In vivo and in vitro recruitment of an IkappaBalpha-ubiquitin ligase to IkappaBalpha phosphorylated by IKK, leading to ubiquitination.
Activation of the transcriptional factor NF-kappaB is triggered by signal-dependent degradation of its inhibitor protein IkappaB through the ubiquitin (Ub)-proteasome pathway. We found here that a phosphorylated IkappaBalpha immunoprecipitated (IP-pIkappaBalpha) from the crude extract of HeLa cells which had been treated with tumor necrosis factor-alpha (TNFalpha) caused a dramatic ubiquitination ... of itself, termed autoubiquitination, when incubated with ATP, Ub, and E1-activating and E2-conjugating enzymes. IP-pIkappaBalpha also catalyzed ubiquitination of an in vitro synthesized 35S-IkappaBalpha previously phosphorylated by IkappaB-kinase (IKK) which is referred to as transubiquitination. No appreciable activity of auto- and transubiquitination was observed in an unphosphorylated IP-IkappaBalpha. Moreover, the putative IkappaBalpha-Ub ligase (IkappaBalpha-E3) present in HeLa cell cytosol associated in vitro with an IKK-phosphorylated recombinant IkappaBalpha, a process independent of NF-kappaB binding to IkappaBalpha or TNFalpha stimulation. Replacement of the two Ser residues at positions 32 and 36 corresponding to IKK phosphorylation sites by Ala resulted in almost complete prevention of binding of an IkappaBalpha-E3 to IkappaBalpha. These results indicate that phosphorylation of IkappaBalpha is necessary and sufficient for recruitment of this IkappaBalpha-E3 to associate with IkappaBalpha.
Mesh Terms:
Adenosine Triphosphate, Amino Acid Substitution, Animals, Blotting, Western, DNA-Binding Proteins, Enzyme Activation, Hela Cells, Humans, I-kappa B Kinase, I-kappa B Proteins, Ligases, NF-kappa B, Phosphorylation, Phosphoserine, Precipitin Tests, Protein Binding, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Signal Transduction, Time Factors, Transcription Factor RelA, Tumor Necrosis Factor-alpha, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitins
Adenosine Triphosphate, Amino Acid Substitution, Animals, Blotting, Western, DNA-Binding Proteins, Enzyme Activation, Hela Cells, Humans, I-kappa B Kinase, I-kappa B Proteins, Ligases, NF-kappa B, Phosphorylation, Phosphoserine, Precipitin Tests, Protein Binding, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Signal Transduction, Time Factors, Transcription Factor RelA, Tumor Necrosis Factor-alpha, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitins
Biochem. Biophys. Res. Commun.
Date: Mar. 05, 1999
PubMed ID: 10066434
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