Independent regulation of adherens and tight junctions by tyrosine phosphorylation in Caco-2 cells.
To study the role of tyrosine phosphorylation in the control of intercellular adhesion of intestinal cells, we have generated several clones of Caco-2 cells that express high levels of pp60v-src only after addition of butyrate. Expression of this oncogene in cells 5 days after confluence induced beta-catenin and p120-ctn tyrosine phosphorylation, redistribution of E-cadherin to the cytosol and disassembly of adherens junctions. However, tight junctions of Caco-2 cells at 5 days after confluence were not altered by expression of pp60v-src. Similar results were obtained when Caco-2 cells were incubated with phosphotyrosine phosphatase inhibitor orthovanadate. Although addition of this compound to postconfluent cells disrupt adherens junctions, tight junctions remain unaltered, as determined measuring monolayer permeability to mannitol or hyperphosphorylation of Triton-insoluble occludin. Modifications in tight junction permeability of Caco-2 were only observed at high concentrations of orthovanadate (1 mM). Interestingly, this tyrosine phosphorylation-refractory state was achieved after confluence since early postconfluent cells (day 2) showed a limited but significant response to low doses of orthovanadate. These results suggest that tight junctions of differentiated Caco-2 cells are uncoupled from adherens junctions and are insensitive to regulation by tyrosine phosphorylation.
Gómez
S
S
Unitat de Biologia Cel.lular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, C/Dr. Aiguader 80, 08003, Barcelona, Spain.
del Mont Llosas
M
M
Verdú
J
J
Roura
S
S
Lloreta
J
J
Fabre
M
M
García de Herreros
A
A
D018938:Caco-2 Cells; D015820:Cadherins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008854:Microscopy, Electron; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D015820:Cadherins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D014638:Vanadates; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src)