The carboxy-terminus of I kappaB alpha determines susceptibility to degradation by the catalytic core of the proteasome.
The Rel/NF-kappaB family of transcription factors controls the expression of a wide variety of genes that are implicated in immune and inflammatory responses and cellular proliferation. Disregulation of NF-kappaB is associated with cellular transformation and the maintenance of a high anti-apoptotic threshold in transformed cells. NF-kappaB activity is in turn regulated by its sequestration in the cytoplasm by the inhibitor I kappaB. I kappaB alpha, the most abundant and well-characterized member of the I kappaB multiprotein family, is rapidly degraded in response to multiple physiologic stimuli. In the present study we show that not only the amino-terminus, but also the carboxy-terminus of I kappaB alpha contain transferable signals that must be simultaneously present in an unrelated protein to render it susceptible to activation-induced, proteasome-mediated degradation. We show here that I kappaB alpha amino-terminal modifications occur independently of the carboxy-terminus. Moreover, we present evidence indicating a critical role for the carboxy-terminal region in facilitating proteolysis by the catalytic core of the proteasome. When incubated with 20S proteasome extracted from rat liver, I kappaB alpha was quickly degraded while a deletion mutant lacking the carboxy-terminus was resistant to proteolysis. Likewise, chimeric proteins of beta-galactosidase with the I kappaB alpha carboxy-terminus were degraded in vitro independently of the presence of the I kappaB alpha amino-terminus, whereas chimeric proteins lacking the I kappaB alpha carboxy-terminus were stable. Our results identify the carboxy-terminus of I kappaB alpha as a domain critical for degradation through interaction with an as yet unidentified component of the proteasome.
Kroll
M
M
Unité d'Immunologie Virale, Institut Pasteur, Paris, France.
Conconi
M
M
Desterro
M J
MJ
Marin
A
A
Thomas
D
D
Friguet
B
B
Hay
R T
RT
Virelizier
J L
JL
Arenzana-Seisdedos
F
F
Rodriguez
M S
MS
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex