Pyk2-dependent phosphorylation of LSR enhances localization of LSR and tricellulin at tricellular tight junctions.

Daiki Nakatsu,Masayuki Murata,Naeko Shinozaki-Narikawa,Fumi Kano

doi:10.1371/journal.pone.0223300

Abstract

Tight junctions (TJs) are cellular junctions within the mammalian epithelial cell sheet that function as a physical barrier to molecular transport within the intercellular space. Dysregulation of TJs leads to various diseases. Tricellular TJs (tTJs), specialized structural variants of TJs, are formed by multiple transmembrane proteins (e.g., lipolysis-stimulated lipoprotein receptor [LSR] and tricellulin) within tricellular contacts in the mammalian epithelial cell sheet. However, the mechanism for recruiting LSR and tricellulin to tTJs is largely unknown. Previous studies have identified that tyrphostin 9, the dual inhibitor of Pyk2 (a nonreceptor tyrosine kinase) and receptor tyrosine kinase platelet-derived growth factor receptor (PDGFR), suppresses LSR and tricellulin recruitment to tTJs in EpH4 (a mouse mammary epithelial cell line) cells. In this study, we investigated the effect of Pyk2 inhibition on LSR and tricellulin localization to tTJs. Pyk2 inactivation by its specific inhibitor or repression by RNAi inhibited the localization of LSR and downstream tricellulin to tTJs without changing their expression level in EpH4 cells. Pyk2-dependent changes in subcellular LSR and tricellulin localization were independent of c-Jun N-terminal kinase (JNK) activation and expression. Additionally, Pyk2-dependent LSR phosphorylation at Tyr-237 was required for LSR and tricellulin localization to tTJs and decreased epithelial barrier function. Our findings indicated a novel mechanism by which Pyk2 regulates tTJ assembly and epithelial barrier function in the mammalian epithelial cell sheet.

Highlights

We previously identified tyrphostin 9, which is a dual inhibitor of Pyk2 and platelet-derived growth factor receptor (PDGFR), treatment suppresses LSR and tricellulin localization to Tricellular TJs (tTJs) in the mammalian epithelial cell sheet, while AG-370, AG-494, AG-1296, tyrphostin 46, and tyrphostin 51, which are inhibitors of PDGFR, have little or no effect on LSR localization to tTJs [15]
To test whether Tyr-237 dephosphorylation is critical for LSR and tricellulin localization, we examined the localization of green fluorescent protein (GFP)-conjugated LSR(Y237F), a mutant with Tyr-237 substituted with phenylalanine, using confocal microscopy
We assumed that Pyk2 is responsible for phosphorylation at Tyr-237 and dephosphorylation of other tyrosine sites. These results suggested that LSR and tricellulin localization to tTJs is regulated by tyrosine phosphorylation of LSR at Tyr-237, which is mediated by Pyk2

Summary

Introduction

The mammalian epithelial cell sheet contains at least six types of cellular junctions: tight junctions (TJs), adherens junctions, desmosomes, hemidesmosomes, focal adhesions, and gap junctions [1,2,3]. Dysregulation of any of these cellular junctions causes mammalian epithelial cell sheet dysfunction, which, in turn, causes various diseases [2]. In the mammalian epithelial cell sheet, TJs regulate molecular transport within the intercellular space and separate compartments of proteins and lipids localized to apical and basolateral membranes [4,5].

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