title: HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity.
abstract: The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.
authors.affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
authors.forename: Gary T
authors.initials: GT
authors.lastname: ZeRuth
authors.affiliation: Mass Spectrometry Group, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
authors.forename: Jason G
authors.initials: JG
authors.lastname: Williams
authors.affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
authors.forename: Yasemin C
authors.initials: YC
authors.lastname: Cole
authors.affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
authors.forename: Anton M
authors.initials: AM
authors.lastname: Jetten
mesh_terms: D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics*; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D054875:Ubiquitination / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C512851:GLIS3 protein, human; C583314:GLIS3 protein, rat; D008565:Membrane Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases