Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia.
Polarized monolayers of strain II Madin-Darby canine kidney cells (MDCK II) were treated with vanadate/H2O2, known inhibitors of protein tyrosine phosphatase activity. Vanadate/H2O2 treatment resulted in a rapid increase in paracellular permeability as revealed by decreased transepithelial resistance and increased permeability to inulin. These alterations in epithelial barrier function coincided with increased phosphotyrosine immunofluorescence in the vicinity of intercellular junctions and with redistribution of F-actin, the adherens junction protein E-cadherin and the tight junction protein ZO-1. The effects of vanadate/H2O2 on intercellular junction permeability and protein distribution were completely blocked by the specific protein tyrosine kinase (PTK) inhibitor tyrphostin 25 and partially inhibited by the alternative PTK inhibitor genistein. The relative potency of these two inhibitors in blocking the effects of vanadate/H2O2 on intercellular junctions correlated with their abilities to inhibit tyrosine phosphorylation. The potent ser/thr protein kinase inhibitor staurosporine had only a small influence on the vanadate/H2O2-induced increase in paracellular permeability and did not affect the observed redistribution of intercellular junction proteins or phosphotyrosine immunofluorescence. The relative potencies of these distinct protein kinase inhibitors in reversing the effects of vanadate/H2O2 indicate that these effects are directly related to tyrosine phosphorylation. In conclusion, our data provide evidence that enhanced tyrosine phosphorylation of intercellular junction proteins in MDCK epithelia increases paracellular permeability and can also induce prominent reorganization of the junctional complex.
Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School, UK. collares@obelix.unicamp.br
C B
CB
Collares-Buzato
M A
MA
Jepson
N L
NL
Simmons
B H
BH
Hirst
D000199:Actins / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D004285:Dogs; D019833:Genistein / Q000494:pharmacology; D007150:Immunohistochemistry; D007444:Inulin / Q000378:metabolism; D007668:Kidney; D008565:Membrane Proteins / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010545:Peroxides / Q000494:pharmacology; D010590:Phalloidine / Q000031:analogs & derivatives; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012235:Rhodamines; D019311:Staurosporine / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D020032:Tyrphostins; D014638:Vanadates / Q000494:pharmacology; D062826:Zonula Occludens-1 Protein
D000199:Actins; D015820:Cadherins; D008565:Membrane Proteins; D009570:Nitriles; D010545:Peroxides; D010750:Phosphoproteins; D012235:Rhodamines; D020032:Tyrphostins; D062826:Zonula Occludens-1 Protein; C079836:tyrphostin 25; D010590:Phalloidine; D019000:Phosphotyrosine; D014638:Vanadates; C041085:tetramethylrhodaminylphalloidine; D007444:Inulin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D019311:Staurosporine