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J. Biol. Chem., Vol. 283, Issue 35, 23701-23710, August 29, 2008

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Unique D Box and KEN Box Sequences Limit Ubiquitination of Acm1 and Promote Pseudosubstrate Inhibition of the Anaphase-promoting Complex^*

Eunyoung Choi, J. Michael Dial¹, Dah-Eun Jeong², and Mark C. Hall^¶3

From the Biochemistry Department and ^¶Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907 and the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599

The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APC^Cdh1) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APC^Cdh1 inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APC^Cdh1 inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APC^Cdh1 in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.

Received for publication, May 14, 2008 , and in revised form, July 1, 2008.

^* This work was supported by an American Cancer Society Institutional Research Grant to the Purdue Cancer Center and by an American Heart Association Scientist Development Grant (to M. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ SVB Financial Group, 5915 Farrington Road, Suite 201, Chapel Hill, NC 27517.

² Gladstone Institute, 1650 Owens St., Box 1230, San Francisco, CA 94158-2261.

³ To whom correspondence should be addressed: Dept. of Biochemistry, Purdue University, 175 S. University St., West Lafayette, IN 47907. Tel.: 765-494-0714; Fax: 765-494-7897; E-mail: mchall{at}purdue.edu.

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