RESUMO
The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms that lead to lung-associated macrophages' functional and developmental adaptation remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal ß-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonary macrophage adaptation. Employing single-cell transcriptomics, high-dimensional imaging and flow cytometric characterization paired with in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of apolipoprotein E (ApoE)-dependent monocyte-derived alveolar macrophages (ApoE+CD11b+ AMs). ApoE+CD11b+ AMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic, highly phagocytic and produced large amounts of interleukin-6 upon restimulation. Functional differences were cell intrinsic, and myeloid cell-specific ApoE ablation inhibited Ly6c+ monocyte to ApoE+CD11b+ AM differentiation dependent on macrophage colony-stimulating factor secretion, promoting ApoE+CD11b+ AM cell death and thus impeding ApoE+CD11b+ AM maintenance. In vivo, ß-glucan-elicited ApoE+CD11b+ AMs limited the bacterial burden of Legionella pneumophilia after infection and improved the disease outcome in vivo and ex vivo in a murine lung fibrosis model. Collectively these data identify ApoE+CD11b+ AMs generated upon environmental cues, under the control of ApoE signaling, as an essential determinant for lung adaptation enhancing tissue resilience.
Assuntos
Apolipoproteínas E , Lectinas Tipo C , Macrófagos Alveolares , Camundongos Endogâmicos C57BL , beta-Glucanas , Animais , Camundongos , Adaptação Fisiológica/imunologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Antígeno CD11b/metabolismo , Diferenciação Celular , Lectinas Tipo C/metabolismo , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismoRESUMO
Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2-/- embryonic stem cells failed to maintain gamma-irradiation-induced arrest in the G2 phase of the cell cycle. Chk2-/- thymocytes were resistant to DNA damage-induced apoptosis. Chk2-/- cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.
Assuntos
Dano ao DNA , Interfase , Proteínas Nucleares , Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Quinase do Ponto de Checagem 2 , Proteínas de Ligação a DNA , Fase G1 , Fase G2 , Raios gama , Regulação da Expressão Gênica , Marcação de Genes , Genes Supressores de Tumor , Genes p53 , Humanos , Camundongos , Fosforilação , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Células-Tronco/citologia , Células-Tronco/metabolismo , Linfócitos T/citologia , Transcrição Gênica , Proteínas Supressoras de TumorRESUMO
Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of ß-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3ß to oncogenic CARMA1. Recruitment of the ß-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of ß-catenin. The ß-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated ß-catenin expression. In line, ß-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased ß-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, ß-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that ß-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and ß-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Carcinogênese , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/patologia , Mutação , Estabilidade Proteica , Fatores de Transcrição TCF/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Hypoxia-inducible factor 1α (Hif1α) is a key regulator of cellular adaptation and survival under hypoxic conditions. In pancreatic ductal adenocarcinoma (PDAC), it has been recently shown that genetic ablation of Hif1α accelerates tumour development by promoting tumour-supportive inflammation in mice, questioning its role as the key downstream target of many oncogenic signals of PDAC. Likely, Hif1α has a context-dependent role in pancreatic tumorigenesis. To further analyse this, murine PDAC cell lines with reduced Hif1α expression were generated using shRNA transfection. Cells were transplanted into wild-type mice through orthotopic or portal vein injection in order to test the in vivo function of Hif1α in two major tumour-associated biological scenarios: primary tumour growth and remote colonization/metastasis. Although Hif1α protects PDAC cells from stress-induced cell deaths in both scenarios-in line with the general function Hif1α-its depletion leads to different oncogenic consequences. Hif1α depletion results in rapid tumour growth with marked hypoxia-induced cell death, which potentially leads to a persistent tumour-sustaining inflammatory response. However, it simultaneously reduces tumour colonization and hepatic metastases by increasing the susceptibility to anoikis induced by anchorage-independent conditions. Taken together, the role of Hif1α in pancreatic tumorigenesis is context-dependent. Clinical trials of Hif1α inhibitors need to take this into account, targeting the appropriate scenario, for example palliative vs adjuvant therapy.
RESUMO
Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-XL/-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34(+) cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Síndromes Mielodisplásicas/tratamento farmacológico , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Células-Tronco/efeitos dos fármacos , Sulfonamidas/farmacologia , Células Cultivadas , Humanos , Síndromes Mielodisplásicas/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/análise , Piperazinas/farmacologiaRESUMO
T lymphocyte non-Hodgkin's lymphoma (T-NHL) represents an aggressive and largely therapy-resistant subtype of lymphoid malignancies. As deregulated apoptosis is a frequent hallmark of lymphomagenesis, we analyzed gene expression profiles and protein levels of primary human T-NHL samples for various apoptotic regulators. We identified the apoptotic regulator MCL-1 as the only pro-survival BCL-2 family member to be highly expressed throughout all human T-NHL subtypes. Functional validation of pro-survival protein members of the BCL-2 family in two independent T-NHL mouse models identified that the partial loss of Mcl-1 significantly delayed T-NHL development in vivo. Moreover, the inducible reduction of MCL-1 protein levels in lymphoma-burdened mice severely impaired the continued survival of T-NHL cells, increased their susceptibility to chemotherapeutics and delayed lymphoma progression. Lymphoma viability remained unaffected by the genetic deletion or pharmacological inhibition of all alternative BCL-2 family members. Consistent with a therapeutic window for MCL-1 treatment within the context of the whole organism, we observed an only minimal toxicity after systemic heterozygous loss of Mcl-1 in vivo. We conclude that re-activation of mitochondrial apoptosis by blockade of MCL-1 represents a promising therapeutic strategy to treat T-cell lymphoma.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose , Linfoma de Células T/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/análise , Animais , Proteínas Reguladoras de Apoptose/análise , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Linfoma de Células T/patologia , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
The development of lymphomas and leukemias is frequently caused by chromosomal translocations that deregulate cellular pathways of differentiation, proliferation or survival. The molecules that are involved in these aberrations provide rational targets for selective drug therapies. Recently, several disease specific translocations have been identified in human MALT lymphoma. These aberrations either upregulate the expression of BCL10 or MALT1 or induce the formation of API2-MALT1 fusion proteins. Genetic and biochemical experiments identified BCL10 and MALT1 as central components of an oligomerization-ubiquitinylation-phosphorylation cascade that activates the transcription factor NF-kappaB in response to antigen receptor ligation. Deregulation of the signaling cascade is directly associated with antigen independent MALT lymphoma growth. Here we provide an overview of the physiological and pathological functions of BCL10/MALT1 signal transduction and discuss the potential of this pathway as a drug target.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Caspases/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Proteína 10 de Linfoma CCL de Células B , Inibidores de Caspase , Humanos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/antagonistas & inibidores , Transdução de Sinais/fisiologiaRESUMO
The membrane currents of chicken embryo fibroblasts (CEFs) transformed by Rous sarcoma virus (RSV) were compared with the currents of their nontransformed counterparts by using the whole-cell patch-clamp technique. In nontransformed CEFs, the main membrane current is a delayed outward K+ current that is sensitive to tetraethylammonium ion but insensitive to 4-aminopyridine. This K+ current is almost independent of the intracellular Ca2+ concentration and becomes completely inactivated at positive membrane potentials with a time constant of about 10 s at +30 mV. In contrast, transformed CEFs exhibit a noninactivating K+ current that strongly depends on the intracellular Ca2+ concentration. This Ca(2+)-dependent K+ current is blocked by the scorpion toxin charybdotoxin with an IC50 value of 19 nM, whereas the K+ current of normal CEFs is insensitive to charybdotoxin (up to 300 nM). The K+ current properties of transformed CEFs were also found after microinjection of purified, enzymatically active pp60v-src into normal CEFs but not after infection of CEFs with the Rous-associated virus RAV5, which lacks the v-src oncogene. Our results suggest that the oncogene product pp60v-src modulates existing K+ channel proteins, leading to profound electrophysiological and pharmacological alterations of the K+ current properties in RSV-transformed CEFs. Furthermore, our experiments identify for the first time K+ channels as possible substrates of pp60v-src.
Assuntos
Vírus do Sarcoma Aviário/genética , Transformação Celular Neoplásica , Proteína Oncogênica pp60(v-src)/metabolismo , Canais de Potássio/fisiologia , Animais , Apamina/farmacologia , Células Cultivadas , Charibdotoxina , Embrião de Galinha , Venenos Elapídicos/farmacologia , Fibroblastos/fisiologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Microinjeções , Proteína Oncogênica pp60(v-src)/administração & dosagem , Canais de Potássio/efeitos dos fármacos , Venenos de Escorpião/farmacologiaRESUMO
The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse-chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células 3T3 , Animais , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Ligases/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/metabolismoRESUMO
Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.
Assuntos
Apoptose , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Células 3T3 , Androstadienos/farmacologia , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Immunoblotting , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Fatores de Tempo , Transfecção , Wortmanina , Proteína bcl-XRESUMO
Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101-/- embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101-/- embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21(WAF-1/CIP-1), was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101-/- embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Perda do Embrião/metabolismo , Proteínas Nucleares , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal , Endoderma/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Expressão Gênica , Marcação de Genes , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Sistema Nervoso Central/anormalidades , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Defeitos do Tubo Neural/fisiopatologia , Receptores de Antígenos/metabolismo , Animais , Anisomicina/farmacologia , Formação de Anticorpos/imunologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Apoptose/efeitos da radiação , Proteína 10 de Linfoma CCL de Células B , Linfócitos B/imunologia , Divisão Celular/imunologia , Sistema Nervoso Central/fisiologia , Cisplatino/farmacologia , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Genes Letais/imunologia , Imunidade Celular/imunologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , Proteínas de Neoplasias/metabolismo , Defeitos do Tubo Neural/imunologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Receptores de Antígenos/imunologia , Transdução de Sinais/imunologia , Estaurosporina/farmacologia , Linfócitos T/imunologiaRESUMO
The hexosamine pathway provides UDP-N:-acetylhexosamine donor substrates used in cytosolic and Golgi-mediated glycosylation of proteins and for formation of glycosylphosphatidylinositol (GPI) anchors, which tether proteins to the outer plasma membrane. We have recently identified the murine glucosamine-6-phosphate (GlcN6P) acetyltransferase, EMeg32, as a developmentally regulated enzyme on the route to UDP-N:-acetylglucosamine (UDP-GlcNAc). Here we describe embryos and cells that have the EMeg32 gene inactivated by homologous recombination. Homozygous mutant embryos die at around embryonic day (E) 7.5 with a general proliferative delay of development. In vitro differentiated EMeg32(-/-) ES cells show reduced proliferation. Mouse embryonic fibroblasts (MEFs) deficient for EMeg32 exhibit defects in proliferation and adhesiveness, which could be complemented by stable re-expression of EMeg32 or by nutritional restoration of intracellular UDP-GlcNAc levels. Reduced UDP-GlcNAc levels predominantly translated into decreased O-GlcNAc modifications of cytosolic and nuclear proteins. Interestingly, growth-impaired EMeg32(-/-) MEFs withstand a number of apoptotic stimuli and express activated PKB/AKT. Thus, EMeg32-dependent UDP-GlcNAc levels influence cell cycle progression and susceptibility to apoptotic stimuli.
Assuntos
Acetiltransferases/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Proteínas de Membrana/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Acetiltransferases/genética , Animais , Apoptose , Divisão Celular , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Perda do Embrião/enzimologia , Perda do Embrião/metabolismo , Embrião de Mamíferos , Desenvolvimento Embrionário e Fetal/genética , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Glucosamina 6-Fosfato N-Acetiltransferase , Glicosilação , Glicosilfosfatidilinositóis/biossíntese , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da PolimeraseRESUMO
Bcl10 and MALT1, products of distinct chromosomal translocations in mucosa-associated lymphoid tissue lymphoma, cooperate in activating NF-kappaB. Mice lacking Bcl10 demonstrate severe immunodeficiency associated with failure of lymphocytes to activate nuclear factor kappaB (NF-kappaB) in response to antigen receptor stimulation and protein kinase C activation. We characterize Bimp1, a new signaling protein that binds Bcl10 and activates NF-kappaB. Bimp1-mediated NF-kappaB activation requires Bcl10 and IkappaB kinases, indicating that Bimp1 acts upstream of these mediators. Bimp1, Bcl10, and MALT1 form a ternary complex, with Bcl10 bridging the Bimp1/MALT1 interaction. A dominant negative Bimp1 mutant inhibits NF-kappaB activation by anti-CD3 ligation, phorbol ester, and protein kinase C expression. These results suggest that Bimp1 links surface receptor stimulation and protein kinase C activation to Bcl10/MALT1, thus leading to NF-kappaB induction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfoma de Zona Marginal Tipo Células B , NF-kappa B/biossíntese , Proteínas de Neoplasias/fisiologia , Núcleosídeo-Fosfato Quinase/fisiologia , Proteína Quinase C/fisiologia , Sequência de Aminoácidos , Proteína 10 de Linfoma CCL de Células B , Caspases , DNA Complementar/análise , Ativação Enzimática , Guanilato Quinases , Humanos , Quinase I-kappa B , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
PTEN is a tumor suppressor with sequence homology to protein tyrosine phosphatases and the cytoskeletal protein tensin. mPTEN-mutant mouse embryos display regions of increased proliferation. In contrast, mPTEN-deficient immortalized mouse embryonic fibroblasts exhibit decreased sensitivity to cell death in response to a number of apoptotic stimuli, accompanied by constitutively elevated activity and phosphorylation of protein kinase B/Akt, a crucial regulator of cell survival. Expression of exogenous PTEN in mutant cells restores both their sensitivity to agonist-induced apoptosis and normal pattern of PKB/Akt phosphorylation. Furthermore, PTEN negatively regulates intracellular levels of phosphatidylinositol (3,4,5) trisphosphate in cells and dephosphorylates it in vitro. Our results show that PTEN may exert its role as a tumor suppressor by negatively regulating the PI3'K/PKB/Akt signaling pathway.