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 transcrip tion in higher eukaryotic cells in response to mainly pathogenic stimu li, 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 en ter the nucleus but is stored in a latent cytoplasmic form. Upon stimu lation of cells I kappa B is released, which allows the activation of NF-kappa B. We have analyzed the molecular mechanism underlying the re moval of I kappa B-alpha. Distinct extracellular stimuli lead to a pho sphorylation of I kappa B-alpha on serines 32 and 36 by a yet unidenti fied kinase. These modifications do not directly dissociate I kappa B from NF-kappa B but render the inhibitor highly susceptible for proteo lytic degradation by, presumably, the proteasome. In this paper, we re port 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 overexp ressing I kappa B-alpha, suggesting the covalent modification of I kap pa 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 indic ating that the conjugation of I kappa B-alpha with ubiquitin was the r ate-limiting step in I kappa B-alpha degradation, and not its phosphor ylation or proteolysis. Our data suggest that conjugation of I kappa B -alpha with ubiquitin is an intermediate reaction in the phosphorylati on-controlled degradation of I kappa B-alpha and the subsequent activa tion of NF-kappa B.