation of G(q)- and G(i)-mediated pathways is sustained. In addition to G(13)alpha, we demonstrate that G(q)alpha is capable of promoting neurite retraction. G(q)-mediated retraction is RhoA-independent and is likely mediated via a mechanism involving protein kinase C and calcium flux. Additionally, we provide evidence that activation of adenylyl cyclase via G(s) inhibits RhoA-mediated neurite retraction via protein kinase A-mediated inhibition of RhoA action. Taken together, we hypothesize that LPA promotes neurite retraction via RhoA-dependent and -independent pathways involving G(13) and G(q), respectively, and that agonists that activate G(s) inhibit the RhoA-dependent pathway.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/farmacologia , Neuritos/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , Forma Celular , Colforsina/farmacologia , Dimetil Sulfóxido/farmacologia , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Neuritos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Isoformas de Proteínas/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals, including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon phospholipase D to modulate cellular actions, such as cell growth, shape, and secretion. We examined the kinetics of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Calpha, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. PKCalpha activation of PLD1 involves N- and C-terminal PLD domains. Rho GTPases were binding activators, enhancing the catalytic efficiency of a purified PLD1 catalytic domain via effects on Km. Arf-1, a catalytic activator, stimulated PLD1 by enhancing the catalytic constant, kcat. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. Synergy was observed only when PLD1 was simultaneously stimulated by a binding activator and a catalytic activator. Surprisingly, synergistic activation was steeply dependent on phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Together, these findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane.
Assuntos
Fosfatos de Fosfatidilinositol/química , Fosfolipase D/química , Proteína Quinase C/química , Fator 1 de Ribosilação do ADP/metabolismo , Difosfato de Adenosina/química , Sítio Alostérico , Animais , Células COS , Catálise , Membrana Celular/metabolismo , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/química , Vetores Genéticos , Cinética , Modelos Químicos , Fases de Leitura Aberta , Fosfatidilcolinas/química , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Transdução de Sinais , Sacarose/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Previous research showed that protein kinase C alpha (PKC alpha) translocated to the perinuclear region and activated phospholipase D1, but the mechanism involved was not clear. Here, we provide evidence that Phe 663 (the 10th amino acid from C-terminus) of PKC alpha is essential for its translocation. A point mutation (F663D) completely blocked PKC alpha's binding to and activation of phospholipase D1. Further studies showed that deletion of the C-terminal nine amino acids of PKC alpha did not alter its translocation to the perinuclear region but deletion of the C-terminal 10 amino acids and the F663D mutation abolished this translocation. The F663D mutant was found to be resistant to dephosphorylation, which might account for its inability to translocate to the perinuclear region and activate PLD1, since dephosphorylation of PKC alpha is required for its relocation from plasma membrane to the perinuclear region.
Assuntos
Fenilalanina/genética , Fosfolipase D/metabolismo , Mutação Puntual , Proteína Quinase C/metabolismo , Animais , Células COS , Ativação Enzimática , Microscopia de Fluorescência , Proteína Quinase C/genética , Proteína Quinase C-alfa , Transporte ProteicoRESUMO
The phosphorylation sites in phospholipase D2 (PLD2) induced by activation of protein kinase Calpha (PKCalpha) in COS 7 cells were analyzed by mass spectrometry. Ser134, 146, and 243, and Thr72, 99/100, and 252 were identified. These sites were mutated to Ala and the double mutation of Ser243 and Thr252 eliminated the phosphorylation. However, the PLD2 activity, and the binding between PKCalpha and PLD2 were unaffected by the mutations. We conclude that phosphorylation of these residues is not required for PLD2 activation by PKCalpha, and that protein-protein interaction between PLD2 and PKCalpha is sufficient to activate PLD2.
Assuntos
Fosfolipase D/química , Fosfolipase D/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Proteína Quinase C-alfa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the activation of PLD1 by phorbol ester is associated with the binding of PKCalpha to a site in the N-terminus of PLD1. The purpose of the present study was to determine this site more precisely. Immunoprecipitation with a series of four PLD1 deletion mutants confirmed that PKCalpha strongly interacted with the amino acid sequence 1-318 at the N-terminus of PLD1 and weakly with the sequence 841-1036 at the C-terminus. Further immunoprecipitation studies with deletion mutants of the 1-318 and 1-215 PLD1 fragments revealed that there were binding sites in the 1-49 N-terminal sequence and also in the 216-318 sequence containing the PH domain. Studies of N-terminal deletion mutants of full-length PLD1 confirmed the presence of a binding site in the 1-49 sequence and a further site in the 1-318 sequence. Both deletion mutants showed impaired activation by PKCalpha in vivo, but unchanged activation by active V(14)RhoA. These findings identify the 1-49 sequence is a major binding/activation site for PKCalpha on PLD1, but also indicate involvement of the PH domain.
Assuntos
Fosfolipase D/metabolismo , Proteína Quinase C-alfa/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Ativação Enzimática , Dados de Sequência Molecular , Mutação , Fosfolipase D/genética , Mapeamento de Interação de Proteínas , Proteína Quinase C-alfa/genética , Estrutura Terciária de Proteína , Acetato de Tetradecanoilforbol/farmacologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Phospholipase D (PLD) and ADP-ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co-localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP-tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans-Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co-expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans-Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative ARF6 selectively inhibited PLD2. In contrast, dominant active ARF1 or Rac1 stimulated both PLD isozymes but the ARF1 effect on PLD2 was very small. Cytochalasin D did not affect the activation of PLD by phorbol ester. The localizations of PLD and ARF6 were also analyzed by fractionation after methyl-beta-cyclodextrin extraction to deplete cholesterol. The results showed that all PLD isoforms and ARF6 mutants existed in the membrane fraction, but only wild type ARF6 was dependent on the presence of cholesterol. These experiments showed that wild type ARF6 had a similar location with PLD isoforms on cell staining, but it did not colocalize with PLD isoforms in fractionation experiments. It is proposed that activated ARF6 translocates to the cholesterol independent microdomain and then activates PLD2 there. It is further concluded that PLD2 is selectively activated by ARF6 in vivo and that disruption of the actin cytoskeleton does not affect this activation.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Fosfolipase D/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Citoesqueleto/genética , Citoesqueleto/metabolismo , Células HeLa , Humanos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Ratos , TransfecçãoRESUMO
The relationship between tyrosine phosphorylation and activation of phospholipase D1 (PLD1) by v-Src was examined. Co-expression of v-Src and PLD1 in COS-7 cells resulted in increased activity and marked tyrosine phosphorylation of PLD1. PLD activity was increased in membranes or immunoprecipitates prepared from these cells. Dephosphorylation of the immunoprecipitated enzyme by tyrosine phosphatase or phosphorylation by c-Src produced no changes in its activity. Tyrosine phosphorylation induced by v-Src caused a shift of the enzyme from the Triton-soluble to the Triton-insoluble fraction. v-Src and PLD1 could be co-immunoprecipitated from cells co-expressing these and were co-localized in the perinuclear region as assessed by immunofluorescence. Mutation of the palmitoylation sites of PLD1 significantly reduced tyrosine phosphorylation by v-Src. It is concluded that tyrosine phosphorylation of PLD1 by v-Src does not per se alter its activity. It is proposed that activation of PLD1 by v-Src in vivo may involve association/colocalization of the two proteins.
Assuntos
Proteína Oncogênica pp60(v-src)/metabolismo , Fosfolipase D/metabolismo , Tirosina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática , Imunoprecipitação , Ácidos Palmíticos/metabolismo , Fosfolipase D/análise , Fosfolipase D/química , FosforilaçãoRESUMO
The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-RasV12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-RasV12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.
Assuntos
Transformação Celular Neoplásica , Neoplasias Colorretais/patologia , Genes ras , Fosfolipase D/metabolismo , Animais , Linhagem Celular , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Humanos , Mucosa Intestinal/enzimologia , Camundongos , Camundongos Nus , RatosRESUMO
1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.
Assuntos
1-Butanol/farmacologia , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Substituição de Aminoácidos , Animais , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/farmacologia , Mutagênese Sítio-Dirigida , Fosfolipase D/genética , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C-alfa , Ratos , Estaurosporina/farmacologia , Fatores de TempoRESUMO
Arfaptin 1 and 2 were identified as targets for GTP bound ADP-ribosylation factors (Arfs). Arfaptin 1 had no significant effects on guanine nucleotide binding to Arfs, nor enzymatic activities of guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) acting on Arfs. However, arfaptin 1 inhibited Arf activation of cholera toxin and phospholipase D (PLD) in a dose-dependent manner. Only GTP-bound forms of Arf1, 5, and 6 interacted with arfaptin 1 and 2, but GTP-Arf1 showed the strongest binding to the arfaptins. In contrast to the binding of Arfs to arfaptins, GDP-Rac1 or dominant negative Rac1-N17N bound to arfaptin 2, whereas GTP-Rac1 or dominant active Rac1-Q61L did not bind to arfaptin 2. Neither GTP-Rac1 nor GDP-Rac1 bound to arfaptin 1. Based on our observation, we propose that arfaptin 2 is a target for GDP-Rac1 and for GTP-Arf1, and is involved in interactions between the Rac1 and Arfs signaling pathways. This chapter describes methods for investigating the interactions of arfaptins 1 and 2 with GTP- or GDP-liganded Arfs and Rac1.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Glutationa Transferase/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Humanos , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tionucleotídeos/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
In this study, we have explored the roles of ADP-ribosylation factors (ARFs), phospholipase D (PLD) isozymes, and arfaptins in phorbol ester (PMA)-induced membrane ruffling in HeLa cells. PMA stimulation induced ruffling and translocated cortactin to the plasma membrane. The cortactin translocation was inhibited by dominant negative (DN)-ARF6, DN-ARF1, and DN-Rac1, but not by DN-RhoA and DN-Cdc42. The inability of DN-forms of ARF6, ARF1, and Rac1 to affect PLD activity in response to PMA indicated that this enzyme was not activated via these small G proteins and that its activation was not essential for the induction of ruffling. Endogenous-ARF1, -ARF6, and -Rac1 existed in the ruffling region along with cortactin after PMA stimulation. DN-ARF1 had no effect on the ruffling induced by DA-ARF6 or DA-Rac1, and DN-ARF6 had no effect on that induced by DA-ARF1 or DA-Rac1. On the other hand DN-Rac1 suppressed the effect of DA-ARF6 but not that of DA-ARF1. These results suggest that PMA causes membrane ruffling via an ARF6-Rac1 pathway and also an ARF1 pathway operating in parallel. Overexpression of PLD1 and PLD2 inhibited PMA-induced cortactin translocation and actin-cortactin complex formation, supporting the view that these enzymes are not required for ruffling, but actually suppress it. We conclude that PMA-induced membrane ruffling is caused via ARF6-Rac1 and ARF1 pathways operating in parallel and that PLD may be inhibitory.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Fosfolipase D/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/farmacologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Membrana Celular/metabolismo , Cortactina , Genes Dominantes , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Distribuição Tecidual , Transfecção , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/farmacologiaRESUMO
Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.
Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , MAP Quinase Quinase 1/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Genes Dominantes , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Plasmídeos/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Frações Subcelulares , Serina-Treonina Quinases TOR , Sais de Tetrazólio/farmacologia , Tapsigargina/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Transcrição Gênica , Tunicamicina/farmacologia , Regulação para Cima , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
The inhibition of phorbol ester activation of phospholipase D1 (PLD1) by protein kinase C (PKC) inhibitors has been considered proof of phosphorylation-dependent activation of PLD1 by PKCalpha. We studied the effect of the PKC inhibitors Ro-31-8220 and bisindolylmaleimide I on PLD1 activation and found that they inhibited the activation by interfering with PKCalpha binding to PLD1. Further studies showed that only unphosphorylated PKCalpha could bind to and activate PLD1 and that both inhibitors induced phosphorylation of PKCalpha. The phosphorylation status of either PLD1 or PKCalpha per se did not affect PLD1 activation in vitro. Immunofluorescence studies showed that PLD1 remained in the perinuclear region after phorbol ester treatment, whereas PKCalpha translocated from cytosol to both plasma membrane and perinuclear regions. Both Ro-31-8220 and bisindolylmaleimide I blocked the translocation of PKCalpha to the perinuclear region but not to the plasma membrane. Studies with okadaic acid suggested that phosphorylation regulated the relocation of PKCalpha from the plasma membrane to the perinuclear region. It is proposed that localization and interaction of PKCalpha with PLD1 in the perinuclear region is required for PLD1 activation and that PKC inhibitors inhibit this through phosphorylation of PKCalpha, which blocks its translocation.
Assuntos
Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-alfa , Transporte Proteico , RatosRESUMO
It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, Gö 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfolipase D/biossíntese , Proteína Quinase C/metabolismo , Animais , Células COS , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Indóis/farmacologia , Maleimidas/farmacologia , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteína Quinase C-alfa , Serina/química , Frações Subcelulares , Acetato de Tetradecanoilforbol/farmacologia , Treonina/química , Fatores de Tempo , TransfecçãoRESUMO
Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.