title: Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process.
abstract: Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.
authors.affiliation: Deutsches Krebsforschungszentrum, Im Neuenheimer Feld-242, 69120 Heidelberg, Germany. k.leykauf@dkfz.de
authors.forename: Kerstin
authors.initials: K
authors.lastname: Leykauf
authors.forename: Mojibrahman
authors.initials: M
authors.lastname: Salek
authors.forename: Jörg
authors.initials: J
authors.lastname: Bomke
authors.forename: Matthias
authors.initials: M
authors.lastname: Frech
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Lehmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Dürst
authors.forename: Angel
authors.initials: A
authors.lastname: Alonso
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018031:Connexin 43; D056827:Endosomal Sorting Complexes Required for Transport; D010455:Peptides; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases