RESUMO
The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.
Assuntos
Doença de Alzheimer/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/genética , Mutação , Transativadores , Doença de Alzheimer/metabolismo , Transporte Biológico/genética , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Presenilina-1 , Presenilina-2 , Ligação Proteica , Transdução de Sinais/genética , beta CateninaRESUMO
AIMS/HYPOTHESIS: Glycogen synthase kinase 3ß (GSK-3ß) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3ß in mice would result in enhanced beta cell mass and function. METHODS: Mice with beta cell deficiency of GSK-3ß (ß-Gsk-3ß [also known as Gsk3b](-/-)) were generated by breeding Gsk-3ß (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting. RESULTS: On a normal diet ß-Gsk-3ß ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet ß-Gsk-3ß (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation. CONCLUSIONS/INTERPRETATION: These results are consistent with a mechanism whereby endogenous GSK-3ß activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.
Assuntos
Diabetes Mellitus Experimental/etiologia , Gorduras na Dieta/efeitos adversos , Resistência a Medicamentos/genética , Quinase 3 da Glicogênio Sintase/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/crescimento & desenvolvimento , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Dieta Aterogênica , Ingestão de Alimentos/fisiologia , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Tamanho do Órgão/genética , Especificidade de Órgãos/genética , Ratos , Regulação para Cima/genéticaRESUMO
The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. We have generated transgenic mice expressing a constitutively active form of PKB (gag-PKB) to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB displayed increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolonged the survival of CD4(+)CD8(+) double positive (DP) thymocytes in fetal thymic organ culture, but was unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I-restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen-specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB displayed elevated levels of the antiapoptotic molecule Bcl-X(L). In addition, the activation of peripheral T cells led to enhanced nuclear factor (NF)-kappaB activation via accelerated degradation of the NF-kappaB inhibitory protein IkappaBalpha. Our data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules (PKB, Bcl-X(L), and NF-kappaB) in vivo in T lymphocytes.
Assuntos
Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Animais , Apoptose , Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Feminino , Expressão Gênica , Genes gag , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Gravidez , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína bcl-XRESUMO
Glycogen synthase kinase (GSK)-3 is a protein serine/threonine kinase that regulates differentiation and cell fate in a variety of organisms. This study examined the role of GSK-3 in antigen-specific T cell responses. Using resting T cells from P14 T cell receptor (TCR)-transgenic mice (specific for the lymphocytic choriomeningitis virus and H-2D(b)), we demonstrated that GSK-3beta was inactivated by serine phosphorylation after viral peptide-specific stimulation in vitro. To further investigate the role of GSK-3, we have generated a retroviral vector that expresses a constitutively active form of GSK-3beta that has an alanine substitution at the regulatory amino acid, serine 9 (GSK-3betaA9). Retroviral transduction of P14 TCR-transgenic bone marrow stem cells, followed by reconstitution, led to the expression of GSK-3betaA9 in bone marrow chimeric mice. T cells from chimeric mice demonstrate a reduction in proliferation and interleukin (IL)-2 production. In contrast, in vitro assays done in the presence of the GSK-3 inhibitor lithium led to dramatically prolonged T cell proliferation and increased IL-2 production. Furthermore, in the presence of lithium, we show that nuclear factor of activated T cells (NF-AT)c remains in the nucleus after antigen-specific stimulation of T cells. Together, these data demonstrate that GSK-3 negatively regulates the duration of T cell responses.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Interleucina-2/biossíntese , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Células 3T3 , Animais , Células Apresentadoras de Antígenos/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Linhagem Celular , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/metabolismo , Linfócitos T Citotóxicos/imunologia , TransfecçãoRESUMO
The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.
Assuntos
Antígenos CD28/metabolismo , Interleucina-2/biossíntese , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Tirosina Quinases/deficiência , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Quimera , Primers do DNA/genética , Centro Germinativo/citologia , Centro Germinativo/imunologia , Região de Troca de Imunoglobulinas , Proteínas Quinases JNK Ativadas por Mitógeno , Ativação Linfocitária , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Recombinação Genética , Transdução de Sinais , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidadeRESUMO
Protein kinase B (PKB, also termed Akt) is a phosphatidylinositol 3' kinase (PI3'K)-dependent enzyme implicated in survival signaling and human tumorigenesis. To identify potential targets of this protein kinase, we employed a genetic screen in Drosophila. Among several genes that genetically interacted with PKB was trachealess (trh), which encodes a bHLH-PAS domain transcription factor required for development of the trachea and other tubular organs. Trh activates expression of the fibroblast growth factor receptor Breathless, which, in turn, is required for directed migration of all tracheal branches. Using a combination of biochemical and transgenic approaches, we show that direct phosphorylation of Trh by PKB at serine 665 is essential for nuclear localization and functional activation of this regulator of branching morphogenesis.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Traqueia/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Genes Reporter , Humanos , Masculino , Morfogênese/fisiologia , Mapeamento de Peptídeos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Testes de Precipitina , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes de Fusão/metabolismo , Traqueia/anatomia & histologia , Fatores de Transcrição/genéticaRESUMO
Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cardiomegalia/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares , Animais , Animais Recém-Nascidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endotelina-1/metabolismo , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Mutação , Miocárdio/citologia , Fatores de Transcrição NFATC , Fenilefrina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Interaction of the p55 tumor necrosis factor receptor 1 (TNF-R1)-associated signal transducer TRADD with FADD signals apoptosis, whereas the TNF receptor-associated factor 2 protein (TRAF2) is required for activation of the nuclear transcription factor nuclear factor kappa B. TNF-induced activation of the stress-activated protein kinase (SAPK) was shown to occur through a noncytotoxic TRAF2-dependent pathway. TRAF2 was both sufficient and necessary for activation of SAPK by TNF-R1; conversely, expression of a dominant-negative FADD mutant, which blocks apoptosis, did not interfere with SAPK activation. Therefore, SAPK activation occurs through a pathway that is not required for TNF-R1-induced apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Acetilcisteína/farmacologia , Apoptose , Proteínas de Transporte/metabolismo , Linhagem Celular , Dactinomicina/farmacologia , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas , Sequestradores de Radicais Livres/farmacologia , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fator 1 Associado a Receptor de TNF , Fator 2 Associado a Receptor de TNF , TransfecçãoRESUMO
A ubiquitous protein-serine kinase, initially implicated in glycogen regulation, has surfaced unexpectedly in the fields of nuclear oncogenes and fruitfly development. This unusual linkage may reflect the role of this kinase in phosphorylating proteins normally activated by dephosphorylation, thus providing a priming function. Loss of such a primer would result in constitutive activation of substrates, a scenario concordant with the dramatic and pleiotropic phenotype observed in Drosophila null mutants.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glicogênio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Proteínas de Ligação a DNA/genética , Drosophila/genética , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-jun , Proteínas Proto-Oncogênicas c-myb , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
Podocytes are key cells in maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Glycogen synthase kinase 3 (GSK3) is a multi-functional serine/threonine kinase existing as two distinct but related isoforms (α and ß). In the podocyte it has previously been reported that inhibition of the ß isoform is beneficial in attenuating a variety of glomerular disease models but loss of both isoforms is catastrophic. However, it is not known what the role of GSK3α is in these cells. We now show that GSK3α is present and dynamically modulated in podocytes. When GSK3α is transgenically knocked down specifically in the podocytes of mice it causes mild but significant albuminuria by 6-weeks of life. Its loss also does not protect in models of diabetic or Adriamycin-induced nephropathy. In vitro deletion of podocyte GSK3α causes cell death and impaired autophagic flux suggesting it is important for this key cellular process. Collectively this work shows that GSK3α is important for podocyte health and that augmenting its function may be beneficial in treating glomerular disease.
RESUMO
Albuminuria affects millions of people, and is an independent risk factor for kidney failure, cardiovascular morbidity and death. The key cell that prevents albuminuria is the terminally differentiated glomerular podocyte. Here we report the evolutionary importance of the enzyme Glycogen Synthase Kinase 3 (GSK3) for maintaining podocyte function in mice and the equivalent nephrocyte cell in Drosophila. Developmental deletion of both GSK3 isoforms (α and ß) in murine podocytes causes late neonatal death associated with massive albuminuria and renal failure. Similarly, silencing GSK3 in nephrocytes is developmentally lethal for this cell. Mature genetic or pharmacological podocyte/nephrocyte GSK3 inhibition is also detrimental; producing albuminuric kidney disease in mice and nephrocyte depletion in Drosophila. Mechanistically, GSK3 loss causes differentiated podocytes to re-enter the cell cycle and undergo mitotic catastrophe, modulated via the Hippo pathway but independent of Wnt-ß-catenin. This work clearly identifies GSK3 as a critical regulator of podocyte and hence kidney function.
Assuntos
Albuminúria/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Nefropatias/metabolismo , Rim/fisiologia , Podócitos/metabolismo , Albuminúria/sangue , Albuminúria/patologia , Albuminúria/urina , Animais , Ciclo Celular , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Deleção de Genes , Inativação Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Via de Sinalização Hippo , Estimativa de Kaplan-Meier , Rim/patologia , Nefropatias/sangue , Nefropatias/patologia , Nefropatias/urina , Masculino , Camundongos , Podócitos/enzimologia , Podócitos/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Ratos Wistar , Insuficiência Renal , Verteporfina/farmacologia , beta Catenina/metabolismoRESUMO
BACKGROUND: Exposing eukaryotic cells to lithium ions (Li+) during development has marked effects on cell fate and organization. The phenotypic consequences of Li+ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases. RESULTS: We investigated the effect of Li+ on the activity of the GSK-3 family. At physiological doses, Li+ inhibits the activity of human GSK-3 beta and Drosophila Zw3Sgg, but has no effect on other protein kinases. The effect of Li+ on GSK-3 is reversible in vitro. Treatment of cells with Li+ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li+ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/beta-catenin, demonstrating that Li+ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3. CONCLUSIONS: Li+ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li+ action on development and differentiation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas de Drosophila , Cloreto de Lítio/farmacologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Linhagem Celular , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Proteínas Associadas aos Microtúbulos/genética , Células PC12 , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Proteína Wnt1RESUMO
Recent evidence for cross-talk between protein kinase B (PKB) and the Raf-1 and NF-kappaB signalling pathways has provided new hints to the complex roles that PKB may play in regulating gene transcription and also raised questions about where and when these targets are relevant.
Assuntos
Regulação Enzimológica da Expressão Gênica , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Humanos , Ligantes , Modelos Biológicos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transcrição GênicaRESUMO
Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun [1] [2] [3]. This mutation markedly enhances v-Jun oncogenicity [4] [5]; however, its transcriptional consequences have not been resolved. In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly [6] [7] or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation [8]. Using a two-hybrid approach, we show that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate. Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation. Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control. This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonise SAPK/JNK-dependent gene expression.
Assuntos
Regulação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Proteína Oncogênica p65(gag-jun)/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Humanos , Proteína Quinase 12 Ativada por Mitógeno , Mutação , Proteína Oncogênica p65(gag-jun)/genética , Oncogenes , Proteínas Quinases/genética , Ratos , Proteínas Recombinantes de FusãoRESUMO
BACKGROUND: Stimuli that stress cells, including inflammatory cytokines, ultra-violet irradiation, DNA-damaging chemotherapeutic drugs and heat shock, stimulate a recently identified cytoplasmic signaling system that is structurally related to the mitogen-activated protein kinase pathway. This pathway consists of a cascade of protein kinases including stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), and two kinases that activate it, MEKK and SEK/MKK4. Despite rapid progress in delineating the components of this pathway, the cellular consequence of its activation remains to be defined. RESULTS: We have screened cells for defects in SAPK signaling and identified a cell line, previously characterized for its thermotolerance properties, as being more refractive to SAPK activation induced by heat stress than its thermosensitive parental line. Stable expression of dominant inhibiting SEK mutants in thermosensitive parental cells specifically and effectively blocked SAPK activation after heat shock. These lines also became markedly resistant to the cytocidal effects of thermal stress, confirming the phenotype of the thermotolerant line. These cell lines defective in SAPK activation were also resistant to the lethal effects of the DNA-damaging drug cis-platinum. CONCLUSIONS: Experimentally induced stable blockade of SAPK activation in cells with normal thermosensitivity is sufficient to confer resistance to cell death induced by diverse stimuli including heat and the chemotherapeutic agent cis-platinum. These results suggest that activation of the SAPK pathway by diverse cell stressors plays a critical part in mediating the toxicity of these treatments and inducing cell death. SAPK activation in this context could broadly influence cellular response to stress, modulate apoptosis during development or determine the clinical response of tumor cells to cytotoxic therapies.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Morte Celular , Cisplatino/farmacologia , Temperatura Alta , Proteínas Quinases Ativadas por Mitógeno , Raios Ultravioleta , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular , Proteínas Quinases JNK Ativadas por Mitógeno , Transdução de SinaisRESUMO
The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.
Assuntos
Proteínas de Drosophila , Drosophila/enzimologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Apoptose , Bovinos , Drosophila/embriologia , Drosophila/genética , Feminino , Regulação da Expressão Gênica , Proteínas Inibidoras de Apoptose , Proteínas de Insetos/genética , Masculino , Neuropeptídeos/genética , Peptídeos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Coelhos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+.
Assuntos
Encéfalo/enzimologia , Isoenzimas/isolamento & purificação , Proteína Quinase C/isolamento & purificação , Aminoácidos/análise , Animais , Anticorpos/isolamento & purificação , Complexo Antígeno-Anticorpo , Cromatografia de Afinidade , Isoenzimas/imunologia , Mapeamento de Peptídeos , Proteína Quinase C/biossíntese , Proteína Quinase C/imunologia , Ratos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
p36, a major in vivo substrate of protein-tyrosine kinases, is shown to be phosphorylated at serine 25, a site very close to the major site of tyrosine phosphorylation by pp60v-src, tyrosine 23 (J. R. Glenney, Jr., and B. F. Tack, Proc. Natl. Acad. Sci. USA 82:7884-7888, 1985). We present evidence suggesting that protein kinase C mediates phosphorylation of serine 25.
Assuntos
Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Anexinas , Bovinos , Fosfoproteínas/análise , Especificidade por SubstratoRESUMO
Activation of Akt by the phosphatidylinositol 3'-OH kinase (PI3K) results in the inhibition of proapoptotic signals and the promotion of survival signals (L. P. Kane et al., Curr. Biol. 9:601-604, 1999; G. J. Kops et al., Nature 398:630-634, 1999). Evidence supporting the importance of the PI3K/Akt signaling pathway in tumorigenesis stems from experiments with transgenic mice bearing polyomavirus middle T antigen under the control of the mouse mammary tumor virus long terminal repeat promoter. Mammary epithelium-specific expression of polyomavirus middle T antigen results in the rapid development of multifocal metastatic mammary tumors, whereas transgenic mice expressing a mutant middle T antigen decoupled from the phosphatidylinositol 3'-OH kinase (MTY315/322F) develop extensive mammary gland hyperplasias that are highly apoptotic. To directly assess the role of Akt in mammary epithelial development and tumorigenesis, we generated transgenic mice expressing constitutively active Akt (HAPKB308D473D or Akt-DD). Although expression of Akt-DD interferes with normal mammary gland involution, tumors were not observed in these strains. However, coexpression of Akt-DD with MTY315/322F resulted in a dramatic acceleration of mammary tumorigenesis correlated with reduced apoptotic cell death. Furthermore, coexpression of Akt-DD with MTY315/322F resulted in phosphorylation of the FKHR forkhead transcription factor and translational upregulation of cyclin D1 levels. Importantly, we did not observe an associated restoration of wild-type metastasis levels in the bitransgenic strain. Taken together these observations indicate that activation of Akt can contribute to tumor progression by providing an important cell survival signal but does not promote metastatic progression.
Assuntos
Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Sobrevivência Celular/fisiologia , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Epitélio/metabolismo , Epitélio/patologia , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Hiperplasia/metabolismo , Proteínas I-kappa B/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.