title: Association of connexin43 with E3 ubiquitin ligase TRIM21 reveals a mechanism for gap junction phosphodegron control.
abstract: Gap junctions (GJs) are sites of direct cell-to-cell communication formed by the connexin (Cx) family of ion channel proteins. The aberrant intercellular communication mediated by GJs is associated with a variety of hereditary and acquired human diseases. GJs utilize a highly interconnected network that is indispensible for synthesis, trafficking and degradation of their constituent proteins. By unbiased proteomic examination and network enrichment, we identified interacting components of the ubiquitin proteasome system associated with Cx43. LC-MS/MS identification and quantification of tryptic peptides from IP materials revealed a variety of interacting candidates, including the E3 ligase TRIM21 and ubiquitin. The interaction of Cx43 with TRIM21 was confirmed by confocal microscopy and coimmunoprecipitation of these proteins from C6 rat glioma and mouse primary astrocyte cultures. To gain a better understanding of this interaction, complexes isolated by high-resolution size-exclusion chromatography revealed signal integration by phosphorylation, ubiquitylation and proteolytic turnover within complexes of Cx43/TRIM21. Cx43/TRIM21 is also responsive to E1 UBE1 and E2 UbcH5a, with the interruption of this activity being an effective inhibitor of in vitro ubiquitin-conjugation. Mathematical models of these complexes demonstrated a mechanism for the switch-like degradation of GJs that were validated in EGF-stimulated cell cultures. Our finding of the interaction of Cx43 with TRIM21 provides mechanisms for the down-regulation of GJ intercellular communication that are known to impact a variety of physiological processes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Chen
authors.forename: Anders R
authors.initials: AR
authors.lastname: Kristensen
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: Christian C
authors.initials: CC
authors.lastname: Naus
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002450:Cell Communication; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005910:Glioma / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D010766:Phosphorylation; D061251:Primary Cell Culture; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D021381:Protein Transport; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods; D014162:Transfection; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000099208:TRIM21 Protein
chemical_list: D018031:Connexin 43; D004815:Epidermal Growth Factor; D046988:Proteasome Endopeptidase Complex; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases; C580592:Gja1 protein, rat; C576159:Trim21 protein, rat; D000099208:TRIM21 Protein; C061553:ATP dependent 26S protease