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.
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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)