RESUMO
(-)-Deprenyl and structurally related propargylamines increase neuronal survival independently of monoamine oxidase B (MAO-B) inhibition, in part by decreasing apoptosis. We found that deprenyl and two other propargylamines, one of which does not inhibit monoamine oxidase B, increased survival in trophically withdrawn 6-day nerve growth factor (NGF)- and 9-day NGF-differentiated PC-12 cells but not in NGF naive or 3-day NGF-differentiated PC-12 cells. Four days of prior NGF exposure were required for the propargylamine-mediated antiapoptosis. Studies using actinomycin D, cycloheximide, and camptothecin revealed that the maintenance of both transcription and translation, particularly between 2 and 6 h after trophic withdrawal, was required for propargylamine-mediated antiapoptosis. Metabolic labeling of newly synthesized proteins for two-dimensional protein gel autoradiography and scintillation counting showed that the propargylamines either increased or reduced the levels of new synthesis or induced de novo synthesis of a number of different proteins, most notably proteins in the mitochondrial and nuclear subfractions. Western blotting for whole cell or subcellular fraction lysates showed that the timing of new protein synthesis changes or subcellular redistribution of apoptosis-related proteins induced by the propargylamines were appropriate to antiapoptosis. The apoptosis-related proteins included superoxide dismutases (SOD1 and SOD2), glutathione peroxidase, c-JUN, and glyceraldehyde-3-phosphate dehydrogenase. Most notable were the prevention of apoptotic decreases in BCL-2 levels and increases in mitochondrial BAX levels. In general, (-)-deprenyl-related propargylamines appear to reduce apoptosis by altering the levels or subcellular localization of proteins that affect mitochondrial membrane permeability, scavenge oxidative radicals, or participate in specific apoptosis signaling pathways.
Assuntos
Apoptose/fisiologia , Fator de Crescimento Neural/metabolismo , Pargilina/análogos & derivados , Pargilina/farmacologia , Propilaminas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Meios de Cultura Livres de Soro , Células PC12 , Inibidores da Síntese de Proteínas/farmacologia , RatosRESUMO
Monocyte differentiation induced by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor alpha (RARalpha) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D(3) receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)(2)D(3) activation from both a minimal VDR responsive reporter and the promoter of p21(WAF1/CIP1), a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)(2)D(3)-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)(2)D(3) to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)(2)D(3) induces monocytic differentiation in hematopoietic cells.
Assuntos
Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Monócitos/efeitos dos fármacos , Receptores de Calcitriol/efeitos dos fármacos , Fatores de Transcrição/farmacologia , Sítios de Ligação , Núcleo Celular/química , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like , Linfoma Difuso de Grandes Células B , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células U937 , Dedos de ZincoRESUMO
The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RAR alpha fusion protein and, in a similar manner, inhibits RAR alpha target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RAR alpha and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia. (Blood. 2000;96:3939-3947)
Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Promielocítica Aguda/genética , Proteínas de Fusão Oncogênica/farmacologia , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Transcrição Gênica/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Transcrição Kruppel-Like , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/genética , Leucemia Promielocítica Aguda/etiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Matriz Nuclear/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Ligação Proteica , Proteína 1 Parceira de Translocação de RUNX1 , Proteínas Repressoras/metabolismo , Proteínas Repressoras/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Arginina/química , Linhagem Celular , Dicroísmo Circular , DNA Complementar/metabolismo , Dimerização , Escherichia coli/metabolismo , Imunofluorescência , Genes Reporter , Glutamina/química , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Mutação Puntual , Proteína com Dedos de Zinco da Leucemia Promielocítica , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Temperatura , Tripsina/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Translocação Genética , Doença Aguda , Animais , Células COS , Humanos , Fatores de Transcrição Kruppel-Like , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteína 1 Parceira de Translocação de RUNX1 , Transfecção , Dedos de ZincoRESUMO
Antisense oligonucleotides against the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are able to reduce some forms of apoptosis. In those forms, overall GAPDH levels increase and the enzyme accumulates in the nucleus. The monoamine oxidase B (MAO-B) inhibitor, (-)-deprenyl (DEP), its metabolite (-)-desmethyldeprenyl, and a tricyclic DEP analog, CGP3466, can reduce apoptosis independently of MAO-B inhibition and have been found to bind to GAPDH. We used neuronally differentiated PC12 cells to show that DEP, DES, and CGP3466 reduce apoptosis caused by serum and nerve growth factor withdrawal over the concentration range of 10(-) to 10(-13) M. We provide evidence that the DEP-like compounds bind to GAPDH in the PC12 cells and that they prevent both the apoptotic increases in GAPDH levels and nuclear accumulation of GAPDH. In vitro, the compounds enhanced the conversion of NAD(+) to NADH by GAPDH in the presence of AUUUA-rich RNA and converted GAPDH from its usual tetrameric form to a dimeric form. Using cell lysates, we found a marked increase in rates of NAD(+) to NADH conversion in early apoptosis, which was returned toward control values by the DEP-like compounds. Accordingly, the DEP-like compounds appear to decrease glycolysis by preventing the GAPDH increases in early apoptosis. GAPDH dimer may not have the capacity to contribute to apoptosis in a similar manner to the tetramer, which might account for the antiapoptotic capacity of the compounds. These actions on GAPDH, rather than MAO-B inhibition, may contribute to the improvements in Parkinson's and Huntington's diseases found with DEP treatment.
Assuntos
Apoptose , Proteínas Sanguíneas/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Fator de Crescimento Neural/fisiologia , Anfetaminas/farmacologia , Animais , Proteínas Sanguíneas/deficiência , Dimerização , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/efeitos dos fármacos , Inibidores da Monoaminoxidase/farmacologia , Fator de Crescimento Neural/deficiência , Oxepinas/farmacologia , Células PC12 , Conformação Proteica , Ratos , Selegilina/farmacologiaRESUMO
Increased expression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are early, critical events in several forms of apoptosis. In order to investigate the subcellular trafficking of GAPDH in vivo, the localization of a GAPDH-green fluorescent protein (GFP) fusion was studied in PC12, HEK 293 and COS-1 cells. In control cells, fusion protein autofluorescence was largely restricted to the cytoplasm, rather than the nuclear concentration favored by GFP alone. In contrast, as early as 30 min after an insult, nuclear fluorescence increased in all cell lines studied. The fusion protein redistribution paralleled the dynamics of endogenous GAPDH. These data suggest that some nuclear GAPDH observed during apoptosis represents protein previously resident in the cytosol. This construct provides an in vivo monitor for an early change in apoptosis.
Assuntos
Apoptose/fisiologia , Núcleo Celular/metabolismo , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Transporte Biológico/fisiologia , Células COS , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Células PC12 , Ratos , Frações Subcelulares/metabolismo , Fatores de Tempo , Distribuição Tecidual/fisiologiaRESUMO
MPP+ inhibits mitochondrial complex I and alpha-ketoglutarate dehydrogenase causing necrosis or apoptosis of catecholaminergic neurons. Low glucose levels or glycolytic blockade has been shown to potentiate MPP+ toxicity. We found that MPP+ caused concentration-dependent apoptosis of neuronally differentiated PC12 cells and that glucose, but not pyruvate, supplementation reduced apoptosis. Oligomycin concentrations sufficient to inhibit ATP synthase blocked the decreased apoptosis afforded by glucose supplementation. Laser-scanning confocal microscope imaging of chloromethyl-tetramethylrosamine methyl ester fluorescence to estimate DeltaPsiM showed that MPP+ and atractyloside reduced DeltaPsiM, while cyclosporin A (CSA) and glucose supplementation reversed decreases in DeltaPsiM caused by MPP+. Oligomycin blocked the effect of glucose supplementation on DeltaPsiM. These findings show that (i) MPP+-induced and atractyloside-induced apoptosis are associated with reduced DeltaPsiM; (ii) CSA maintains DeltaPsiM and reduces MPP+-induced apoptosis; and (iii) glucose supplementation maintains DeltaPsiM, likely by glycolytic ATP-dependent proton pumping at ATP synthase and reduces MPP+-induced apoptosis.
Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Apoptose/efeitos dos fármacos , Glucose/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , 1-Metil-4-fenilpiridínio/antagonistas & inibidores , Animais , Atractilosídeo/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glucose/antagonistas & inibidores , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Microscopia Confocal , Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Fatores de Crescimento Neural/farmacologia , Oligomicinas/farmacologia , Células PC12 , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ácido Pirúvico/farmacologia , Ratos , Fatores de TempoRESUMO
The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH-PML interaction may be involved in the regulation of apoptosis.
Assuntos
Núcleo Celular/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Fibroblastos , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Humanos , Camundongos , Proteínas de Neoplasias/isolamento & purificação , Proteína da Leucemia Promielocítica , Ribonucleases , Fatores de Transcrição/isolamento & purificação , Proteínas Supressoras de TumorRESUMO
The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.
Assuntos
Vírus da Coriomeningite Linfocítica/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Protozoários , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Dedos de Zinco , Células 3T3 , Animais , Fracionamento Celular , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Citoplasma/metabolismo , Células HeLa , Humanos , Camundongos , Proteína da Leucemia Promielocítica , Frações Subcelulares , Transfecção , Proteínas Supressoras de Tumor , Proteínas Virais/genéticaRESUMO
Studies in non-neural cells have suggested that a fall in mitochondrial membrane potential (DeltaPsiM) is one of the earliest events in apoptosis. It is not known whether neural apoptosis caused by nerve growth factor (NGF) and serum withdrawal involves a decrease in DeltaPsiM. We used epifluorescence and laser confocal microscopy with the mitochondrial potentiometric dyes chloromethyl-tetramethylrosamine methyl ester and 5,5',6, 6'-tetrachloro-1,1',3,3'-tetraethybenzimidazol carbocyanine iodide to estimate DeltaPsiM. PC12 cells were differentiated in media containing serum and NGF for 6 d before withdrawal of trophic support. After washing, the cells were incubated with media containing serum and NGF (M/S+N), media without serum and NGF, or media with the "trophic-like" monoamine oxidase B inhibitor, (-)-deprenyl. Mitochondria in cells without trophic support underwent a progressive shift to lower DeltaPsiM values that was significant by 3 hr after washing. The percentages of cells with nuclear chromatin condensation or nuclear DNA fragmentation were not significantly increased above those for cells in M/S+N until 6 hr after washing. Replacement of cells into M/S+N or treatment with (-)-deprenyl markedly reduced the proportion of mitochondria with decreased DeltaPsiM. Measurements of cytoplasmic peroxyl radical levels with 2',7'-dihydrodichlorofluorescein fluorescence and intramitochondrial Ca2+ with dihydro-rhodamine-2-acetylmethyl ester indicated that cytoplasmic peroxyl radical levels were not increased until after 6 hr, whereas increases in intramitochondrial Ca2+ paralleled the decreases in DeltaPsiM. (-)-Deprenyl appeared to alter the relationship between intramitochondrial Ca2+ levels and DeltaPsiM, possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.
Assuntos
Apoptose/fisiologia , Proteínas Sanguíneas/farmacologia , Mitocôndrias/metabolismo , Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Selegilina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Cromatina/fisiologia , Citoplasma/metabolismo , Fragmentação do DNA , Radicais Livres/metabolismo , Processamento de Imagem Assistida por Computador , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Células PC12 , Peróxidos/metabolismo , RatosRESUMO
The Tpl-2 protein serine/threonine kinase was originally identified, in a C-terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats. The kinase domain of Tpl-2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl-2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl-2 and Tpl-2deltaC (a C-terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK-1 and SEK-1, in vitro. Furthermore, transfection of Tpl-2 into COS-1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK-1 and SAP kinase (JNK), which are substrates for MEK-1 and SEK-1, respectively. Tpl-2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl-2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK-1.
Assuntos
Proteínas Fetais/metabolismo , MAP Quinase Quinase 4 , MAP Quinase Quinase Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Primers do DNA/química , Ativação Enzimática , Humanos , MAP Quinase Quinase 1 , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-raf , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor EphA4 , Transdução de Sinais , Linfócitos TRESUMO
Two molecules involved in signal transduction via the T cell antigen receptor, namely the protein-tyrosine kinase ZAP-70 and the proto-oncoprotein Vav, were found to be constitutively associated with tubulin in Jurkat T cells. Both were able to bind to tubulin independently of one another, as determined by transient transfection into COS-7 cells. The ZAP-70 associated with tubulin was preferentially tyrosine-phosphorylated after T cell antigen receptor stimulation of Jurkat T cells, suggesting that this interaction was functionally significant. Vav was also found to co-immunoprecipitate with ZAP-70 from cell extracts depleted of tubulin. This raised the possibility that Vav might be a substrate for ZAP-70 protein-tyrosine kinase activity. However, tyrosine phosphorylation of Vav preceded that of ZAP-70, indicating that Vav was unlikely to be a downstream target of ZAP-70. The association of ZAP-70 and Vav with tubulin implies that the microtubules may be involved in the signaling function of these two molecules, perhaps by targeting them to their appropriate intracellular location.
Assuntos
Proteínas de Ciclo Celular , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Citosol/metabolismo , Humanos , Imunossupressores/farmacologia , Muromonab-CD3/farmacologia , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-vav , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Linfócitos T , Transfecção , Tubulina (Proteína)/isolamento & purificação , Células Tumorais Cultivadas , Proteína-Tirosina Quinase ZAP-70RESUMO
The purpose of the present study was to develop a technique to identify, isolate and partially purify these membrane bound compartments for further characterizations of their Ca2+ transport and storage mechanisms. We 45Ca(2+)-loaded the agonist-sensitive Ca2+ stores in rat pancreatic acini. The loading was accomplished by first depleting the stores with carbachol stimulation followed by the addition of 45Ca2+ and atropine to the extracellular media. After homogenization of the 45Ca(2+)-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca2+ fluxes were minimized during these procedures by inclusion in the media of LaCl3. Five subcellular fractions were identified that specifically accumulated 45Ca2+ after carbachol stimulation. Electron microscopic observations of the fractions demonstrated that three of the fractions consisted of rough membrane vesicles; that one consisted of a mixture of rough and smooth membrane vesicles; and that one consisted of smooth membrane vesicles. All fractions were enriched in glucose-6-phosphatase. All 5 fractions demonstrated ATP dependent 45Ca2+ uptake. By Western blot analysis, all fractions contained calnexin, p58, sarcoplasmic reticulum type Ca(2+)-ATPase, and IP3 receptor. These results demonstrated that the 45Ca(2+)-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca2+ store in the pancreatic acinar cell.