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.
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
Panagiotis S
PS
Kabouridis
Maemunah
M
Hasan
Justine
J
Newson
Derek W
DW
Gilroy
Toby
T
Lawrence
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