Self-association of PAR-3-mediated by the Conserved N-terminal Domain Contributes to the Development of Epithelial Tight Junctions

Keiko Mizuno,Atsushi Suzuki,Tomonori Hirose,Koichi Kitamura,Koichi Kutsuzawa,Masaaki Futaki,Yoshiko Amano,Shigeo Ohno

doi:10.1074/jbc.m303593200

Keiko Mizuno, Atsushi Suzuki + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m303593200

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Abstract

PAR-3 is a scaffold-like PDZ-containing protein that forms a complex with PAR-6 and atypical protein kinase C (PAR-3-atypical protein kinase C-PAR-6 complex) and contributes to the establishment of cell polarity in a wide variety of biological contexts. In mammalian epithelial cells, it localizes to tight junctions, the most apical end of epithelial cell-cell junctions, and contributes to the formation of functional tight junctions. However, the mechanism by which PAR-3 localizes to tight junctions and contributes to their formation remains to be clarified. Here we show that the N-terminal conserved region, CR1-(1-86), and the sequence 937-1,024 are required for its recruitment to the most apical side of the cell-cell contact region in epithelial Madin-Darby canine kidney cells. We also show that CR1 self-associates to form an oligomeric complex in vivo and in vitro. Further, overexpression of CR1 in Madin-Darby canine kidney cells disturbs the distribution of atypical protein kinase C and PAR-6 as well as PAR-3 and delays the formation of functional tight junctions. These results support the notion that the CR1-mediated self-association of the PAR-3-containing protein complex plays a role during the formation of functional tight junctions.

Highlights

Cell polarity plays essential roles in cell functions and in development and tissue maintenance
We previously demonstrated that the overexpression of atypical protein kinase C (aPKC)␭kn or PAR-3 in MDCK cells affects the formation of tight junctions (TJ) (10, 21), these effects were observed only when ectopic proteins were expressed during the course of cell polarization but not after the cells were fully polarized
The CR1 domain contributes to the localization of PAR-3 on the apical side of the cell-cell contact region in polarized cells, because a PAR-3 mutant lacking CR1 failed to concentrate in the cell-cell contact region (Fig. 3, E and F), and the overexpression of the CR1 domain attenuated the localization of endogenous PAR-3 in the cell-cell contact region (Figs. 4 and 5D)

Summary

Introduction

Cell polarity plays essential roles in cell functions and in development and tissue maintenance. PAR-3-(1–936) lacking the Cterminal domain showed a diffused distribution in the cytosol in addition to weak staining throughout the cell surface (Fig. 3D), suggesting that residues 937–1,024 are required for the localization of PAR-3/sPAR-3 on the most apical side of the cell-cell contact region.

Results

Conclusion