Evidence that increased tyrosine phosphorylation causes disassembly of adherens junctions but does not perturb paracellular permeability in Caco-2 cells

In this study, we report on the apparent effect of increased tyrosine phosp horylation events on the assembly and integrity of adherens junctions (AJs) and on paracellular permeability in Caco-2 cells. Cell monolayers were inc ubated with the phosphotyrosine phosphatase inhibitor vanadate/H2O2. Additi on of this compound to monolayer resulted in disruption of the AJs, as reve aled by electron microscopy and by a loss of membrane association of the AJ -associated protein uvomorulin/E-cadherin (U/E-c). However, tight junctions (TJs) were unaltered, as determined by measuring the transepithelial resis tance (Rt), by ruthenium red labeling, as seen by transmission electron mic roscopy, and the distribution of TJ strands as seen in freeze-fracture repl icas and by hyperphosphorylation of triton-insoluble occludin. Also examina tion of vanadate/H2O2 treated cells indicated a specific increase in AJ-ass ociated phosphotyrosine residues as evaluated by immunofluorescence microsc opy, but no modification of F-actin distribution, as revealed by confocal l aser scanning microscopy analysis. To verify that modulation of AJs was ind eed related to tyrosine phosphorylation, we tested a range of distinct prot ein kinase inhibitors. Of the three inhibitors tested (tyrphostin 25, genis tein and staurosporine), tyrphostin 25 completely blocked the effects of va nadate/H2O2 on assembly and integrity of AJs, redistribution of U/E-c and p hosphotyrosine labeling. Our results indicate that, after addition of vanad ate/H2O2 to Caco-2 monolayers, specific tyrosine phosphorylation of protein s cause disruption of AJs, but no modifications of the TJs' structure and f unctionality. These observations suggest that, in contrast to what happens with epithelial cells, TJs and AJs of Caco-2 cells are regulated by indepen dent mechanisms. (C) 2001 Harcourt Publishers Ltd.