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Molecular and Cellular Biology, October 2007, p. 7041-7052, Vol. 27, No. 20
0270-7306/07/$08.00+0     doi:10.1128/MCB.00812-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Human Cdc34 Employs Distinct Sites To Coordinate Attachment of Ubiquitin to a Substrate and Assembly of Polyubiquitin Chains{triangledown}

Stefan Gazdoiu, Kosj Yamoah, Kenneth Wu, and Zhen-Qiang Pan*

Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, New York 10029-6574

Received 8 May 2007/ Returned for modification 25 June 2007/ Accepted 30 July 2007

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 I{kappa}B{alpha} 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 I{kappa}B{alpha}, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34E108A/E112A abolished both poly- and monoubiquitination of I{kappa}B{alpha}. 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.

* Corresponding author. Mailing address: Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, NY 10029-6574. Phone: (212) 659-5500. Fax: (212) 849-2446. E-mail: zhen-qiang.pan{at}mssm.edu

{triangledown} Published ahead of print on 13 August 2007.

Molecular and Cellular Biology, October 2007, p. 7041-7052, Vol. 27, No. 20
0270-7306/07/$08.00+0     doi:10.1128/MCB.00812-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yamoah, K., Oashi, T., Sarikas, A., Gazdoiu, S., Osman, R., Pan, Z.-Q. (2008). Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail. Proc. Natl. Acad. Sci. USA 105: 12230-12235 [Abstract] [Full Text]  


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