Snail Destabilizes Cell Surface Crumbs3a

Abstract

During Epithelial to Mesenchymal Transition (EMT), cells modulate expression of proteins resulting in loss of apical-basal polarity. Effectors of this EMT switch target the polarity protein Crumbs3a, a small transmembrane protein that is essential for generation of the apical membrane and tight junctions of mammalian epithelial cells. We previously showed that the Crumbs3 gene is a direct target of transcriptional regulation by Snail, a potent inducer of EMT. However, Snail has also been shown to have multiple non-transcriptional roles, including regulation of cell adhesion, proliferation and survival. Using SNAP-tag labeling, we determined that cell surface Crumbs3a has a half-life of approximately 3 hours and that this cell surface half-life is significantly reduced when EMT is induced by Snail. We further observe that Snail induces differential glycosylation of Crumbs3a, including sialylation, suggesting a mechanism by which Crumbs3a may be destabilized. These results indicate that Crumbs3a is a post-translational target of Snail, in addition to being a transcriptional target. We conclude that Snail’s ability to post-translationally modify and destabilize Crumbs3a augments the depolarizing process of EMT.

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

“…Like the native Crumbs3a protein, SNAP-Crumbs3a is distributed over the entire apical surface and is more highly concentrated at or near the tight junctions (Harder et al, 2012; Fig. S2 A).…”

“…To examine surface delivery of another apical protein, we used a previously characterized MDCK cell line expressing SNAP-tagged Crumbs3a (Harder et al, 2012). Like the native Crumbs3a protein, SNAP-Crumbs3a is distributed over the entire apical surface and is more highly concentrated at or near the tight junctions (Harder et al, 2012; Fig.…”

Membrane-Initiated Estradiol Signaling of Epithelial-Mesenchymal Transition-Associated Mechanisms Through Regulation of Tight Junctions in Human Breast Cancer Cells

“…To show that MCF-7 cells have these features, we evaluated the effects of E2 on the expression of CRB3, a novel epithelial marker [15], and N-cadherin, a mesenchymal marker. CRB3 is the human ortholog of the crumbs (CRB) protein and is required to establish the apical membrane, define the basolateral plasma domain, and has a fundamental function in the establishment of morphogenesis of 3D epithelial cell cultures [43].…”

“…CRB3 is the human ortholog of the crumbs (CRB) protein and is required to establish the apical membrane, define the basolateral plasma domain, and has a fundamental function in the establishment of morphogenesis of 3D epithelial cell cultures [43]. CRB3 is a tumor suppressor during the transformation and progression of mammalian epithelial cells; its loss favors tumor cell growth, metastasis, and EMT [15,44]. We found that E2 downregulates CRB3 expression in MCF-7 cells; therefore, it is possible that CRB3 could be used as a novel marker in EMT, and its role in breast carcinogenesis has just begun to be unveiled.…”

“…Some evidence suggests that TJ protein phosphorylation plays an important role in the regulation of cell-cell adhesion [13,14]. It is worth noting that occludin and CRB3, intercellular binding proteins, have been used as epithelial and polarity markers in EMT studies [15,16]. In our study, to understand the complex process of tumor progression, we attempted to demonstrate that estradiol induces EMT-associated mechanisms, including c-Src phosphorylation, p-Src/ZO-1 complex formation, ZO-1/ZONAB nuclear translocation, increased permeability, downregulation of occludin and CRB3 expression, increased N-cadherin expression, and motility of breast cancer cells, through a novel pathway involving TJ proteins.…”