RESUMO
Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Complexo Antígeno-Anticorpo/análise , Linhagem Celular , Receptores ErbB/genética , Soros Imunes , Cinética , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-raf , TransfecçãoRESUMO
SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Células 3T3 , Animais , Catálise , Divisão Celular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/uso terapêutico , Glioma/irrigação sanguínea , Humanos , Indóis/uso terapêutico , Camundongos , Pirróis/uso terapêutico , Ratos , Receptores Mitogênicos/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular , Células Tumorais CultivadasRESUMO
A number of growth factor receptor tyrosine kinases have been implicated in angiogenesis, including epidermal growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vascular endothelial growth factor (VEGF), is expressed exclusively in endothelial cells. Using dominant-negative methods, Flk-1 was shown to play a role in angiogenesis and the growth of a variety of tumor types. Because of this, a drug discovery effort was established to identify Flk-1 kinase inhibitors. For initial screening, an ELISA in, a 96-well format was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells. Compounds that inhibited ligand-induced receptor autophosphorylation were confirmed by antiphosphotyrosine immunoblotting. Inhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using these methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine kinase activity, endothelial cell mitogenesis, and blood vessel formation in the chorioallantoic membrane assay have been found.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Células 3T3/efeitos dos fármacos , Células 3T3/enzimologia , Animais , Moléculas de Adesão Celular/análise , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Estimulação Química , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We examined the effect of overexpression of growth factor-regulated second messenger enzymes, alone and in combination, on transformation of NIH3T3 cells. Signal transducers included phospholipase C-gamma (PLC-gamma), protein kinase C-gamma (PKC-gamma), and two proto-oncogenes, c-H-ras and c-raf-1. Three of these proteins, PLC-gamma, PKC-gamma and Raf-1, did not transform NIH3T3 cells alone or in combination. c-H-ras, which under its own promoter control has low transforming activity, also did not cooperate with PLC-gamma or PKC-gamma. In contrast, the combination of normal or oncogenic p21 H-Ras with the Raf-1 kinase dramatically increased transformation efficiency. The level of Ras protein required for transformation was reduced in Raf-1 co-transfectants, implying that, at low levels of p21 Ras, p74 Raf-1 is rate limiting. As transformation by Ras depends on jun-mediated transcriptional events, we also examined H-ras and c-raf-1 cooperation in transcriptional transactivation of TPA-responsive element (TRE)-dependent reporters. Like the H-ras/c-raf-1 cooperation in transformation, we observed this synergistic stimulation of TRE-dependent transcription. This pathway for transformation and transcriptional activation by increased levels of normal Ras and Raf may be important in tumors that show overexpression but lack mutationally activated forms of these two proto-oncogenes.
Assuntos
Transformação Celular Neoplásica/genética , Genes ras , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/genética , Células 3T3 , Animais , Expressão Gênica , Substâncias de Crescimento/fisiologia , Técnicas In Vitro , Camundongos , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf , Sistemas do Segundo Mensageiro , Transcrição Gênica , Transfecção , Fosfolipases Tipo C/fisiologiaRESUMO
A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.
Assuntos
Genes ras , Proteína Quinase C/fisiologia , Proteínas Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Fenômenos Fisiológicos Sanguíneos , Citosol/metabolismo , Dexametasona/farmacologia , Receptores ErbB/fisiologia , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
3-Substituted indolin-2-ones have been designed and synthesized as a novel class of tyrosine kinase inhibitors which exhibit selectivity toward different receptor tyrosine kinases (RTKs). These compounds have been evaluated for their relative inhibitory properties against a panel of RTKs in intact cells. By modifying the 3-substituted indolin-2-ones, we have identified compounds which showed selective inhibition of the ligand-dependent autophosphorylation of various RTKs at submicromolar levels in cells. Structure-activity analysis for these compounds and their relative potency and selectivity to inhibit particular RTKs has determined that (1) 3-[(five-membered heteroaryl ring)methylidenyl]indolin-2-ones are highly specific against the VEGF (Flk-1) RTK activity, (2) 3-(substituted benzylidenyl)indolin-2-ones containing bulky group(s) in the phenyl ring at the C-3 position of indolin-2-ones showed high selectivity toward the EGF and Her-2 RTKs, and (3) the compound containing an extended side chain at the C-3 position of the indolin-2-one (16) exhibited high potency and selectivity when tested against the PDGF and VEGF (Flk-1) RTKs. Recent published crystallographic data for two of these 3-substituted indolin-2-ones provides a rationale to suggest that these compounds may bind in the ATP binding pocket of RTKs. The structure-activity analysis supports the use of subsets of these compounds as specific chemical leads for the development of RTK-specific drugs with broad application for the treatment of human diseases.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas , Células Cultivadas , Inibidores Enzimáticos/química , Receptores ErbB/antagonistas & inibidores , Humanos , Indóis/química , Espectroscopia de Ressonância Magnética , Fosforilação , Receptor ErbB-2/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of 3-indoleacrylonitrile tyrphostins, 2-chloro-3-phenylquinolines, and 3-arylquinoxalines were prepared and tested for inhibition of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK) activity. The potency of the inhibitors was found to be quinoxalines > quinolines > indoles. Lipophilic groups (methyl, methoxy) in the 6 and 7 positions and phenyl at the 3 position of quinoxalines and quinolines were essential for potency, in contrast to the hydrophilic catechol group in tyrphostins active against EGFR kinase inhibition at different sites. The inhibitors showed selectivity for PDGF and were not active against EGF receptor and HER-2/c-ErbB-2 receptor.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nitrilas/química , Nitrilas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Células 3T3 , Animais , Membrana Celular/metabolismo , Inibidores Enzimáticos/síntese química , Fator de Crescimento Epidérmico/farmacologia , Indicadores e Reagentes , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Camundongos , Estrutura Molecular , Nitrilas/síntese química , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Quinolinas/síntese química , Quinolinas/química , Quinolinas/farmacologia , Quinoxalinas/síntese química , Quinoxalinas/química , Quinoxalinas/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Five new asterriquinone analogs (2-4, 6, 7), together with previously identified neoasterriquinone (1) and isoasterriquinone (5), were isolated from a fermentation broth of the fungus Aspergillus candidus and purified by HSCCC (high speed counter current chromatography) followed by HPLC. The structures were determined by 1D and 2D NMR and MS/MS techniques. All seven showed inhibitory activity against the binding of a recombinant protein containing the SH2 protein domain of Grb-2 to the tyrosine phosphorylated form of the EGF receptor tyrosine kinase. Some of these asterriquinones exhibited specific inhibition of Grb-2 binding compared to Grb-7 and PLC-gamma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antibióticos Antineoplásicos/farmacologia , Aspergillus/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Receptores ErbB/metabolismo , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Fermentação , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Indóis/isolamento & purificação , Indóis/metabolismo , Indóis/farmacologia , Espectroscopia de Ressonância Magnética , Fosforilação , Ligação Proteica , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria Ultravioleta , Domínios de Homologia de srcRESUMO
The activity of yeast trehalase when assayed at pH 7 in a crude extract was found to increase 2- to 3-fold upon incubation with 0.1% (v/v) polyethyleneimine or other polycations such as polylysine (0.075-mMol) and calf thymus histones (0.08 mMol). Incubation with 3 mM-Mn2+ and 5 mM-Ca2+ also led to 3- and 1.6-fold increases in trehalase activity, respectively. The activities of 11 other enzymes assayed in the crude yeast extract did not increase after addition of polyethylene imine. At concentrations of polyethyleneimine that maximally stimulated trehalase activity, 97% of the total RNA present in the crude extract, 40% of total protein, and 60% of the polyphosphate (assayed as inorganic phosphate liberated during 7 min incubation at 95 degrees C and pH O) were found to be precipitated. A similar finding was made with trehalase-stimulating concentrations of Mn2+. Activation of trehalase by polyethylene imine rendered this enzyme susceptible to inhibition by a preparation of total yeast RNA, inorganic polyphosphates, and related polyanions. We present further evidence that the removal of a distinct RNA and/or polyphosphate is the basic principle of polyethyleneimine-induced activation of trehalase. A more pronounced stimulation of trehalase activity (4-fold) could be obtained by enzymatic phosphorylation with ATP in the presence of cyclic AMP and Mg2+ as described by van Solingen and van der Plaat (1975) [9].(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Polifosfatos/metabolismo , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/enzimologia , Trealase/metabolismo , Trifosfato de Adenosina/análise , Glucose Oxidase , Concentração de Íons de Hidrogênio , Metais/farmacologia , Fosfatos/análise , Salicilatos/análise , Fatores de TempoRESUMO
Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.
Assuntos
Mutação , Saccharomyces cerevisiae/enzimologia , Trealase/isolamento & purificação , Fosfatase Alcalina/metabolismo , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Ativação Enzimática , Reativadores Enzimáticos , Escherichia coli/enzimologia , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Técnicas de Imunoadsorção , Peso Molecular , Fosforilação , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Trealose/metabolismoRESUMO
Several growth factors and mitogens have been shown to activate the proto-oncogene product Raf-1 protein kinase in murine fibroblasts, apparently through a direct agonist-stimulated tyrosine phosphorylation of the Raf-1 protein. We investigated the possibility that insulin could also activate the Raf-1 kinase, since its receptor also contains an intrinsic insulin-activated protein tyrosine kinase activity. In several cell lines expressing relatively large numbers of insulin receptors, insulin rapidly stimulated the phosphorylation of immunoreactive Raf-1 protein. In H35 cells, a line of well differentiated rat hepatoma cells, the effect of insulin was maximal by 6 min and at 7 nM insulin and occurred normally in cells virtually completely depleted of protein kinase C activity. The insulin-stimulated increase in Raf-1 protein phosphorylation occurred concurrently with a 3-fold increase in Raf-1 protein kinase activity. However, phosphoamino acid analysis showed that only phosphoserine and a trace of phosphothreonine were present in the Raf-1 protein after insulin stimulation of the cells. This was true even when investigated at shorter times (4 min) after insulin stimulation and despite the use of phosphotyrosine phosphatase inhibitors. We conclude that insulin can rapidly activate the Raf-1 kinase in some insulin-sensitive cell types but that this activation probably occurs through a mechanism distinct from direct phosphorylation of the Raf-1 protein by the insulin receptor protein tyrosine kinase.
Assuntos
Insulina/farmacologia , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Camundongos , Fosforilação , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas c-rafRESUMO
The serine/threonine kinase RAF-1 is phosphorylated in intact macrophages in response to CSF-1 at 37 degrees C The augmented phosphorylation of RAF-1 and a concomitant increase in RAF-1 associated serine/threonine kinase activity are kinetically later events than CSF-1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF-1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF-1 does not react with anti-phosphotyrosine antibodies. In contrast to CSF-1 induced protein tyrosine phosphorylation, RAF-1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF-1 with the CSF-1 receptor. Thus, while RAF-1 is not a direct substrate for the CSF-1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF-1 response.
Assuntos
Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Fosfotirosina , Fator de Crescimento Derivado de Plaquetas/farmacologia , Testes de Precipitina , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-raf , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Temperatura , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Interleukin 2 (IL-2) is a lymphokine, produced by T cells upon antigenic or mitogenic stimulation, that is a critical regulator of T-cell proliferation. Although the binding of IL-2 to its receptor has been well characterized, the molecular mechanisms by which IL-2 transmits its signal from the membrane to the interior of the cell are poorly understood. Like most other growth factors, IL-2 causes rapid phosphorylation of proteins within its target cells. Unlike many other growth factors, however, the known subunits of the IL-2 receptor lack tyrosine-specific kinase activity, and little is known about the kinases whose activities are regulated by IL-2. Here we show that IL-2 (but not IL-4) induces rapid phosphorylation of the p72-74 serine/threonine-specific kinase encoded by the c-Raf-1 protooncogene in an IL-2-dependent murine T-cell line, CTLL-2, and that this phosphorylation is associated with increased kinase activity in p72-74 Raf-1-containing immune complexes. The concentration dependence of IL-2-mediated elevations in Raf-1 kinase activity correlated well with IL-2-stimulated proliferation of CTLL-2 cells. Furthermore, much of the IL-2-stimulated phosphorylation of p72-74 Raf-1 occurred on tyrosines. To our knowledge, the Raf-1 kinase represents the first endogenous substrate of an IL-2-regulated tyrosine kinase to be identified.
Assuntos
Interleucina-2/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Interleucina-4/farmacologia , Cinética , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-raf , Proto-Oncogenes , Especificidade por Substrato , Linfócitos T Citotóxicos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Potent 4-anilido-substituted quinazolines which potently inhibit epidermal growth factor receptor (EGFR) kinase were prepared. Structure-activity relationship studies reveal high sensitivity to substitution at the aniline ring.
Assuntos
Compostos de Anilina/química , Receptores ErbB/antagonistas & inibidores , Quinazolinas/química , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Estaurosporina/farmacologia , Relação Estrutura-AtividadeRESUMO
The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.
Assuntos
Proteínas Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Ativação Enzimática , Camundongos , Mitógenos/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-raf , Transdução de Sinais , TransfecçãoRESUMO
A recently described tumor-derived glycoprotein, designated 90K, has been shown to have positive effects on the generation of cytotoxic effector cells (NK/LAK) from human PBMC. To determine the mechanism of these effects, we have examined the effects of 90K on cytokine production by human PBMC. A culture of normal PBMC with 90K alone did not result in IL-2 secretion; however, in coculture with suboptimal doses of ConA, 90K increased IL-2 secretion by PBMC. Coculture of PBMC with 90K and ConA also resulted in increased production of the cytokines IL-1, IL-6, GM-CSF, and TNF alpha. T cells depleted of accessory cells failed to respond to ConA alone, 90K alone, or the combination of ConA and 90K, suggesting that this protein does not have a direct effect on T cells. However, 90K alone was sufficient to induce cytokine production by unfractionated PBMC (IL-1, IL-6, GM-CSF, and TNF alpha) or by CD14-enriched PBMC (IL-1 and IL-6). In addition, expression of ICAM-1 was increased on a human monocytic cell line cultured with purified 90K in the absence of any other stimulus. This 90K-induced upregulation of ICAM-1 expression was accompanied by an increased accessory function of the monocytes, demonstrated by their ability to support ConA-induced activation of peripheral blood T cells. Based on the current data, we propose a model in which 90K activates accessory cells, resulting in the secretion of cytokines and the expression of adhesion molecules, which in turn act as costimulatory signals for T-cell activation. Activated T cells then produce cytokines such as IL-2, which lead to a more vigorous cell-mediated immune response to tumor cells and virus-infected cells. Thus, 90K shows promise as an immunotherapeutic reagent for diseases such as cancer and viral infection.