Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains.
The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.
Gazdoiu
Stefan
S
Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Yamoah
Kosj
K
Wu
Kenneth
K
Pan
Zhen-Qiang
ZQ
D064173:Anaphase-Promoting Complex-Cyclosome; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; D000072000:NF-KappaB Inhibitor alpha; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
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