RESUMO
The NF-kappaB family of transcription factors functions broadly in the host control of immunoregulatory gene expression, inflammation, and apoptosis. Using Jurkat T cells engineered to inducibly express a transdominant repressor of IkappaBalpha, we examined the role of NF-kappaB in the regulation of cytokine and apoptotic gene expression. In this T cell model, as well as in primary T lymphocytes, expression of TNF-related apoptosis-inducing ligand (TRAIL) apoptotic signaling protein was dramatically down-regulated by inhibition of NF-kappaB binding activity. TRAIL acts through membrane death receptors to induce apoptosis of activated T lymphocytes and can be up-regulated by a variety of physiological and pharmacological inducers. However, regulation of TRAIL gene expression has not been defined. Treatment with TCR mimetics (PMA/ionomycin, PHA, and anti-CD3/CD28 Abs) resulted in a rapid increase in the expression of TRAIL mRNA and cell surface TRAIL protein. Induction of the transdominant repressor of IkappaBalpha dramatically down-regulated surface expression of TRAIL, indicating an essential role for NF-kappaB in the regulation of TRAIL. The induced expression of TRAIL was linked to a c-Rel binding site in the proximal TRAIL promoter at position -256 to -265; mutation of this site or an adjacent kappaB site resulted in a complete loss of the inducibility of the TRAIL promoter. The regulation of TRAIL expression by NF-kappaB may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis in T lymphocytes.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas I-kappa B , Glicoproteínas de Membrana/biossíntese , NF-kappa B/fisiologia , Linfócitos T/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Substituição de Aminoácidos , Anticorpos Monoclonais/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose , Antígenos CD28/imunologia , Complexo CD3/imunologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ionomicina/farmacologia , Células Jurkat , Glicoproteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fito-Hemaglutininas/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-rel/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
As with the neutrophil NADPH oxidase, the B lymphocyte NADPH oxidase consists of a membrane-bound flavocytochrome b and regulatory factors including Rac and the cytosolic phox protein triad p67phox, p47phox, and p40phox. Here we demonstrate by phosphoamino acid analysis and the use of the potent PKC inhibitor GFX that, in response to stimulation of B lymphocytes with sodium orthovanadate and H(2)O(2), the p40phox component of the cytosolic phox triad is selectively phosphorylated on serine and threonine residues by a PKC-type protein kinase. The pattern of p40phox phosphorylation was closely related to the kinetics of tyrosine phosphorylation of PKC-delta, the main PKC isotype of B lymphocytes. Blocking H(2)O(2)-dependent tyrosine phosphorylation of PKC by genistein resulted in inhibition of p40phox phosphorylation. The correlation between the tyrosine phosphorylation of PKC-delta and the serine/threonine phosphorylation of p40phox, together with the inhibition of p40phox phosphorylation by rottlerin, a selective inhibitor of PKC-delta, makes the activated PKC-delta a likely candidate in the process of the oxidant-dependent phosphorylation of p40phox in B cells.
Assuntos
Linfócitos B/metabolismo , Oxidantes/farmacologia , Fosfoproteínas/metabolismo , Acetofenonas/farmacologia , Linfócitos B/efeitos dos fármacos , Benzopiranos/farmacologia , Fracionamento Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Peróxido de Hidrogênio/farmacologia , Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Substâncias Macromoleculares , Maleimidas/farmacologia , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Fosforilação , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C-delta , Vanadatos/farmacologiaRESUMO
The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).
Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Fragmentos de Peptídeos/fisiologia , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Cromatografia em Gel , Citosol/enzimologia , Dimerização , Células HL-60 , Humanos , Peso Molecular , Nêutrons , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Espalhamento de Radiação , SoluçõesRESUMO
The human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4(+) T lymphocytes, and is also associated with a neurological demyelinating disease, tropical spastic paraparesis. The oncogenic potential of HTLV-1 resides in the 353-aa, 40-kDa viral Tax oncoprotein, a positive regulator of viral gene transcription. A novel member of the interferon regulatory factor (IRF) family of transcription factors, IRF-4, was shown to be constitutively produced in HTLV-1-infected cells. IRF-4 is transiently expressed in anti-CD3 and PMA/ionomycin-stimulated T lymphocytes but not in continuous non-Tax-expressing T cell lines. In transient coexpression assays, HTLV-1 Tax protein induced the 1. 2-kb IRF-4 promoter, indicating that Tax functions as an indirect trans-activator of the IRF-4 gene. Furthermore, IRF-4 levels in HTLV-1-infected cells appear to be proportional to the level of Tax expression, suggesting a role for IRF-4 in T cell transformation. In an effort to further characterize IRF-4 function, we identified a novel interaction between IRF-4 and FKBP52, a 59-kDa member of the immunophilin family with peptidyl-prolyl isomerase activity (PPIase). IRF-4-FKBP52 association inhibited the interaction between IRF-4 and its DNA-binding partner PU.1, as well as the trans-activation function of IRF-4/PU.1. FKBP52 association resulted in a structural modification of IRF-4, detectable by immunoblot analysis and by IRF-4 partial proteolysis. These results demonstrate a novel posttranslational mechanism of transcriptional control, mediated through the interaction of an immunophilin with a transcriptional regulator.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Linfócitos T/virologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Linhagem Celular Transformada , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Produtos do Gene tax/genética , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/metabolismo , Humanos , Fatores Reguladores de Interferon , Ativação Linfocitária , Regiões Promotoras Genéticas , Conformação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.
Assuntos
Antígenos Nucleares , Linfócitos B/imunologia , Linfócitos B/metabolismo , DNA Helicases , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Sítios de Ligação/genética , Células COS , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Complementar/metabolismo , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/genética , Imunofluorescência , Humanos , Autoantígeno Ku , NADPH Oxidases , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67(phox) (phagocyte oxidase), p47(phox), and p40(phox), which translocate to the membrane upon activation. In a previous paper, we reported that p40(phox) undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531-539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40(phox), we show that p40(phox) is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40(phox) in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40(phox). Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40(phox) are phosphorylated during activation of the NADPH oxidase.