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J. Biol. Chem., Vol. 281, Issue 50, 38738-38747, December 15, 2006

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The Ubiquitin Ligase Itch Is Auto-ubiquitylated in Vivo and in Vitro but Is Protected from Degradation by Interacting with the Deubiquitylating Enzyme FAM/USP9X^*

Rania Mouchantaf¹, Bilal A. Azakir¹, Peter S. McPherson², Susan M. Millard^¶||3, Stephen A. Wood^¶3, and Annie Angers⁴

From the Départment de sciences biologiques, Université de Montréal, Montreal, Quebec H3C 3J7, Canada, the Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada, the ^¶Child Health Research Institute, 72 King William Road, North Adelaide SA 5006, Australia, and the ^||School of Molecular and Biomedical Sciences, University of Adelaide, North Adelaide SA 5005, Australia

Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins.

Received for publication, June 21, 2006 , and in revised form, September 20, 2006.

^* This work was supported in part by Natural Sciences and Engineering Research Council of Canada Discovery Grant 288238 and Fonds de Recherche sur la Nature et les Technologies of Quebec (FQRNT) Young Investigator Grant SC-94269 (to A. A.). 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 These authors contributed equally to this work.

² A Canadian Institutes of Health Research Investigator and a McGill University William Dawson Scholar.

³ Both authors were supported by the National Health and Medical Research Council, Australia.

⁴ Supported by a FQRNT young investigator award. To whom correspondence should be addressed: Départment de Sciences Biologiques, Universitéde Montréal, P. O. Box 6128, Station Centre-Ville, Montreal, Quebec H3C 3J7, Canada. Tel.: 514-343-7012; Fax: 514-343-2293; E-mail: Annie.Angers{at}umontreal.ca.

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