Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions

BMC Cell Biology, Sep 2013

Background Epithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified. Results This study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin. Conclusions PP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://www.biomedcentral.com/content/pdf/1471-2121-14-42.pdf

Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions

Ohama et al. BMC Cell Biology 0 Center for Cell Signaling and Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine , Box 800577, West Complex MSB 7225, Charlottesville, Virginia 22908 , USA Ohama et al. - Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions Open Access Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions Takashi Ohama1,2, Lifu Wang1, Erin M Griner1 and David L Brautigan1* Background: Epithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified. Results: This study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin. Conclusions: PP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells. Background The physiological function and physical integrity of epithelial tissues depends on contacts between cells that include adherens junctions and tight junctions (for review [1]). Adherens junctions are cell-cell attachments of epithelial cells that are required for the subsequent construction of tight junctions and maintenance of cell polarization. Adherens junctions are based on Ca2+-dependent homotypic interactions of the extracellular domains of E-cadherin between neighboring cells. The cytosolic portion of the transmembrane E-cadherin is best known to bind to -catenin, but as many as 170 proteins are reported to colocalize and/or associate with cadherin in adherens junctions in what has been called the Cadhesome [2]. E-cadherin mediated cell-cell junctions play an important role in contact inhibition of cell growth, and loss of E-cadherin expression occurs during tumor progression and metastasis [3,4]. This makes it important to understand the mechanisms that govern E-cadherin expression and distribution to the cell surface. Wnt signaling plays a role in the distinctive switching of gene expression in epithelial cells. Wnt activity is dramatically decreased after formation of cell-cell contacts [5]. The E-cadherin/catenin complex physically associates with the Wnt co-receptor and is necessary for downstream -catenin activation [6,7]. Phosphorylation is an established mechanism for regulating cadherin, and E-cadherin/catenin complexes are regulated by Ser phosphorylation [8]. Casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) phosphorylate human E-cadherin at Ser838, Ser853, and Ser855, which enhances the interaction with -catenin [9]. On the other hand, CK1 phosphorylation of human Ser844 (mouse Ser846) in the same region of E-cadherin disrupts cell-cell contacts with internalization of E-cadherin [10]. These phosphoSer sites in E-cadherin constitute a recognition site for Skp2, leading to ubiquitination and degradation of E-cadherin, with an increase in cell migration and tumorigenesis [11]. The protein Ser/Thr phosphatases that regulate E-cadherin by countering CK1 and CK2 phosphorylation are unknown. Here, we discovered protein phosphatase-6 (PP6), which is highly expressed in epithelial cells, localizes to adherens junctions and associates with E-cadherin/catenin complexes in human epithelial cells. Levels of PP6c mRNA and protein are significantly increased in confluent cells and inducible RNAi to deplete PP6c in confluent monolayers results in internalization of E-cadherin. The effects of PP6c knockdown are prevented by chemical inhibition of CK1 or by mutation of Ser846 in mouse E-cadherin to a nonphosphorylated Ala, showing PP6c requires this site in E-cadherin to regulate localization in adherens junctions. Our results show PP6 is a key regulator of E-cadherin and suggests feedback up-regulation of PP6 to support E-cadherin localization. Results Protein phosphatase-6 in human epithelial cells Protein phosphatase-6 catalytic subunit (PP6c) expression is highest in the gastrointestinal tract and in hematopoietic cells, based on tissue a (...truncated)


This is a preview of a remote PDF: http://www.biomedcentral.com/content/pdf/1471-2121-14-42.pdf

Takashi Ohama, Lifu Wang, Erin M Griner, David L Brautigan. Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions, BMC Cell Biology, 2013, pp. 42, 14, DOI: 10.1186/1471-2121-14-42