Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium

Abstract

SummaryLittle is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKCf. We recently reported that Eya1 phosphatase functions through aPKCf-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCf phosphorylation levels, aPKCf-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKCf in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1 2/2 lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKCf-Notch1-Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.

Multiple Functions of the Eya Phosphotyrosine Phosphatase

“…As discussed above for H2AX, the work by El-Hashash and colleagues (17)(18)(19) has opened up new directions for exploring Eya function and regulation. Of particular significance to the field, this is the first demonstration of a cytoplasmic function for Eya during animal development.…”

“…Genetic rescue assays using overexpressed phosphatase-dead Eya transgenes suggested a requirement for cytoplasmic tyrosine phosphatase activity, leading to a model in which Abl-mediated tyrosine phosphorylation recruits Eya phosphatase activity to cytoplasmic signaling centers, and autocatalytic dephosphorylation returns Eya to the nucleus to regulate gene expression (56). Somewhat ironically, although the relevance of these phenomena to Drosophila physiology has since been called into question (55), as discussed below, the prediction of cytoplasmic Eya tyrosine phosphatase activity has been validated in mammals (17,18).…”

“…To date, three protein substrates of the mammalian Eya phosphotyrosine phosphatase have been identified: the histone H2A variant H2AX, the atypical protein kinase C zeta (aPKC), and the estrogen receptor beta (ER␤) (17,18,(64)(65)(66) (Fig. 3).…”

“…The first cytoplasmic Eya tyrosine phosphatase substrate, aPKC, emerged from exploration of the requirement for Eya1 in lung epithelial morphogenesis (17,18). Briefly, apically localized cytoplasmic Eya1 protein was found to overlap both the aPKC-Par polarity complex and the tight junction proteins ZO-1, occludin, and claudin in mouse embryonic lung epithelia.…”

“…In contrast to Drosophila Eya, which appears constitutively nuclear (13), mammalian Eya proteins show significant cytoplasmic accumulation and rely on binding to Six for nuclear recruitment and retention (11,(13)(14)(15)(16)(17)(18)(19)(20). Although initially dismissed as reflecting two slightly different mechanisms for regulating Eyamediated transcriptional events, in retrospect, as discussed later in this minireview, this observation seems to have foretold a much more profound functional divergence between mammalian and fly Eya proteins.…”