Abstract
The transcription factor NF-kappaB is regulated by the IkappaB family of proteins. The nonphosphorylatable, nondegradable superrepressor IkappaBalpha (srIkappaBalpha) mutant is a potent inhibitor of NF-kappaB activity when expressed in cells. We generated a form of srIkappaBalpha in which its N terminus is fused to the protein transduction domain of HIV TAT (TAT-srIkappaBalpha). Purified TAT-srIkappaBalpha protein rapidly and efficiently entered HeLa or Jurkat T cells. TAT-srIkappaBalpha, when exogenously added to HeLa cells, inhibited in a dose-dependent manner TNF-alpha- or IL-1beta-induced NF-kappaB activation and binding of NF-kappaB to its consensus DNA sequence. TAT-srIkappaBalpha was coimmunoprecipitated with the p65 subunit of NF-kappaB, and this interaction was resistant to stimulation with IL-1beta. Therefore, TAT-srIkappaBalpha-mediated inhibition could result from its nonreversible binding and sequestration of endogenous NF-kappaB. In contrast, exogenously added TAT-srIkappaBalpha did not inhibit IL-1beta-induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 mitogen-activated protein kinases or the phosphorylation and degradation of endogenous IkappaBalpha. These results identify a novel way for direct regulation of NF-kappaB activity in diverse cell types that may be useful for therapeutic purposes.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Binding, Competitive / genetics
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Biological Transport / genetics
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Cell Membrane / genetics
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Cell Membrane / metabolism
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Consensus Sequence
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DNA-Binding Proteins / antagonists & inhibitors
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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DNA-Binding Proteins / physiology*
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Gene Products, tat / genetics
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Gene Products, tat / metabolism
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Gene Products, tat / physiology
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HeLa Cells
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Humans
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I-kappa B Proteins*
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Jurkat Cells
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MAP Kinase Signaling System / genetics
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Mitogen-Activated Protein Kinases / antagonists & inhibitors
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Mitogen-Activated Protein Kinases / metabolism
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Mutation*
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NF-KappaB Inhibitor alpha
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NF-kappa B / antagonists & inhibitors*
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NF-kappa B / metabolism*
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Recombinant Fusion Proteins / antagonists & inhibitors
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Recombinant Fusion Proteins / physiology*
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Repressor Proteins / antagonists & inhibitors
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Repressor Proteins / physiology*
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Transcription Factor RelA
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Transcription, Genetic
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Transduction, Genetic*
Substances
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DNA-Binding Proteins
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Gene Products, tat
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I-kappa B Proteins
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NF-kappa B
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NFKBIA protein, human
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Recombinant Fusion Proteins
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Repressor Proteins
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Transcription Factor RelA
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NF-KappaB Inhibitor alpha
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Mitogen-Activated Protein Kinases