Occludin Localizes to Centrosomes and Modifies Mitotic Entry

E Aaron Runkle,Jeffrey M Sundstrom,Kristin B Runkle,Xuwen Liu,David A Antonetti

doi:10.1074/jbc.m111.262857

Abstract

Proper control of cell cycle progression and barrier function are essential processes to the maintenance of epithelial cell homeostasis. The contribution of tight junction proteins to barrier function is well established, whereas their contribution to cell cycle control is only beginning to be understood. Centrosomes are the principal microtubule organizing centers in eukaryotic cells and centrosome duplication and separation are linked to the cell cycle and mitotic entry. Here we demonstrate that occludin localizes with centrosomes in Madin-Darby canine kidney cells. Immunocytochemistry and biochemical fractionation studies reveal occludin localizes with centrosomes during interphase and occludin Ser-490 phosphorylation at centrosomes increases with mitotic entry. Stable expression of aspartic acid phosphomimetic (S490D) results in centrosomal localization of occludin and increases cell numbers. Furthermore, we provide evidence that occludin regulates centrosome separation and mitotic entry as the nonphosphorylatable alanine mutation (S490A) impedes centrosome separation, delays mitotic entry, and reduces proliferation. Collectively, these studies demonstrate a novel location and function for occludin in centrosome separation and mitosis.

Highlights

Occludin was the first membrane spanning Tight junctions (TJs) protein identified and is composed of four membrane spanning domains and an N and C terminus within the cytoplasm [7]
In confluent MDCK cells, occludin is maintained at the cell border co-localizing with zonula occludens (ZO)-1 and addition of platelet-derived growth factor (PDGF) induced occludin Ser-490 phosphorylation in a time- and dose-dependent manner
30 min of PDGF (250 ng/ml) treatment induced an increase in intracellular puncta of occludin phospho-Ser-490 as observed by confocal microscopy suggesting endocytosis of occludin as was observed in endothelial cells treated with vascular endothelial growth factor (VEGF) [18] and was previously reported in MDCK cells in response to PDGF [42]

Summary

Introduction

Occludin was the first membrane spanning TJ protein identified and is composed of four membrane spanning domains and an N and C terminus within the cytoplasm [7]. These data indicate that occludin contributes to centrosomal separation and mitotic entry through Ser-490 phosphorylation and demonstrate a novel function for occludin.

Results

Conclusion