title: Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.
abstract: Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.
authors: Unit of Virus Host Cell Interactions (UVHCI), UJF Grenoble1-EMBL-CNRS UMI 3265, 6 rue Jules Horowitz, BP 181, F-38042 Grenoble Cedex 9, France.
authors: Audrey
authors: A
authors: Conti
authors: Charlotte
authors: C
authors: Sueur
authors: Julien
authors: J
authors: Lupo
authors: Xavier
authors: X
authors: Brazzolotto
authors: Wim P
authors: WP
authors: Burmeister
authors: Evelyne
authors: E
authors: Manet
authors: Henri
authors: H
authors: Gruffat
authors: Patrice
authors: P
authors: Morand
authors: Véronique
authors: V
authors: Boyer
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D022005:Adherens Junctions / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008969:Molecular Sequence Data; D015398:Signal Transduction; D019108:Tight Junctions / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D017868:Cyclic AMP-Dependent Protein Kinases