Inhibition of NF-kappa B activity by a membrane-transducing mutant of I kappa B alpha.
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.
Kabouridis
Panagiotis S
PS
Bone and Joint Research Unit and Department of Experimental Pathology, Barts and London School of Medicine and Dentistry, London, United Kingdom. p.s.kabouridis@qmul.ac.uk
Hasan
Maemunah
M
Newson
Justine
J
Gilroy
Derek W
DW
Lawrence
Toby
T
D001667:Binding, Competitive / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D019169:Jurkat Cells; D020935:MAP Kinase Signaling System / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D020928:Mitogen-Activated Protein Kinases