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J Cell Sci Suppl. 1995;19:79-84.

Appearance of apparently ubiquitin-conjugated I kappa B-alpha during its phosphorylation-induced degradation in intact cells.

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1
Institute of Biochemistry, Albert-Ludwigs-University, Freiburg, Germany.

Abstract

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.

PMID:
8655651
[Indexed for MEDLINE]
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