RESUMO
NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/patologia , Linfoma de Zona Marginal Tipo Células B/patologia , Células Mieloides/patologia , NF-kappa B/metabolismo , Receptores Notch/metabolismo , Animais , Linfócitos B/metabolismo , Transformação Celular Neoplásica/metabolismo , Feminino , Humanos , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , NF-kappa B/genética , Receptores Notch/genética , Transdução de SinaisRESUMO
The role of TRAF2 and TRAF5 in TNFα-induced NF-κB activation has become complicated owing to the accumulation of conflicting data. Here, we report that 7-day-old TRAF2-knockout (KO) and TRAF2 TRAF5 double KO (TRAF2/5-DKO) mice exhibit enhanced canonical IκB kinase (IKK) and caspase-8 activation in spleen and liver, and that subsequent knockout of TNFα suppresses the basal activity of caspase-8, but not of IKK. In primary TRAF2 KO and TRAF2/5-DKO cells, TNFα-induced immediate IKK activation is impaired, whereas delayed IKK activation occurs normally; as such, owing to elevated basal and TNFα-induced delayed IKK activation, TNFα stimulation leads to significantly increased induction of a subset of NF-κB-dependent genes in these cells. In line with this, both TRAF2 KO and TRAF2/5-DKO mice succumb to a sublethal dose of TNFα owing to increased expression of NF-κB target genes, diarrhea and bradypnea. Notably, depletion of IAP1 and IAP2 (also known as BIRC2 and BIRC3, respectively) also results in elevated basal IKK activation that is independent of autocrine TNFα production and that impairs TNFα-induced immediate IKK activation. These data reveal that TRAF2, IAP1 and IAP2, but not TRAF5, cooperatively regulate basal and TNFα-induced immediate IKK activation.
Assuntos
Caspase 8/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/genética , Proteína 3 com Repetições IAP de Baculovírus , Células Cultivadas , Quimiocina CCL5/metabolismo , Ativação Enzimática/genética , Quinase I-kappa B/genética , Proteínas Inibidoras de Apoptose/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/genética , Fator de Necrose Tumoral alfa/genética , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.
Assuntos
Anoikis/fisiologia , Proteínas de Transporte/metabolismo , Neoplasias do Colo/metabolismo , Inflamação/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Ratos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OLs). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified BM CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single-cell RNA-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.
Assuntos
Reabsorção Óssea , Mieloma Múltiplo , Osteólise , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular/fisiologia , Cistatina M/metabolismo , Humanos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Osteoclastos/metabolismo , Osteólise/genética , Osteólise/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ligante RANK/genética , Ligante RANK/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismoRESUMO
CD40 plays an important role in immune responses by activating the c-Jun N-terminal protein kinase (JNK) and NF-κB pathways; however, the precise mechanisms governing the spatiotemporal activation of these two signaling pathways are not fully understood. Here, using four different TRAF2-deficient cell lines (A20.2J, CH12.LX, HAP1, and mouse embryonic fibroblasts [MEFs]) reconstituted with wild-type or phosphorylation mutant forms of TRAF2, along with immunoprecipitation, immunoblotting, gene expression, and immunofluorescence analyses, we report that CD40 ligation elicits TANK-binding kinase 1 (TBK1)-mediated phosphorylation of TRAF2 at Ser-11. This phosphorylation interfered with the interaction between TRAF2's RING domain and membrane phospholipids and enabled translocation of the TRAF2 complex from CD40 to the cytoplasm. We also observed that this cytoplasmic translocation is required for full activation of the JNK pathway and the secondary phase of the NF-κB pathway. Moreover, we found that in the absence of Ser-11 phosphorylation, the TRAF2 RING domain interacts with phospholipids, leading to the translocation of the TRAF2 complex to lipid rafts, resulting in its degradation and activation of the noncanonical NF-κB pathway. Thus, our results provide new insights into the CD40 signaling mechanisms whereby Ser-11 phosphorylation controls RING domain-dependent subcellular localization of TRAF2 to modulate the spatiotemporal activation of the JNK and NF-κB pathways.
Assuntos
Antígenos CD40/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Citoplasma/metabolismo , Citosol/metabolismo , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Transdução de Sinais/fisiologia , Fator 2 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Drug resistance remains the key problem in cancer treatment. It is now accepted that each myeloma patient harbors multiple subclones and subclone dominance may change over time. The coexistence of multiple subclones with high or low chromosomal instability (CIN) signature causes heterogeneity and drug resistance with consequent disease relapse. In this study, using a tandem affinity purification-mass spectrometry (TAP-MS) technique, we found that NEK2, a CIN gene, was bound to the deubiquitinase USP7. Binding to USP7 prevented NEK2 ubiquitination resulting in NEK2 stabilization. Increased NEK2 kinase levels activated the canonical NF-κB signaling pathway through the PP1α/AKT axis. Newly diagnosed myeloma patients with activated NF-κB signaling through increased NEK2 activity had poorer event-free and overall survivals based on multiple independent clinical cohorts. We also found that NEK2 activated heparanase, a secreted enzyme, responsible for bone destruction in an NF-κB-dependent manner. Intriguingly, both NEK2 and USP7 inhibitors showed great efficacy in inhibiting myeloma cell growth and overcoming NEK2-induced and -acquired drug resistance in xenograft myeloma mouse models.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Glucuronidase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Modelos Biológicos , Mieloma Múltiplo/patologia , NF-kappa B/metabolismo , Quinases Relacionadas a NIMA/antagonistas & inibidores , Prognóstico , Ligação Proteica , Estabilidade Proteica , Transdução de Sinais , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Ligante de CD40/fisiologia , Caspase 8/metabolismo , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Doença de Hodgkin/metabolismo , Humanos , Células Jurkat , Camundongos Knockout , Dados de Sequência Molecular , Proteólise , UbiquitinaçãoRESUMO
The death receptor (DR) ligand TRAIL is being evaluated in clinical trials as an anti-cancer agent; however, many studies have found that TRAIL also enhances tumor progression by activating the NF-κB pathway in apoptosis-resistant cells. Although RIP1, cFLIP and caspase-8 have been implicated in TRAIL-induced JNK and NF-κB activation, underlying mechanisms are unclear. By examining the kinetics of pathway activation in TRAIL-sensitive lymphoma cells wild-type or deficient for RIP1, TRAF2, cIAP1/2 or HOIP, we report here that TRAIL induces two phases of JNK and NF-κB activation. The early phase is activated by TRAF2- and cIAP1-mediated ubiquitination of RIP1, whereas the delayed phase is induced by caspase-dependent activation of MEKK1 independent of RIP1 and TRAF2 expression. cFLIP overexpression promotes the early phase but completely suppresses the delayed phase of pathway activation in lymphoma cells, whereas Bcl-2 overexpression promotes both the early and delayed phases of the pathways. In addition, stable overexpression of cFLIP in RIP1- or TRAF2-deficient cells confers resistance to apoptosis, but fails to mediate NF-κB activation. HOIP is not essential for, but contributes to, TRAIL-induced NF-κB activation in cFLIP-overexpressing cells. These findings not only elucidate details of the mechanisms underlying TRAIL-induced JNK and NF-κB activation, but also clarify conflicting reports in the field.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína 3 com Repetições IAP de Baculovírus , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Células Jurkat , MAP Quinase Quinase Quinase 1/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator 2 Associado a Receptor de TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein-Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.
Assuntos
NF-kappa B/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Regulação para Baixo/fisiologia , Células HEK293 , Humanos , Interleucina-8/metabolismo , Domínios RING Finger/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteínas da Matriz ViralRESUMO
TNFα is a pleotropic cytokine that plays a central role in the inflammatory response by activating the NF-κB signaling pathway, and is targeted in a range of chronic inflammatory diseases, underscoring the therapeutic importance of understanding its underlying molecular mechanisms. Although K63-linked ubiquitination of RIP1 by TRAF2/5 and cIAP1/2 was thought to serve as a scaffold to activate the NF-κB pathway, the recent accumulation of conflicting results has challenged the necessity of these proteins in NF-κB activation. In addition, several serine/threonine kinases have been implicated in TNFα-induced IKK activation; however, the targeted disruption of these kinases had no effect on transient IKK activation. The recent discovery of RIP1-dependent and -independent activation of the early and delayed phases of IKK and TRAF2 phosphorylation-dependent activation of the prolonged phase of IKK offers a reconciliatory model for the interpretation of contradictory results in the field. Notably, the TNFα-induced inflammatory response is not exclusively controlled by the NF-κB pathway but is subject to regulatory crosstalk between NF-κB and other context-dependent pathways. Thus further elucidation of these spatiotemporally-coordinated signaling mechanisms has the potential to provide novel molecular targets and therapeutic strategies for NF-κB intervention.
Assuntos
Quinase I-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/química , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismoRESUMO
Tumor necrosis factor alpha (TNF-α)-induced NF-κB activation has been believed to depend on TRAF2- and cIAP1-mediated RIP1 ubiquitination. However, recent findings have challenged the notion that these proteins play essential roles in NF-κB activation. Here, by assessing the kinetics and amplitude of IκB kinase (IKK) activation, we report that TNF-α-induced immediate and robust activation of IKK requires K63-linked and linearly linked ubiquitination of RIP1 and that in the absence of RIP1 expression, TRAF2 and cIAP1 cooperatively induce delayed IKK activation by recruiting LUBAC to TNFR1. Knockdown of HOIP (a component of LUBAC) in RIP1-deficient cells completely impairs the recruitment and activation of IKK but does not affect K63-linked ubiquitination of TRAF2 and recruitment of TAK1 to TNFR1, suggesting that the K63-linked ubiquitin chain is not capable of recruiting IKK in vivo. We also demonstrate that TRAF2 and cIAP1 together, but not either one alone, directly catalyze linearly linked ubiquitination of RIP1. Importantly, in embryonic hepatocytes, TNF-α activates NF-κB through a RIP1-independent pathway. Thus, our findings clarify molecular details of this important signaling mechanism by providing evidence for the existence of two phases of IKK activation: the immediate phase, induced by TRAF2/cIAP1-mediated ubiquitination of RIP1, and the delayed phase, activated by TRAF2/cIAP1-dependent recruitment of LUBAC.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Quinase I-kappa B/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular , Ativação Enzimática , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Hepatócitos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Ligação Proteica , Transporte Proteico , Proteólise , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismo , UbiquitinaçãoRESUMO
NF-κB transcription factors are central regulators of inflammation and when dysregulated contribute to malignant transformation. IκB kinase ε (IKKε; IKKi, encoded by IKBKE) is a breast oncogene that is amplified in 30% of breast cancers and drives transformation in an NF-κB-dependent manner. Here we demonstrate that IKKε interacts with and phosphorylates tumor necrosis factor receptor-associated factor 2 (TRAF2) at Ser11 in vitro and in vivo. This activity promotes Lys63-linked TRAF2 ubiquitination and NF-κB activation and is essential for IKKε transformation. Breast cancer cells that depend on IKKε expression for survival are also dependent on TRAF2. This work defines TRAF2 phosphorylation to be one key effector of IKKε-induced mammary epithelial cell transformation.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Quinase I-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Sequência de Aminoácidos , Animais , Mama/citologia , Mama/imunologia , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/imunologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Enzima Desubiquitinante CYLD , Regulação para Baixo , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Células HEK293 , Humanos , Quinase I-kappa B/imunologia , Camundongos , NF-kappa B/imunologia , Células NIH 3T3 , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Fator 2 Associado a Receptor de TNF/química , Fator 2 Associado a Receptor de TNF/imunologia , UbiquitinaçãoRESUMO
Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.
Assuntos
Apoptose , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Estresse Oxidativo , Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Técnicas de Inativação de Genes , Células HeLa , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Immunoblotting , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Mutação , NF-kappa B/genética , Células NIH 3T3 , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genéticaRESUMO
Members of the nuclear factor-κB (NF-κB) family of transcription factors play critical roles in regulating the expression of genes whose products are involved in inflammation, the immune response, cell proliferation, and the suppression of both death receptor- and stress-induced apoptosis. Abnormal NF-κB activation has been observed in various inflammatory diseases and many types of cancers. Gene knockout studies have clearly demonstrated that most of the physiologically relevant stimuli that activate NF-κB converge on inhibitor of κB kinase (IKK). Although the mechanism by which IKK activates NF-κB is well established, the upstream signaling mechanisms-those that underlie IKK activation by IKK kinases (IKK-Ks)-are not yet fully understood. The current belief is that members of the TNF receptor-associated factor (TRAF) family function as ubiquitin E3 ligases that catalyze non-canonical polyubiquitination of adaptor proteins, and that the ubiquitinated adaptor proteins in turn serve as platforms to recruit IKK and IKK-Ks, facilitating IKK activation through proximity-mediated phosphorylation. This review will focus on the most recent findings relating to the role of TRAFs-mediated protein ubiquitination in regulating IKK activation, and highlight the newly emerging complexity of protein ubiquitination in receptor-induced NF-κB activation.
RESUMO
Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of kappaB kinase (IKK), as well as in inhibiting apoptosis induced by TNFalpha. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFalpha-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-DeltaR) has been widely used as a dominant negative in transient overexpression systems to block TNFalpha-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-DeltaR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFalpha-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells efficiently inhibited the TNFalpha-induced later phase of prolonged JNK activation, yet failed to inhibit TNFalpha-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-DeltaR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFalpha-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFalpha and is essential for suppressing the noncanonical nuclear factor kappaB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFalpha-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFalpha-induced cell death.
Assuntos
Morte Celular , NF-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , NF-kappa B/imunologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Fator 2 Associado a Receptor de TNF/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an adaptor protein that modulates the activation of the c-Jun NH(2) terminal kinase (JNK)/c-Jun and IkappaB kinase (IKK)/nuclear factor-kappaB (NF-kappaB) signaling cascades in response to TNFalpha stimulation. Although many serine/threonine kinases have been implicated in TNFalpha-induced IKK activation and NF-kappaB-dependent gene expression, most of them do not directly activate IKK. Here, we report that protein kinase Czeta phosphorylates TRAF2 at Ser(55), within the RING domain of the protein, after TNFalpha stimulation. Although this phosphorylation event has a minimal effect on induction of the immediate/transient phase of IKK and JNK activation by TNFalpha, it promotes the secondary/prolonged phase of IKK activation and inhibits that of JNK. Importantly, constitutive TRAF2 phosphorylation increased both basal and inducible NF-kappaB activation and rendered Ha-Ras-V12-transformed cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some malignant cancer cell lines and Hodgkin's lymphoma. These results reveal a new level of complexity in TNFalpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation is one of the events that are responsible for elevated basal NF-kappaB activity in certain human cancers.
Assuntos
Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Apoptose/fisiologia , Ativação Enzimática , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , MAP Quinase Quinase 4/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação , Domínios RING Finger , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IkappaB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-alpha) stimulation. Although the downstream events in TNF-alpha signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-alpha and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-kappaB target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-alpha-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-alpha stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-alpha-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-kappaB activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-alpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-kappaB activity in certain human cancers.
Assuntos
Apoptose , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Linfoma , Camundongos , NF-kappa B/metabolismo , Células NIH 3T3 , Estresse Oxidativo , Fosforilação , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Serina/metabolismo , Fator 2 Associado a Receptor de TNF/genéticaRESUMO
Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFalpha-induced activation of the c-Jun N-terminal kinase and nuclear factor kappaB (NF-kappaB) pathways. Currently, TNFalpha-induced NF-kappaB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IkappaB kinase (IKK) activity and elevated expression of NF-kappaB-dependent genes in unstimulated conditions. Although TNFalpha-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFalpha stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-kappaB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFalpha-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFalpha can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFalpha-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFalpha-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFalpha-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-kappaB pathway.
Assuntos
Quinase I-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , NF-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Activation of the Jun-N-terminal kinase (JNK) signaling cascade by phorbol esters (TPA) or protein kinase C (PKC) is well documented, although the underlying mechanism is not known. Here, we demonstrate that the receptor for activated C kinase 1 (RACK1) serves as an adaptor for PKC-mediated JNK activation. Phosphorylation of JNK by PKC occurs on Ser129 and requires the presence of RACK1. Ser129 phosphorylation augments JNK phosphorylation by MKK4 and/or MKK7 and is required for JNK activation by TPA, TNFalpha, UV irradiation, and PKC, but not by anisomycin or MEKK1. Inhibition of RACK1 expression by siRNA attenuates JNK activation, sensitizes melanoma cells to UV-induced apoptosis, and reduces their tumorigenicity in nude mice. In finding the role of RACK1 in activation of JNK by PKC, our study also highlights the nature of crosstalk between these two signal-transduction pathways.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Quinase C/metabolismo , Animais , Anisomicina/farmacologia , Anticorpos Fosfo-Específicos/metabolismo , Apoptose , Sítios de Ligação , Carbazóis/farmacologia , Catálise/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Citoplasma/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP , Deleção de Genes , Humanos , Indóis/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase 7/genética , MAP Quinase Quinase 7/metabolismo , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/genética , Fosfopeptídeos/análise , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C beta , RNA Interferente Pequeno/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Raios UltravioletaRESUMO
TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF-kappaB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFalpha-induced activation of JNK but not of p38 or NF-kappaB. Intact RING and zinc finger domains are required for TNFalpha-induced TRAF2 ubiquitination, which is also dependent on Ubc13. TRAF2 ubiquitination coincides with its translocation to the insoluble cellular fraction, resulting in selective activation of JNK. Inhibition of Ubc13 expression by RNAi resulted in inhibition of TNFalpha-induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in Hodgkin-Reed-Sternberg lymphoma cells, resulting in constitutive NF-kappaB activity but failure to activate JNK. These findings demonstrate that the TRAF2 RING is required for Ubc13-dependent ubiquitination, resulting in translocation of TRAF2 to an insoluble fraction and activation of JNK, but not of p38 or NF-kappaB. Altogether, our findings highlight a novel mechanism of TRAF2-dependent activation of diverse signaling cascades that is impaired in Hodgkin-Reed-Sternberg cells.