MAGUK proteins and junctions

In recent years intriguing evidence has accumulated suggesting that junctional proteins not only exert functions related to structural barrier mechanisms but are also involved in signal transduction. They receive and convert signals from the cell interior to regulate junction assembly and function, and transmit signals to the cell interior to modulate gene expression, cell growth and behavior. Zonula occludens proteins (ZOPs) are multi-domain scaffolding proteins, localizing preferentially at tight junctions. ZOPs, currently comprising ZO-1, ZO-2, and ZO- 3, are structurally similar to the membrane-associated guanylate kinase-like (MAGUK) protein family of signaling molecules. The homology of ZOPs with the Drosophila tumour suppressor protein dlgA has provided a first clue towards a yet undefined role of ZOPs in epithelial cancer development and/or progression. The molecular pathways by which the tight junction-associated ZOPs interfere with cell growth and proliferation are far from being understood. In a recent study we have shown that ZO-2 localizes to the nucleus of epithelial and endothelial cells. Using a yeast-based Two-Hybrid assay and co-immunoprecipitation we have demonstrated that ZO-2 interacts directly with the nuclear scaffold attachment factor-B (SAF-B), a co-repressor of estrogen receptor- alpha, known to regulate transcription of genes involved in proliferation, apoptosis, migration and other cellular processes. The aim of this project is to further explore the biological significance of nuclear ZO-2 in tumour and non-tumour epithelial and endothelial cells. We will elaborate our earlier findings concerning the nuclear accumulation of ZO-2 focusing on upstream and downstream events related to the aberrant localization of ZO-2 at the protein and genomic level. We will further dissect the interaction of ZO-2 with SAF-B and will focus on a possible interplay between ZO-2/SAF-B binding and estrogen receptor-alpha activity. The technical spectrum of the project will comprise cell culture and transfection experiments, confocal microscopy, site-directed mutagenesis, pathway-focused gene profiling, 2D gel electrophoresis, mass spectrometric analysis (MALDI), co-immunoprecipitation and Two-Hybrid assays.

Occluding intercellular contacts (junctions) consist of single- or multi-pass transmembrane components, connected to cytoplasmic proteins, which are collectively referred to as peripheral junctional proteins. The extracellular domains of the integral junctional proteins of two opposing cells interact in a homophilic or heterophilic manner to create a paracelluar "seal". So far, a plethora of proteins locating at the cytoplasmic surface of junctional contact sites have been discovered, the role of which has just partly been unraveled. Zonula occludens proteins (ZOPs) are peripheral, scaffolding proteins which belong to the large family of MAGUK (membrane-associated guanylate kinase-like) proteins. These multidomain proteins, currently comprising ZO-1, -2, and -3, are critically involved in the structural organization of epithelial and endothelial intercellular junctions but are also components of intracellular signaling networks. In earlier studies we have shown that ZO-2 localizes to the nucleus of cultured endothelial and epithelial cells in response to various environmental stress conditions including heat shock or chemical insult. During the last couple of years we have focused on the nuclear targeting of ZO-2 and on its intranuclear activities. To this end, we have generated epithelial and endothelial cell lines, stably overexpressing nuclear ZO-2. Following such experimentally induced nuclear accumulation of ZO-2, a series of experiments have been carried out to identify novel protein-protein interactions of nuclear ZO-2 in epithelial and endothelial cells. Our results provide evidence to suggest that accumulation of nuclear ZO-2 alters gene and protein expression of epithelial and endothelial cells. Proteins found to be increased or decreased following nuclear overexpression of ZO-2 may be related to cell metabolism, barrier function, migration and oxidative stress response. We have further shown that ZO-2 directly associates with a transcriptional regulator, known to be involved in cytoprotection during oxidative stress/hypoxia, and with a multifunctional nucleolar phosphoprotein. In addition, nuclear accumulation of ZO-2 was shown to trigger the down-regulation of a distinctive class of matrix-metalloproteinases, which was also reflected by a substantially reduced migratory activity of cells containing high levels of nuclear ZO-2. Since downregulation of such enzymes is considered to exert protective effects in ischemic/hypoxic conditions, it remains to be clarified whether nuclear targeting of ZO proteins may represent a first step in a signalling cascade elicited by oxidative stress. Taken together, our data support the notion that ZO proteins do exert a "dual "function. They support the structural organization of junctional complexes at the plasma membrane, but are also involved intracellular cell signalling and modulation of gene expression.