Identification of lysine residues required for signal-induced ubiquitination and degradation of I kappa B-alpha in vivo.
Activation of transcription factor NF-kappaB involves signal-induced degradation of the protein inhibitor IkappaB-alpha and release of NF-kappaB which translocates to the nucleus where it influences transcription of responsive genes. Although multiple regions of IkappaB-alpha are involved in this process, the N-terminal region of the protein has been identified as a regulatory region that is required for signal induced phosphorylation and degradation. The sensitivity of IkappaB-alpha degradation to peptide aldehydes which inhibit components of the proteasome and the detection of ubiquitinated forms of IkappaB-alpha indicate that IkappaB-alpha is degraded by the ubiquitin-proteasome pathway. To identify lysine residues that represent the sites of ubiquitin addition, a series of lysine to arginine mutations were introduced into IkappaB-alpha and the mutant proteins tested for their ability to function in vivo. Exposure of COS7 cells, cotransfected with IkappaB-alpha and a TNF-responsive NF-kappaB reporter gene, resulted in stimulation of reporter activity as a consequence of IkappaB-alpha degradation. In contrast, this effect was drastically reduced when an IkappaKB-alpha mutant carrying serine to alanine changes at amino-acids, 32 and 36, which blocks both signal-induced phosphorylation and ubiquitin conjugation of the protein, was co-transfected with the reporter gene. Likewise, a mutant form of IkappaB-alpha containing lysine to arginine changes at positions 21 and 22 (K21R, K22R) severely reduces TNF-induced activation of the NF-kappaB-dependent reporter gene. Examination of the metabolism of mutant IkappaB-alpha molecules reveals that, while the K21R, K22R mutant inhibits the DNA-binding activity of NF-kappaB and undergoes signal induced phosphorylation, it is neither ubiquitinated nor degraded in response to TNF. Thus, it is likely that after signal-induced phosphorylation Of IkappaB-alpha on serine residues 32 and 36, lysine residues 21 and 22 are major sites of ubiquitin ligation which target the protein for rapid degradation by the proteasome.
Rodriguez
M S
MS
Unité d'Immunologie Virale, Institut Pasteur, Paris, France.
Wright
J
J
Thompson
J
J
Thomas
D
D
Baleux
F
F
Virelizier
J L
JL
Hay
R T
RT
Arenzana-Seisdedos
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004988:Ethers, Cyclic / Q000494:pharmacology; D017930:Genes, Reporter; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019319:Okadaic Acid; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004988:Ethers, Cyclic; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid; D004247:DNA; D008239:Lysine