1 WDR 20 regulates activity of the USP 12 / UAF 1 deubiquitinating enzyme complex

The UAF1 (Usp1 Associated Factor 1) protein binds and stimulates three deubiquitinating enzymes (DUB), USP1, USP12, and USP46. While the USP1/UAF1 complex is required for regulation of the Fanconi anemia (FA) DNA repair pathway, less is known about the USP12/UAF1 and the USP46/UAF1 complexes. To further understand the nature of the USP12 and USP46 complexes, we attempted to identify proteins that interact with the USP12 and USP46 DUB complexes. We identified WDR20, a WD40-repeat containing protein, as a common binding partner of UAF1, USP12, and USP46. Further analysis showed that WDR20 exclusively associates with USP12 and USP46, not with USP1. Furthermore, we demonstrate the purification of a ternary USP12/UAF1/WDR20 complex. Interestingly, and consistent with the binding assays, WDR20 stimulated the enzymatic activity of the USP12/UAF1, but not of the USP1/UAF1. Consistent with our previous report that USP12 and USP46 do not regulate the FA pathway, siRNA-mediated depletion of WDR20 protein did not affect the FA pathway or DNA damage responses. We provide a model in which WDR20 serves as a stimulatory subunit for preserving and regulating the activity of the subset of the UAF1/USP complexes. INTRODUCTION Balanced ubiquitination and deubiquitination regulates numerous cellular processes. Increasing reports suggest that deubiquitination of proteins, the reversal process of ubiquitinaton, is an as important step as ubiquitination, for specific regulation of particular cellular pathways 1,2 . For instance, deubiquitinating enzymes are critical regulators of the p53/mdm2 3 , NFκB signaling 4,5 , and the Fanconi Anemia (FA) DNA repair pathways 6 , by directly removing the ubiquitin moieties from their substrates. Deubiquitination either rescues the proteins from proteosome-mediated degradation or alters the activities of the proteins. For instance, USP7/HAUSP regulates the dynamics of p53dependent pathways, by directly deubiquitinating p53 and Mdm2 3 . CYLD suppresses the NFkB pathway by deubiquitinating K63-linked polyubiquitintion of NEMO, a regulatory subunit for IκB kinase and TRAF2/6 E3 ligases. USP1 deubiquitinates monoubiquitianted FANCD2, which is an important regulator of the FA pathway, a step required for the completion of this DNA repair pathway 6 . Inactivation of the deubiquitinating enzymes result in abnormal cellular phenotypes in various organism settings 710 , further suggesting that deubiquitination is a critical step in a variety of biological processes. There are approximately 95 putative DUBs encoded by human cells, and they can be divided into five sub-families 2,11 . Four families belong to cysteine proteases, including ubiquitin C-terminal hydrolases (UCHs), ubiquitin specific proteases (USPs), otubain proteases (OTUs), and Josephine domain proteases (MJDs), with the fifth http://www.jbc.org/cgi/doi/10.1074/jbc.M109.095141 The latest version is at JBC Papers in Press. Published on February 10, 2010 as Manuscript M109.095141 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 6, 2017 hp://w w w .jb.org/ D ow nladed from