HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Su, L. Articles by Fathman, C. G. Search for Related Content PubMed PubMed Citation Articles by Su, L. Articles by Fathman, C. G.

A Novel E3 Ubiquitin Ligase Substrate Screen Identifies Rho Guanine Dissociation Inhibitor as a Substrate of Gene Related to Anergy in Lymphocytes¹

Department of Medicine, Division of Immunology and Rheumatology, Stanford University, Stanford, CA 94305

Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes, including regulation of T cell activation and tolerance. We have previously demonstrated that gene related to anergy in lymphocytes (GRAIL), a ring finger ubiquitin E3 ligase, is required for the induction of T cell anergy; however, the substrate(s) for GRAIL E3 ligase activity is/are unknown. In this study, we report a novel prokaryotic system developed to screen for substrates of E3 ligases. Using this screen, Rho guanine dissociation inhibitor (RhoGDI) was identified as a potential substrate of GRAIL. GRAIL was subsequently demonstrated to bind and ubiquitinate RhoGDI, although GRAIL-mediated ubiquitination of RhoGDI did not result in proteosomal degradation. Expression of GRAIL in T cells resulted in specific inhibition of RhoA GTPase activation; activation of Rac1, cdc42, and Ras GTPases were not affected. Interestingly, stable T cell lines expressing dominant-negative RhoA mimicked the GRAIL-mediated IL-2 inhibition phenotype, and T cells expressing constitutively active RhoA were able to overcome GRAIL-mediated inhibition of IL-2 expression. These findings validate our prokaryotic screen as a method of identifying substrates for ubiquitin E3 ligases and suggest a role for Rho effector molecules in T cell anergy.

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 This work was supported by National Institutes of Health Grant CA65237.

² Address correspondence and reprint requests to Dr. C. Garrison Fathman, Stanford University School of Medicine, Center for Clinical Science and Research Building, Room 2240, 269 West Campus Drive, Stanford, CA 94305. E-mail address: cfathman{at}stanford.edu

³ Abbreviations used in this paper: GRAIL, gene related to anergy in lymphocytes; RhoGDI, Rho guanine dissociation inhibitor; IPTG, isopropyl -D-thiogalactoside; tet, tetracycline; RBD, Rho binding domain; MCS, multiple cloning site.

This article has been cited by other articles:

				N. Knezevic, A. Roy, B. Timblin, M. Konstantoulaki, T. Sharma, A. B. Malik, and D. Mehta GDI-1 Phosphorylation Switch at Serine 96 Induces RhoA Activation and Increased Endothelial Permeability Mol. Cell. Biol., September 15, 2007; 27(18): 6323 - 6333. [Abstract] [Full Text] [PDF]