Abstract
Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Antigens, CD / genetics
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Antigens, CD / immunology
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Antigens, CD / metabolism
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Apoptosis / physiology
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Biological Transport
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Carrier Proteins / immunology
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Carrier Proteins / metabolism
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Cell Line / drug effects
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Cell Nucleus / metabolism
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DNA-Binding Proteins / metabolism*
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Fas-Associated Death Domain Protein
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Humans
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Immunochemistry / methods
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NF-kappa B / drug effects
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NF-kappa B / genetics
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NF-kappa B / metabolism*
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Proteins / genetics
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Proteins / immunology
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Proteins / metabolism*
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Receptor-Interacting Protein Serine-Threonine Kinases
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Receptors, Tumor Necrosis Factor / genetics
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Receptors, Tumor Necrosis Factor / immunology
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Receptors, Tumor Necrosis Factor / metabolism*
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Receptors, Tumor Necrosis Factor, Type I
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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STAT1 Transcription Factor
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Signal Transduction*
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TNF Receptor-Associated Factor 1
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TNF Receptor-Associated Factor 2
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Trans-Activators / metabolism*
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Transcriptional Activation / drug effects
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Tumor Necrosis Factor-alpha / pharmacology
Substances
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Adaptor Proteins, Signal Transducing
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Antigens, CD
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Carrier Proteins
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DNA-Binding Proteins
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FADD protein, human
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Fas-Associated Death Domain Protein
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NF-kappa B
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Proteins
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Receptors, Tumor Necrosis Factor
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Receptors, Tumor Necrosis Factor, Type I
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Recombinant Fusion Proteins
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STAT1 Transcription Factor
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STAT1 protein, human
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TNF Receptor-Associated Factor 1
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TNF Receptor-Associated Factor 2
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Trans-Activators
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Tumor Necrosis Factor-alpha
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RIPK1 protein, human
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Receptor-Interacting Protein Serine-Threonine Kinases