RESUMO
The adenoviral early region 4 open reading frame 4 (E4orf4) death factor induces p53-independent apoptosis in many cell types and appears to kill selectively transformed cells. Here we show that expression of E4orf4 in transformed epithelial cells results in early caspase-independent membrane blebbing, associated with changes in the organization of focal adhesions and actin cytoskeleton. Evidence that E4orf4 can associate with and modulate Src family kinase activity, inhibiting Src-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin while increasing phosphorylation of cortactin and some other cellular proteins, is presented. Furthermore, E4orf4 dramatically inhibited the ability of FAK and c-src to cooperate in induction of tyrosine phosphorylation of cellular substrates, suggesting that E4orf4 can interfere with the formation of a signaling complex at focal adhesion sites. Consistent with a functional role for E4orf4-Src interaction, overexpression of activated c-src dramatically potentiated E4orf4-induced membrane blebbing and apoptosis, whereas kinase dead c-src constructs inhibited E4orf4 effects on cell morphology and death. Moreover treatment of E4orf4-expressing cells with PP2, a selective Src kinase inhibitor, led to inhibition of E4orf4-dependent membrane blebbing and later to a marked decrease in E4orf4-induced nuclear condensation. Taken together, these observations indicate that expression of adenovirus 2 E4orf4 can initiate caspase-independent extranuclear manifestations of apoptosis through a modulation of Src family kinases and that these are involved in signaling E4orf4-dependent apoptosis. This study also suggests that Src family kinases are likely to play a role in the cytoplasmic execution of apoptotic programs.
Assuntos
Adenovírus Humanos/genética , Apoptose/fisiologia , Membrana Celular/ultraestrutura , Proteínas Virais/metabolismo , Quinases da Família src/metabolismo , Caspases/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , Movimento Celular , Fibronectinas , Humanos , Rim , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207-6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J. G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529-535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-XL. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.
Assuntos
Proteínas E4 de Adenovirus/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose , Caspases , Proteína Supressora de Tumor p53/fisiologia , Proteínas E4 de Adenovirus/genética , Animais , Apoptose/efeitos dos fármacos , Células CHO , Caspase 3 , Linhagem Celular , Membrana Celular/química , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Cricetinae , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA , Doxiciclina/farmacologia , Expressão Gênica , Genes Reporter , Genes bcl-2 , Membranas Intracelulares/fisiologia , Potenciais da Membrana , Camundongos , Mitocôndrias/fisiologia , Fosfatidilserinas/análise , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Humanos , Células KB , Masculino , Camundongos , Dados de Sequência Molecular , Miocárdio/citologia , Miocárdio/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína X Associada a bcl-2RESUMO
Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Arabidopsis , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Proteínas de Transporte/genética , Inibidores de Caspase , Caspases/metabolismo , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas Correpressoras , Citoplasma/metabolismo , Dimerização , Proteína de Domínio de Morte Associada a Fas , Proteínas de Choque Térmico/genética , Humanos , Cinética , Microscopia de Fluorescência , Chaperonas Moleculares , Mutação , Fosforilação , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Transporte Proteico , Transdução de Sinais/efeitos dos fármacos , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Citoesqueleto de Actina/ultraestrutura , Animais , Compartimento Celular , Células Cultivadas , Cricetinae , Cricetulus , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
We previously described in the CCL39 hamster fibroblast cell line the inhibition of DNA synthesis reinitiation by agents that elevate cyclic AMP. Here, we show that 8Br-cAMP strongly blocks both the growth factor-induced increase in cyclin D1 protein expression and decrease in p27(KIP1) protein levels, leaving untouched the levels of cyclin D3, cdk2 and cdk4. To assess the role of cyclin D1 in the cAMP-mediated inhibition of DNA synthesis, we overexpressed the cyclin D1 gene in CCL39 and analysed the cAMP response in stable transfectants. We showed that the kinase activities associated to G1 cyclin-cdk complexes are significantly more resistant to cAMP in cyclin D1 transfectants than in their normal counterparts, although the serum-induced p27(KIP1) disparition is still cAMP sensitive in cyclin D1 overexpressors. Interestingly, the mitogen-induced DNA synthesis reinitiation is also much less inhibited by cAMP in cyclin D1 transfectants than in control cells. These data clearly establish that the cAMP-inducible blockade of the G1 phase of the cell cycle can be partially alleviated by overexpression of cyclin D1 in hamster fibroblasts, thus strongly suggesting that cyclin D1 protein is one of the major targets for cAMP inhibitory action in fibroblasts.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , AMP Cíclico/metabolismo , Ciclinas/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Ciclina D1 , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/fisiologia , Replicação do DNA/efeitos dos fármacos , Fibroblastos , Genes Supressores de Tumor , Glutationa Transferase/metabolismo , Cinética , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Oncogênicas/fisiologia , Proteínas Recombinantes/metabolismo , Fase de Repouso do Ciclo Celular , Proteína do Retinoblastoma/metabolismo , Fase S , TransfecçãoRESUMO
Innovative revenue generation by Canadian hospitals is drawing increasing attention. After a critical examination of the literature, we classified these into six areas: clinical/diagnostic insured services, clinical/diagnostic non-insured services, hotel services, retail services, administrative services and financial activities. We concluded that many Canadian hospitals are engaging in innovative revenue generation activities, the success of such activities has been mixed, there are many factors to consider when selecting revenue generation activities, many aspects of innovative revenue generation involve sophisticated business and risk management skills not traditionally required in hospital management, and implementation of many such activities requires support from the hospital board, hospital staff and medical staff.
Assuntos
Administração Financeira de Hospitais/tendências , Departamentos Hospitalares/economia , Renda , Inovação Organizacional , Administração de Linha de Produção/economia , Canadá , Comércio/tendências , Governo , Reestruturação Hospitalar/economia , Técnicas de Planejamento , RiscoRESUMO
The human adenovirus E4orf4 protein, when expressed alone, induces p53-independent death in a wide range of cancer cells. Earlier studies by our groups suggested that although in some cases cell death can be associated with some hallmarks of apoptosis, it is not always affected by caspase inhibitors. Thus it is unlikely that E4orf4-induced cell death occurs uniquely through apoptosis. In the present studies using H1299 human lung carcinoma cells as a model system we found that death is induced in the absence of activation of any of the caspases tested, accumulation of reactive oxygen species, or release of cytochrome c from mitochondria. E4orf4 caused a substantial change in cell morphology, including vigorous membrane blebbing, multiple nuclei in many cells and increased cell volume. Most of these characteristics are not typical of apoptosis, but they are of necrosis. FACS analysis and western blotting for cell cycle markers showed that E4orf4-expressing cells became arrested in G(2)/M and also accumulated high levels of cyclin E. The presence of significant numbers of tetraploid and polyploid cells and some cells with micronuclei suggested that E4orf4 appears to induce death in these cells through a process resulting from mitotic catastrophe.
Assuntos
Apoptose/fisiologia , Núcleo Celular/metabolismo , Neoplasias Pulmonares/metabolismo , Mitose , Proteínas Virais/fisiologia , Trifosfato de Adenosina/metabolismo , Caspases/metabolismo , Ciclo Celular/fisiologia , Senescência Celular/fisiologia , Citocromos c/metabolismo , Ativação Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We recently reported that overexpression of heat shock protein 27 (HSP27) in rodent fibroblasts increases the stability of stress fibers during hyperthermia and partially prevents actin depolymerization during exposure to cytochalasin D (Lavoie, J.N., Gingras-Breton, G., Tanguay, R. M., and Landry, J. (1993) J. Biol. Chem. 268, 3420-3429). Because HSP27 is a ubiquitous target of phosphorylation upon cell stimulation with a variety of growth factors and agents that affect cellular differentiation, we examined the role of HSP27 phosphorylation in regulating actin filament dynamics. Here we show that HSP27 is enriched at the leading edge of polarized fibroblasts. HSP27 is localized in lamellipodia and membrane ruffles where most actin polymerization occurs. We developed Chinese hamster cell lines that constitutively overexpressed either human HSP27 or a nonphosphorylatable mutant form of the protein. Overexpression of HSP27 caused an increased concentration of filamentous actin (F-actin) at the cell cortex and elevated pinocytotic activity. In contrast, overexpression of the non-phosphorylatable mutant form of HSP27 reduced cortical F-actin concentration and decreased pinocytosis activity relative to control cells. Mitogenic stimulation of fibroblasts resulted in a rapid polymerization of submembranous actin filaments. HSP27 enhanced growth factor-induced F-actin accumulation, whereas mutant HSP27 exerted a dominant negative effect and inhibited this response to growth factors. Thus, HSP27 is a component of a signal transduction pathway that can regulate microfilament dynamics.
Assuntos
Actinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Pinocitose , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Cricetulus , Fatores de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Temperatura Alta , Humanos , Fosforilação , Transdução de Sinais , Trombina/farmacologiaRESUMO
The role of hsp27, one of Drosophila melanogaster's small heat shock proteins, in the process of thermotolerance was investigated. The coding sequence of hsp27 was subcloned downstream of the human hsp27 promoter which has been shown to be constitutively expressed in Chinese hamster O23 cells. Cellular resistance to a thermal stress was measured two days after transfection by a survival assay following a 3.5 h heat treatment at 44 degrees C. Expression of Drosophila hsp27 was shown to confer thermal resistance to O23 cells in a manner which was dependent on the level of expression of this hsp. Immunoblot analysis confirmed that the thermal resistance was related to the expression of Drosophila hsp27 as none of the endogeneous hsps showed an increased level under these conditions.
Assuntos
Drosophila/genética , Fibroblastos/metabolismo , Proteínas de Choque Térmico/biossíntese , Transfecção , Adaptação Biológica , Animais , Linhagem Celular , Cricetinae , Cricetulus , Proteínas de Choque Térmico/genética , Temperatura Alta/efeitos adversosRESUMO
We have previously shown that the persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts (Pagès, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., and Pouysségur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323) and postulated that MAPKs control the activation of G1 cyclin-dependent complexes. We examined the mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression were compared in CCL39 cells, using a co-transfection procedure. We found that inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibited expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increased cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) revealed that in absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade showed an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3) together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression was observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhanced cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular , Ciclinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Células Cultivadas , Cricetinae , Cricetulus , Ciclina D1 , Ciclinas/genética , Ativação Enzimática , Expressão Gênica , Humanos , Interleucina-1/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Oncogênicas/genética , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , RNA Mensageiro/genética , Proteínas Recombinantes , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Trombina/farmacologia , Transcrição Gênica , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We have studied the contribution of the individual kinases of the MAP (mitogen-activated protein) kinase family, including ERK (extracellular-signal regulated kinase), JNK/SAPK (c-JUN NH2-terminal kinase/stress-activated protein kinase) and p38, to activation of the HSP27 (heat shock protein 27) kinase MAPKAP kinase-2/3 and to HSP27 phosphorylation in Chinese hamster CCL39 cells stimulated by either growth factors, cytokines or stressing agents. In vitro assays using fractionated cell extracts or immunoprecipitates indicated that only fractions containing ERK or p38, and not those containing JNK/SAPK, had the capacity to activate MAPKAP kinase-2/3. In vivo, however, it appeared that only p38 is an upstream activator of HSP27 phosphorylation after both stress or growth factor stimulation: expression of an interfering mutant of ras, which blocked the activation of ERK by both types of inducers, had no effect on HSP27 phosphorylation and p38 activation; and the cell-permeant specific inhibitor of 038, SB203580, blocked MAPKAP-kinase2/3 activation and HSP27 phosphorylation. HSP27 has been suggested to have a phosphorylation-activated homeostatic function at the actin cytoskeleton level. This raises the possibility that p38 might be directly involved in mediating actin responses to external stimuli. Accordingly, we observed that a prior activation of p38 increased the stability of the actin microfilaments in cells exposed to cytochalasin D. The effect was dependent on the expression of HSP27 and was totally annihilated by blocking the p38 activity with SB203580. The results provide strong support to the idea that activation of p38 during adverse environmental conditions serves a homeostatic function aimed at regulating actin dynamics that would otherwise be destabilized during stress. Its activation during normal agonist stimulation may constitute an additional actin signaling pathway, the importance of which depends on the level of expression of HSP27.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Animais , Linhagem Celular , Cricetinae , Citocalasina D/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Substâncias de Crescimento/farmacologia , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Cell cycle re-entry requires the growth factor-stimulation of at least two distinct classes of protein kinases: (i) the p42/p44 MAP kinases activated by the Ras > Raf > MKK cascade and (ii) the G1 cyclin-dependent protein kinases (CDKs). Specific inactivation of either class of kinase arrests fibroblasts in G1. Growth factors promote nuclear translocation and persistent activation of p42/p44 MAP kinases during the entire G0/G1 period. Here, we demonstrate that induction of cyclin D1, and therefore cdk4/6 activity associated with, is positively controlled by the p42/p44 MAP kinase cascade whereas the parallel cytokines/stress-activated p38MAP kinase cascade is antagonistic. Finally, using an antisense approach we demonstrate that p27Kip1 plays a key role in setting the growth factor-dependency of the G0 state.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Ciclina D1/biossíntese , Substâncias de Crescimento/metabolismo , Proteínas Supressoras de Tumor , Animais , Ciclo Celular/genética , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitógenos/farmacologia , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Heat-shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation-regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15-fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N-acetyl-L-cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by heat shock, H2O2, sodium arsenite, TNF or growth factors was identified by in-gel renaturation and purified approximately 8000-fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen-activated-protein kinase-activated protein (MAPKAP) kinase-2 based on its reactivation in vitro by 42/44-kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54-kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild-type HSP27 but not of a nonphosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54-kDa HSP27 kinase is a common mechanism of HSP27 phosphorylation to which converge both oxyradical-dependent and oxyradical-independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cricetinae , Cricetulus , Ativação Enzimática , Temperatura Alta , Humanos , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Mitógenos/farmacologia , Dados de Sequência Molecular , Estresse Oxidativo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/isolamento & purificação , Espécies Reativas de Oxigênio/farmacologia , Especificidade por Substrato , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Heat shock induces in cells the development of a transient state of thermotolerance thought to result from the induction of heat shock proteins. To assess directly whether a transient overexpression of one of these proteins, HSP27, can contribute to increased cellular resistance, mouse NIH/3T3 cells were cotransfected with a plasmid containing the Chinese hamster HSP27 gene under the control of the metallothionein promoter and a plasmid containing the neo gene. Stable transfectant cell lines were selected for resistance to the antibiotic G418. Analyses of several stable transfectant cell lines indicated that expression of Chinese hamster HSP27 could be selectively induced by exposure to 3 microM CdCl2, a concentration that had no effect on the induction of the endogenous heat shock proteins (HSP). In clone 15, the level of HSP27 increased steadily during the first day of exposure to CdCl2, from a concentration of 1 microgram/mg of total protein to 7 micrograms/mg. After withdrawal of CdCl2, the level of HSP27 returned to normal within the next 5 days. Accumulation of the Chinese hamster HSP27 was accompanied by a progressive development of thermoresistance that attained a level approaching heat shock-induced thermotolerance. After CdCl2 removal, thermal resistance and HSP27 decayed in a coordinated manner. In control cells transfected with the neo gene only, increased thermoresistance was not induced by 3 microM CdCl2; in these cells, an exposure to 20 microM CdCl2 was required to induce a level of thermoresistance comparable to that induced by 3 microM CdCl2 in clone 15. Elevated expression of HSP27 was accompanied by an increased stability of stress fibers during hyperthermia. The protein also partially prevented actin depolymerization during acute exposure to cytochalasin D and reduced cytotoxicity and growth inhibition of chronic exposures to the drug. The results indicated that accumulation of HSP27, as it occurs after a mild heat shock or other inducing treatments, is sufficient for acquisition of thermotolerance that may result in part from a stabilization of actin filaments.
Assuntos
Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Células 3T3 , Animais , Cádmio/farmacologia , Cloreto de Cádmio , Sobrevivência Celular/efeitos dos fármacos , Cloretos/farmacologia , Cricetinae , Cricetulus , Citocalasina D/farmacologia , Relação Dose-Resposta a Droga , Imunofluorescência , Expressão Gênica , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/genética , Camundongos , Plasmídeos , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled phosphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82-Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78-Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress.
Assuntos
Arsenitos , Proteínas de Choque Térmico/metabolismo , Proteínas Quinases/metabolismo , Serina/metabolismo , Compostos de Sódio , Sequência de Aminoácidos , Animais , Arsênio/farmacologia , Western Blotting , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Fatores de Crescimento de Fibroblastos/metabolismo , Células HeLa , Humanos , Mitógenos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas , Acetato de Tetradecanoilforbol/farmacologia , Trombina/metabolismo , Tripsina/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.