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A novel E3 ubiquitin ligase substrate screen identifies Rho guanine dissociation inhibitor as a substrate of gene related to anergy in lymphocytes.

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.

Pubmed ID: 17114425

Authors

  • Su L
  • Lineberry N
  • Huh Y
  • Soares L
  • Fathman CG

Journal

Journal of immunology (Baltimore, Md. : 1950)

Publication Data

December 1, 2006

Associated Grants

  • Agency: NCI NIH HHS, Id: CA 65237
  • Agency: NIAID NIH HHS, Id: T32 AI007290

Mesh Terms

  • Biological Assay
  • Cell Line
  • Enzyme Activation
  • Escherichia coli
  • Genetic Vectors
  • Guanine Nucleotide Dissociation Inhibitors
  • Humans
  • Interleukin-2
  • Plasmids
  • Reverse Transcriptase Polymerase Chain Reaction
  • T-Lymphocytes
  • Transfection
  • Ubiquitin-Protein Ligases
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors