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Molecular and Cellular Biology, August 2007, p. 5860-5870, Vol. 27, No. 16
0270-7306/07/$08.00+0     doi:10.1128/MCB.01555-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

K. Matthew Scaglione,1 Parmil K. Bansal,2 Andrew E. Deffenbaugh,1 Alexi Kiss,1 Johnnie M. Moore,1 Sergey Korolev,1 Ross Cocklin,3 Mark Goebl,3 Katsumi Kitagawa,2 and Dorota Skowyra1*

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{Delta}C is indistinguishable from Cdc34{Delta}C in ubiquitination of the prototype SCFCdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the K0cdc34{Delta}C or the cdc34{Delta}C allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

* Corresponding author. Mailing address: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104. Phone: (314) 977-9280. Fax: (314) 977-9205. E-mail: skowyrad{at}slu.edu

{triangledown} Published ahead of print on 6 June 2007.

Molecular and Cellular Biology, August 2007, p. 5860-5870, Vol. 27, No. 16
0270-7306/07/$08.00+0     doi:10.1128/MCB.01555-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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