title: Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1).
abstract: We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.
authors.affiliation: Unit of Virus Host Cell Interactions, UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lupo
authors.forename: Audrey
authors.initials: A
authors.lastname: Conti
authors.forename: Charlotte
authors.initials: C
authors.lastname: Sueur
authors.forename: Pierre-Alain
authors.initials: PA
authors.lastname: Coly
authors.forename: Yohann
authors.initials: Y
authors.lastname: Couté
authors.forename: Walter
authors.initials: W
authors.lastname: Hunziker
authors.forename: Wim P
authors.initials: WP
authors.lastname: Burmeister
authors.forename: Raphaelle
authors.initials: R
authors.lastname: Germi
authors.forename: Evelyne
authors.initials: E
authors.lastname: Manet
authors.forename: Henri
authors.initials: H
authors.lastname: Gruffat
authors.forename: Patrice
authors.initials: P
authors.lastname: Morand
authors.forename: Véronique
authors.initials: V
authors.lastname: Boyer
mesh_terms: D015815:Cell Adhesion Molecules / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D019073:HT29 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D016601:RNA-Binding Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
chemical_list: C061407:CGN protein, human; D015815:Cell Adhesion Molecules; C483039:MTDH protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C419368:RACK1 protein, human; D016601:RNA-Binding Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C569042:TJP1 protein, human; D014157:Transcription Factors; C406814:UBN1 protein, human; D062826:Zonula Occludens-1 Protein; D019204:GTP-Binding Proteins