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SCF E3-Mediated Autoubiquitination Negatively Regulates Activity of Cdc34 E2 but Plays a Nonessential Role in the Catalytic Cycle In Vitro and In Vivo

Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104,¹ Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794,² Department of Biochemistry and Molecular Biology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202³

Received 21 August 2006/ Returned for modification 13 November 2006/ Accepted 21 May 2007

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless ^K0Cdc34^C is indistinguishable from Cdc34^C in ubiquitination of the prototype SCF^Cdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the ^K0cdc34^C or the cdc34^C allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

Published ahead of print on 6 June 2007.

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