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Mol. Cell. Biol. doi:10.1128/MCB.01555-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

SCF E3 -mediated autoubiquitination negatively regulates activity of the Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo

Edward A. Doisy Dept. of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104; Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794; Department of Biochemistry and Molecular Biology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202

^* To whom correspondence should be addressed. Email: skowyrad{at}slu.edu.

	Abstract

One of the several yet 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 S. cerevisiae we find that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an inter-molecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intra-molecular 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 lysine-less ^K0Cdc34^C is undistinguishable from Cdc34^C in ubiquitination of the prototype SCF^Cdc4 substrate Sic1 in vitro and replacement of CDC34 gene with either ^K0cdc34^C or cdc34^C allele in yeast has no cell cycle phenotype. We discuss implications of these findings on the mechanism of Cdc34 function with SCF.

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