Appearance of apparently ubiquitin-conjugated I kappa B-alpha during its phosphorylation-induced degradation in intact cells.
NF-kappa B is a dimeric protein that serves to initiate gene transcription in higher eukaryotic cells in response to mainly pathogenic stimuli. Its activity is controlled by a third inhibitory subunit, called I kappa B. When I kappa B is bound, NF-kappa B cannot bind to DNA or enter the nucleus but is stored in a latent cytoplasmic form. Upon stimulation of cells I kappa B is released, which allows the activation of NF-kappa B. We have analyzed the molecular mechanism underlying the removal of I kappa B-alpha. Distinct extracellular stimuli lead to a phosphorylation of I kappa B-alpha of serines 32 and 36 by a yet unidentified kinase. These modifications do not directly dissociate I kappa B from NF-kappa B but render the inhibitor highly susceptible for proteolytic degradation by, presumably, the proteasome. In this paper, we report for the first time that higher molecular mass forms of I kappa B-alpha occur under conditions that lead to a phosphorylation of I kappa B-alpha and activation of NF-kappa B. These I kappa B-alpha variants had discrete molecular masses and were most prominent in cells overexpressing I kappa B-alpha, suggesting the covalent modification of I kappa B-alpha by ubiquitin conjugation. The proteasome inhibitor Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), which stabilizes the phospho form of I kappa B-alpha, only slightly increased the amount of conjugates indicating that the conjugation of I kappa B-alpha with ubiquitin was the rate-limiting step in I kappa B-alpha degradation, and not its phosphorylation or proteolysis. Our data suggest that conjugation of I kappa B-alpha with ubiquitin is an intermediate reaction in the phosphorylation-controlled degradation of I kappa B-alpha and the subsequent activation of NF-kappa B.
Traenckner
E B
EB
Institute of Biochemistry, Albert-Ludwigs-University, Freiburg, Germany.
Baeuerle
P A
PA
D003546:Cysteine Endopeptidases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D019319:Okadaic Acid; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D012694:Serine / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009842:Oligopeptides; D014452:Ubiquitins; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid; D012694:Serine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex