Appearance of apparently ubiquitin - conjugated I k B - oc during its phosphorylation - induced degradation in intact cells

NF-kB is a dimeric protein that serves to initiate gene tran­ scription in higher eukaryotic cells in response to mainly pathogenic stimuli. Its activity is controlled by a third inhibitory subunit, called IkB. When IkB is bound, NF-kB cannot bind to DNA or enter the nucleus but is stored in a latent cytoplasmic form. Upon stimulation of cells IkB is released, which allows the activation of NF-kB. We have analyzed the molecular mechanism underlying the removal of IkB-oc. Distinct extracellular stimuli lead to a phospho­ rylation of IkB-oc on serines 32 and 36 by a yet unidentified kinase. These modifications do not directly dissociate IkB from NF-kB 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 IkB-oc occur under conditions that lead to a phosphorylation of IkB-oc and activation of NFkB. These IkB-oc variants had discrete molecular masses and were most prominent in cells overexpressing IkB-oc, suggesting the covalent modification of IkB-oc by ubiquitin conjugation. The proteasome inhibitor Cbz-Ile-Glu(0-tBu)-Ala-leucinal (PSI), which stabilizes the phospho form of IkB-oc, only slightly increased the amount of conjugates indicating that the conjugation of IkB-oc with ubiquitin was the rate-limiting step in IkB-oc degradation, and not its phosphorylation or proteolysis. Our data suggest that con­ jugation of IkB-oc with ubiquitin is an intermediate reaction in the phosphorylation-controlled degradation of IkB-oc and the subsequent activation of NF-kB.