Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/35537
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Type: Journal article
Title: 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
Author: Mouchantaf, Rania
Azakir, Bilal A.
McPherson, Peter S.
Millard, Susan Marie
Wood, Stephen Andrew
Angers, Annie
Citation: Journal of Biological Chemistry, 2006; 281 (50):38738-38747
Publisher: American Society of Biochemistry and Molecular Biology
Issue Date: 2006
ISSN: 0021-9258
School/Discipline: School of Molecular and Biomedical Science
Organisation: Centre for the Molecular Genetics of Development
Statement of
Responsibility: 
Rania Mouchantaf, Bilal A. Azakir, Peter S. McPherson, Susan M. Millard, Stephen A. Wood and Annie Angers
Abstract: 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.
Description: Copyright © 2006 by the American Society for Biochemistry and Molecular Biology
Provenance: Originally published In Press as doi:10.1074/jbc.M605959200 on October 12, 2006
DOI: 10.1074/jbc.M605959200
Published version: http://www.jbc.org/cgi/content/abstract/281/50/38738
Appears in Collections:Centre for the Molecular Genetics of Development publications
Molecular and Biomedical Science publications

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