ZO proteins redundantly regulate the transcription factor DbpA/ZONAB.

The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus.