RESUMO
The zonula occludens (ZO)-2 protein links tight junctional transmembrane proteins to the actin cytoskeleton and associates with splicing and transcription factors in the nucleus. Multiple posttranslational modifications control the intracellular distribution of ZO-2. Here, we report that ZO-2 is a target of the SUMOylation machinery and provide evidence on how this modification may affect its cellular distribution and function. We show that ZO-2 associates with the E2 SUMO-conjugating enzyme Ubc9 and with SUMO-deconjugating proteases SENP1 and SENP3. In line with this, modification of ZO-2 by endogenous SUMO1 was detectable. Ubc9 fusion-directed SUMOylation confirmed SUMOylation of ZO-2 and was inhibited in the presence of SENP1 but not by an enzymatic-dead SENP1 protein. Moreover, lysine 730 in human ZO-2 was identified as a potential modification site. Mutation of this site to arginine resulted in prolonged nuclear localization of ZO-2 in nuclear recruitment assays. In contrast, a construct mimicking constitutive SUMOylation of ZO-2 (SUMO1ΔGG-ZO-2) was preferentially localized in the cytoplasm. Based on previous findings the differential localization of these ZO-2 constructs may affect glycogen-synthase-kinase-3ß (GSK3ß) activity and ß-catenin/TCF-4-mediated transcription. In this context we observed that ZO-2 directly binds to GSK3ß and SUMO1ΔGG-ZO-2 modulates its kinase activity. Moreover, we show that ZO-2 forms a complex with ß-catenin. Wild-type ZO-2 and ZO-2-K730R inhibited transcriptional activity in reporter gene assays, whereas the cytosolic SUMO1ΔGG-ZO-2 did not. From these data we conclude that SUMOylation affects the intracellular localization of ZO-2 and its regulatory role on GSK3ß and ß-catenin signaling activity.
Assuntos
Espaço Intracelular/metabolismo , Sumoilação , Proteína da Zônula de Oclusão-2/metabolismo , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/metabolismo , Cães , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transfecção , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteína da Zônula de Oclusão-2/química , beta Catenina/metabolismoRESUMO
Tight junctions (TJs) regulate the transit of ions and molecules through the paracellular pathway in epithelial cells. Zonula occludens 2 (ZO-2) is a cytoplasmic TJ protein. Here, we studied the ubiquitination of hZO-2 employing mutants of SUMOylation site K730 present in the GuK domain and the putative ubiquitination residues K759 and K992 located at the GuK domain and proline-rich region, respectively. In immunoprecipitation experiments done with MDCK cells transfected with wild-type (WT) hZO-2 or the ubiquitination-site mutants hZO-2-K759R or -K992R, we observed diminished ubiquitination of the mutants, indicating that residues K759 and K992 in hZO-2 are acceptors for ubiquitination. Moreover, using TUBES, we found that residues K759 and K992 of hZO-2 are targets of K48 polyubiquitination, a signal for proteasomal degradation. Accordingly, compared to WT hZO-2, the half-life of hZO-2 mutants K759R and K992R augmented from 19.9 to 37.3 and 23.3 h, respectively. Instead, the ubiquitination of hZO-2 mutant K730R increased, and its half-life diminished to 6.7 h. The lack of these lysine residues in hZO-2 affects TJ sealing as the peak of TER decreased in monolayers of MDCK cells transfected with any of these mutants. These results highlight the importance of ZO-2 ubiquitination and SUMOylation to maintain a healthy and stable pool of ZO-2 molecules at the TJ.
Assuntos
Sumoilação , Junções Íntimas , Proteína da Zônula de Oclusão-2/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Lisina/metabolismo , Fosfoproteínas/metabolismo , Linhagem Celular , Prolina/metabolismoRESUMO
The apical junctional complex (AJC) is a cell-cell adhesion system present at the upper portion of the lateral membrane of epithelial cells integrated by the tight junction (TJ) and the adherens junction (AJ). This complex is crucial to initiate and stabilize cell-cell adhesion, to regulate the paracellular transit of ions and molecules and to maintain cell polarity. Moreover, we now consider the AJC as a hub of signal transduction that regulates cell-cell adhesion, gene transcription and cell proliferation and differentiation. The molecular components of the AJC are multiple and diverse and depending on the cellular context some of the proteins in this complex act as tumor suppressors or as promoters of cell transformation, migration and metastasis outgrowth. Here, we describe these new roles played by TJ and AJ proteins and their potential use in cancer diagnostics and as targets for therapeutic intervention.
Assuntos
Junções Aderentes/genética , Junções Intercelulares/genética , Neoplasias/genética , Junções Íntimas/genética , Transcrição Gênica , Junções Aderentes/metabolismo , Adesão Celular/genética , Diferenciação Celular/genética , Movimento Celular/genética , Polaridade Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Regulação da Expressão Gênica/genética , Humanos , Transdução de Sinais/genética , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+ in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+ sensing receptor (CaSR) with Gd3+ triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+ triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.