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

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.

Tricellulin secures the epithelial barrier at tricellular junctions by interacting with actomyosin

“…These examples suggest that the reversible permeability of cells and substances through tricellular junctions of epithelial cells is regulated by mechanisms still unknown. Because tricellulin undergoes post-translational modifications such as phosphorylation ( Ikenouchi et al, 2005 ), and various kinases are reportedly involved in the regulation of the formation of tTJs ( Kojima et al, 2010 ; Nakatsu et al, 2019 ), it will be important to elucidate how and under what physiological conditions the interaction between α-catenin and tricellulin is regulated in the future studies.…”

Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells

“…This might affect the barrier function and formation as evidenced by Kirschner et al through different knockdowns of claudin-1 and -4, occludin, and ZO-1, causing increased paracellular permeability for ions and larger molecules [37]. Furthermore, the absence of occludin and claudin-1 in both KO clones prevents the specific localization of tricellulin at tricellular contacts and promotes its localization at the bTJ [38,39]. These observations suggested that the proteins functionally influence each other.…”

“…Therefore, it can be concluded that the observed clonal variation of the claudins and tricellulin in sum is balanced and provides a constant barrier function of the bTJ and tTJ. These results suggested that the effects of angulin-1 and tricellulin relocalization from tTJs to bTJs on epithelial barrier function are controversial [39]. Interestingly, claudin-2 was downregulated in the KO 12 clone and upregulated in the KO 32 clone and appeared not to affect the water permeability.…”

“…On the other hand, angulin-1 knockout in HT-29/B6 cells increased the expression of barrier-forming claudins [39] and tricellulin (Figure 2d). Therefore, it can be concluded that the observed clonal variation of the claudins and tricellulin in sum is balanced and provides a constant barrier function of the bTJ and tTJ.…”

“…The effects of angulin-1 knockout on the epithelial barrier function vary by cell type and according to the interdependence between different TJ species affecting cell expression, localization and renewal. The reason could be the differences in angulin-1 interaction partners in bTJs and tTJ [22,39]. Therefore, combinations of proteins influenced by angulin-1 knockout in bTJs and tTJ vary by cell type and tissue, and the resulting epithelial barrier function of the mammalian epithelial cell sheet causes cell specific changes.…”