RESUMO
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.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Células HT29 , Humanos , Proteínas de Membrana/química , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA , Receptores de Quinase C Ativada , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Junções Íntimas/metabolismo , Fatores de Transcrição/química , Proteína da Zônula de Oclusão-1RESUMO
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 14-3-3É 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 14-3-3É ligand binding site are responsible for the binding of 14-3-3É 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 14-3-3É. 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 14-3-3É following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.
Assuntos
Proteínas 14-3-3/metabolismo , Junções Aderentes/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Junções Íntimas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cães , Células HT29 , Humanos , Células Madin Darby de Rim Canino , Camundongos , Dados de Sequência Molecular , Transdução de SinaisRESUMO
We report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1.