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Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IkappaBalpha.

The Journal of biological chemistry (1999-05-18)
H Gonen, B Bercovich, A Orian, A Carrano, C Takizawa, K Yamanaka, M Pagano, K Iwai, A Ciechanover
ABSTRACT

The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
UbcH5b Conjugating Enzyme, 100 µg, The His6-tagged fusion protein of UbcH5b is charge & support ubiquitinylation in vitro. The His6-tagged UbcH5 family members all appear to form thiol ester conjugates with ubiquitin at a similar rate under similar conditions.
Sigma-Aldrich
Anti-CDC34 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Ubiquitin-Carrier Protein H5b human, ≥95% (SDS-PAGE), solution
Sigma-Aldrich
Ubiquitin-Carrier Protein H5c human, ≥95% (SDS-PAGE), recombinant, expressed in E. coli (GST-tagged), solution

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