Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells.
Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca(2+) was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca(2+) resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca(2+) or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca(2+) repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.
Chen
Yan-Hua
YH
Department of Anatomy and Cell Biology, East Carolina University School of Medicine, Greenville, NC 27858, USA. cheny@mail.ecu.edu
Lu
Qun
Q
Goodenough
Daniel A
DA
Jeansonne
Beverly
B
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D062793:Occludin; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051575:Proto-Oncogene Proteins c-yes; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D019108:Tight Junctions; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
D008565:Membrane Proteins; D062793:Occludin; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D011758:Pyrroles; D014443:Tyrosine; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; C119957:CGP 77675; D002118:Calcium