Assuntos
MAP Quinase Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Células 3T3 , Animais , Especificidade de Anticorpos , Benzimidazóis/química , Benzimidazóis/farmacologia , Butadienos/química , Butadienos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Ativação Enzimática , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Células K562 , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Nitrilas/química , Nitrilas/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Functional proteomics provides a powerful method for monitoring global molecular responses following activation of signal transduction pathways, reporting altered protein posttranslational modification and expression. Here we combine functional proteomics with selective activation and inhibition of MKK1/2, in order to identify cellular targets regulated by the MKK/ERK cascade. Twenty-five targets of this signaling pathway were identified, of which only five were previously characterized as MKK/ERK effectors. The remaining targets suggest novel roles for this signaling cascade in cellular processes of nuclear transport, nucleotide excision repair, nucleosome assembly, membrane trafficking, and cytoskeletal regulation. This study represents an application of functional proteomics toward identifying regulated targets of a discrete signal transduction pathway and demonstrates the utility of this discovery-based strategy in elucidating novel MAP kinase pathway effectors.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteoma , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Butadienos/farmacologia , Eletroforese em Gel Bidimensional , Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Humanos , Células K562 , Cinética , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Nitrilas/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas/análise , Proteínas/metabolismo , Proteoma/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.
Assuntos
Megacariócitos/citologia , Megacariócitos/enzimologia , Proteínas Quinases/metabolismo , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/fisiologia , Primers do DNA/genética , Ativação Enzimática/efeitos dos fármacos , Eritropoese/fisiologia , Expressão Gênica , Globinas/genética , Humanos , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/patologia , Megacariócitos/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Proteínas Quinases/genética , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
In mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss-of-function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain-of-function allele, Tumorous-lethal (hopTum-l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum-l mutation. Overexpression of either wild-type hop or hopTum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum-l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum-l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum-l results in greater phosphorylation than overexpression of the wild-type. We conclude that hopTum-l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells.
Assuntos
Transformação Celular Neoplásica , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Tirosina Quinases/genética , Animais , Sequência de Bases , Linhagem Celular , Análise Mutacional de DNA , Proteínas de Drosophila , Drosophila melanogaster/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Genes de Insetos/genética , Genes Letais/genética , Janus Quinases , Larva , Sistema Linfático/patologia , Dados de Sequência Molecular , Fosforilação , Mutação Puntual/genética , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Transcrição , Tirosina/metabolismoRESUMO
The signaling pathways used by interleukin-3 (IL-3) and by active phorbol ester (12-0-tetradecanoyl phorbol-13-acetate, TPA) to stimulate mitogenesis in the growth factor dependent myeloid cell line FDC-P1 were studied by 'reporter' analysis of nuclear proto-oncogene expression. These studies revealed that IL-3 strongly stimulated c-myc expression by a transcriptional mechanism but IL-3 poorly stimulated c-jun expression, a measure of protein kinase C dependent signals. On the other hand, the protein kinase C agonist, TPA, strongly activated c-jun expression but poorly promoted expression (transcription) of c-myc in FDC-P1. These findings appeared to correlate with the poor mitogenic capacity of TPA for FDC-P1. However, stable transfection of FDC-P1 with a c-myc expression vector driven by a human methallothionein IIA promoter containing the TPA responsive element (TRE), led to a cell clone, FDMT myc.A1, in which TPA mediated selective transcription of the transfected TRE driven c-myc vector and down-regulated expression of the endogenous c-myc gene. IL-3 selectively failed to stimulate expression of the TRE driven c-myc vector in FDMT myc.A1. Augmented TPA dependent vector derived c-myc expression was accompanied by enhanced mitogenesis of the cell line FDMT myc.A1 compared with FDC-P1. In addition, TPA mediated expression of the transfected c-myc gene in FDMT myc.A1 was accompanied by augmented transcription of c-jun and c-fos in response to TPA. These studies show the importance of a non-protein kinase C dependent pathway for IL-3 mediated c-myc transcription. However, these studies reveal that protein kinase C mediated pathways can be promitogenic, especially when complemented by unregulated c-myc expression (in this case driven by an alternative, TRE containing promoter).
Assuntos
Interleucina-3/farmacologia , Animais , Northern Blotting , Southern Blotting , Linhagem Celular , Regulação da Expressão Gênica , Genes jun , Genes myc , Vetores Genéticos , Técnicas In Vitro , Camundongos , Mitose/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteína Quinase C/fisiologia , Proto-Oncogene Mas , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transcrição GênicaRESUMO
We examined the role of augmented formation of intracellular cyclic AMP (cAMP) in the mediation of stromal cell growth factor production that occurs constitutively or upon cytokine stimulation. Clonal murine marrow adherent cell lines were stimulated under serum-free conditions by interleukin-1 (IL-1) or lipopolysaccharide (LPS) and one (+/+ -1.LDA11) was found to produce low quantities of granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF identity was confirmed by the ability of supernatants from stromal cells to promote proliferation of the factor-dependent cell line FDC-P1, neutralization of this activity by antiserum to GM-CSF, and by Northern blot analysis. However, optimal concentrations of IL-1 and tumor necrosis factor-alpha (TNF-alpha), in combination, led to synergistic (greater than 5-fold higher quantity) GM-CSF production compared with either stimulus alone in the +/+ -1. LDA11 cell line, capable of GM-CSF production after only single stimulation with IL-1 or LPS. In addition, synergistic stimulation by IL-1 and TNF-alpha led to equivalent high amounts of GM-CSF in another cell line incapable of GM-CSF production after induction with only IL-1 or LPS. Any of several means to raise intracellular cAMP levels, including addition of 8-bromo-cyclic AMP (8Br cAMP) (0.25-1mM), pertussis toxin (20-100 ng/ml), or addition of prostaglandin E1 (PGE1) (1 microM), failed to stimulate GM-CSF production alone and strongly inhibited GM-CSF production in stromal cells stimulated by IL-1, LPS, or the synergistic combination of IL-1 and TNF-alpha. In addition, PGE1 and pertussis intoxication were agonists of adenylate cyclase in membranes of marrow adherent cells, whereas IL-1 and LPS were not. The role for regulators of intracellular cAMP was specific because any of the cAMP agonists alone, or in the presence of cytokine stimulators of stromal cells, strongly enhanced IL-6 production, an event known to be cAMP-responsive. Thus, acute formation of intracellular cAMP is a negative regulator of stromal cell GM-CSF production mediated by cytokines, but positively regulates IL-6 production and may be an important determinant of cytokine-directed marrow microenvironmental function. These findings on the requirement for augmentation versus inhibition of cytokine-mediated production of hemopoietic growth factors might be applied to an analysis of marrow stromal cell heterogeneity.
Assuntos
Fatores Estimuladores de Colônias/biossíntese , AMP Cíclico/metabolismo , Substâncias de Crescimento/biossíntese , Interleucina-1/farmacologia , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Toxina Adenilato Ciclase , Animais , Células da Medula Óssea , Linhagem Celular , Células Cultivadas , AMP Cíclico/fisiologia , Matriz Extracelular/citologia , Matriz Extracelular/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Líquido Intracelular/metabolismo , Camundongos , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The factor-dependent cell line FDC-P1 has been utilized as a model of interleukin 3 (IL-3)-dependent myeloid cell proliferation. However, it has been recently observed that active phorbol esters (e.g., phorbol 12-myristate 13-acetate) may entirely replace IL-3 to promote its proliferation. These observations reveal abnormal regulation of protein kinase C (pkC) (absence of downregulation or overexpression). This property allowed a test of the hypothesis that the T24 RAS (codon 12) oncogene acts by constitutive and persistent pkC activation, driving proliferation. FDC-P1 cells were transfected by electroporation with the T24 RAS-containing vector pAL 8, or with a control vector pSVX Zip Neo, and neomycin-resistant clones were selected. Multiple RAS-transfectant clones were categorized for their growth factor requirement and incorporation of the 6.6-kb human mutant H-RAS genome. IL-3-independent clones had incorporated multiple (more than two) copies of the entire 6.6-kb RAS genome. The incorporation of multiple 6.6-kb RAS genomes was correlated with high-level p21 RAS expression. No evidence for autostimulatory growth factor production by clones containing the RAS oncogene was observed. Thus, acquisition of growth factor independence in myeloid cells by abundant expression of a RAS oncogene is linked, in part, to abnormal regulation of pkC, which acts as a collaborating oncogene.