RESUMO
The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.
Assuntos
Bovinos/genética , Proteínas de Ligação a DNA/genética , Evolução Molecular , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Cromossomo Y/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos , Primers do DNA , Hibridização in Situ Fluorescente , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Pseudogenes/genética , Recombinação Genética/genética , Análise de Sequência de DNA , Proteína da Região Y Determinante do Sexo , Especificidade da EspécieRESUMO
Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95(vav) and phospholipase C-gamma isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Proteínas de Membrana/fisiologia , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Células HeLa , Humanos , Isoenzimas/metabolismo , Células Jurkat , Células K562 , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fosfolipase C gama , Fosfoproteínas/metabolismo , Fosforilação , Testes de Precipitina , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismo , Tirosina/fisiologia , Proteína-Tirosina Quinase ZAP-70 , Domínios de Homologia de src/imunologiaRESUMO
The mitogen-activated protein kinase (MAPK) p38 modulates a variety of cellular functions, including proliferation, differentiation and cell death. However, we report here a novel function for p38, i.e. the regulation of cytotoxic lymphocyte-mediated cytotoxicity. Stimulation of NK cells by either cross-linking of their FcgammaRIII receptors or by binding to NK-sensitive target cells induces the phosphorylation and activation of p38, and also of its upstream regulators MKK3/MKK6. Pharmacologic analyses suggest that Src-family and Syk-family protein tyrosine kinases couple the NK cell surface receptors to p38 activation. The role of p38 in the cytotoxic function of NK cells was tested by treatment of NK cells with the cell-permeable, p38-specific inhibitor SB203580. Interestingly, exposure to the drug reduced both antibody-dependent cellular cytotoxicity and natural cytotoxicity, but maximal inhibitory concentrations resulted in only partial inhibition. Collectively, these results suggest that the p38 MAPK pathway is stimulated during the development of NK cell-mediated cytotoxicity and that efficient killing is influenced by both p38-dependent and p38-independent pathways. More broadly, this study identifies the regulation of cell-mediated killing as a novel role for p38 in cytotoxic lymphocytes.
Assuntos
Citotoxicidade Imunológica/fisiologia , Células Matadoras Naturais/enzimologia , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Androstadienos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/fisiologia , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Maleimidas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologia , Piridinas/farmacologia , Receptores de IgG/imunologia , Quinase Syk , Wortmanina , Proteínas Quinases p38 Ativadas por Mitógeno , Quinases da Família src/fisiologiaRESUMO
Methane exchange with the atmosphere was measured during three seasons at the Rooney Road landfill in Jefferson County, CO. Substantial spatial and temporal variability in exchange rates were observed. Mean fluxes to the atmosphere were 534, 1290, and 538 mg CH4/m2/day, respectively, in the fall of 1994, winter of 1994-1995, and summer of 1995. Median fluxes were 12.42, 8.62, and 5.65 mg CH4/m2/day, respectively, during those seasons. Forty-three of 177 measurements had small negative fluxes, suggesting methanotrophic activity in the landfill cover soils. Despite probable methanotrophic activity in cover soils, landfills without gas collection systems may emit substantial CH4 to the atmosphere, with large spatial and seasonal variability.
Assuntos
Poluentes Atmosféricos , Metano , Eliminação de Resíduos , Poluentes Atmosféricos/análise , Clima , Colorado , Monitoramento Ambiental , Metano/análise , Metano/metabolismo , Estações do Ano , Solo , TemperaturaRESUMO
Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcgammaRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-gamma. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and "natural cytotoxicity," thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Proteínas de Membrana , Proteínas de Transporte/fisiologia , Linhagem Celular , Humanos , Ativação Linfocitária , Fosfoproteínas/fisiologia , Fosfolipases Tipo C/fisiologiaRESUMO
The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell-mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.
Assuntos
Proteínas de Ciclo Celular , Citotoxicidade Imunológica , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células Matadoras Naturais/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Humanos , Células Matadoras Naturais/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-vav , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/metabolismo , Proteínas rac de Ligação ao GTPRESUMO
Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Precursores Enzimáticos/metabolismo , Células Matadoras Naturais/enzimologia , Proteínas Tirosina Quinases/metabolismo , Animais , Cálcio/metabolismo , Genes MHC Classe I , Fosfatos de Inositol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Cinética , Camundongos , Transdução de Sinais , Quinase Syk , Transfecção , Células Tumorais CultivadasRESUMO
Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Proteínas Monoméricas de Ligação ao GTP , Proteínas Tirosina Fosfatases/metabolismo , Receptores Imunológicos/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Complexo Principal de Histocompatibilidade , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores Fc/metabolismo , Transdução de Sinais , Tirosina/metabolismoRESUMO
Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.
Assuntos
Antígenos CD/imunologia , Células Clonais/imunologia , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Glicoproteínas de Membrana/imunologia , Sequência de Aminoácidos , Animais , Citotoxicidade Imunológica , Precursores Enzimáticos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/fisiologia , Ativação Linfocitária , Sarcoma de Mastócitos/patologia , Camundongos , Dados de Sequência Molecular , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Fosfatidilinositol 3-Quinases , Fosfolipase C gama , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais/fisiologia , Quinase Syk , Células Tumorais Cultivadas , Fosfolipases Tipo C/fisiologia , Proteína-Tirosina Quinase ZAP-70 , Quinases da Família src/fisiologiaRESUMO
Ligation of the Fc gamma R on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that Fc gamma R ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early Fc gamma R-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the zeta chain of the Fc gamma R complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-gamma 2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-gamma 2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling Fc gamma R stimulation to critical downstream signaling events. Taken together, our results suggest not only that Fc gamma R stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade.
Assuntos
Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais , Linhagem Celular , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/virologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Fosfolipase C gama , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Recombinantes/metabolismo , Quinase Syk , Fosfolipases Tipo C/metabolismo , Vaccinia virus/genéticaRESUMO
Selective cell death plays a critical role in the development of the immune repertoire and in the elimination of target cells expressing foreign Ags. The apoptosis induced by ligation of the Fas Ag, a member of the TNFR/nerve growth factor receptor superfamily, contributes to both of these modes of cell loss. However, in spite of the molecular cloning of the Fas Ag and the identification of a specific cytoplasmic domain required for its function, it remains unclear as to which Fas-induced second messengers mediate the development of programmed cell death. We, therefore, evaluated Fas-initiated signal transduction in susceptible cell types. We determined that Fas ligation induces the rapid tyrosine phosphorylation of multiple cellular proteins. These phosphorylation events occur within 1 min and decline toward baseline by 30 min. In addition, Fas ligation increases the in vitro protein kinase activity of the tyrosine phosphorylated proteins. Pharmacologic inhibitors of protein tyrosine kinases block, in a concentration-dependent manner, Fas-induced DNA fragmentation and prolong cell survival. These results suggest that protein tyrosine kinase activation is an early and obligatory signal in Fas-induced apoptosis.
Assuntos
Antígenos de Superfície/fisiologia , Apoptose , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/fisiologia , Linhagem Celular , Ativação Enzimática , Humanos , Técnicas In Vitro , Fosfoproteínas/metabolismo , Fosfotirosina , Agregação de Receptores , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/metabolismo , Receptor fasRESUMO
Interleukin-2 (IL-2) potently stimulates natural killer (NK) cell proliferation and cytotoxic function. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p56lck. The present studies indicate that IL-2 induces a rapid (< or = 1 min) increase in the catalytic activity of p56lck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56lck itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56lck containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p56lck. These results suggest that p56lck serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56lck.
Assuntos
Interleucina-2/fisiologia , Células Matadoras Naturais/fisiologia , Proteínas Tirosina Quinases/metabolismo , Células Cultivadas , Ativação Enzimática , Humanos , Técnicas In Vitro , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotirosina , Transdução de Sinais/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.