title: A novel E3 ubiquitin ligase substrate screen identifies Rho guanine dissociation inhibitor as a substrate of gene related to anergy in lymphocytes.
abstract: 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.
authors.affiliation: Department of Medicine, Division of Immunology and Rheumatology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Leon
authors.initials: L
authors.lastname: Su
authors.forename: Neil
authors.initials: N
authors.lastname: Lineberry
authors.forename: Yul
authors.initials: Y
authors.lastname: Huh
authors.forename: Luis
authors.initials: L
authors.lastname: Soares
authors.forename: C Garrison
authors.initials: CG
authors.lastname: Fathman
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: C569638:ARHGDIA protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D007376:Interleukin-2; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D044767:Ubiquitin-Protein Ligases