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Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
After the
simulation of anoxia by ATP depletion of MDCK cell monolayers with
metabolic inhibitors, the tight junction (TJ) is known to become
structurally perturbed, leading to loss of the permeability barrier.
Peripheral TJ proteins such as zonula occludens 1 (ZO-1), ZO-2, and
cingulin become extremely insoluble and associate into large
macromolecular complexes (T. Tsukamoto and S. K. Nigam.
J. Biol. Chem. 272: 16133-16139,
1997). For up to 3 h, this process is reversible by ATP repletion. We
now show that the reassembly process depends on tyrosine
phosphorylation. Recovery of transepithelial electrical resistance in
ATP-replete monolayers was markedly inhibited by the
tyrosine kinase inhibitor, genistein. Indirect immunofluorescence
revealed a decrease in staining of occludin, a membrane component of
the TJ, in the region of the TJ after ATP depletion, which reversed
after ATP repletion; this reversal process was inhibited by genistein.
Examination of the Triton X-100 solubilities of occludin and several
nonmembrane TJ proteins revealed a shift of occludin and nonmembrane TJ
proteins into an insoluble pool following ATP depletion. These changes reversed after ATP repletion, and the movement of insoluble occludin, ZO-1, and ZO-2 back into the soluble pool was again via a
genistein-sensitive mechanism. Rate-zonal centrifugation analyses of
detergent-soluble TJ proteins showed a reversible increase in higher
density fractions following ATP depletion-repletion, although this
change was not affected by genistein. In
32P-labeled cells,
dephosphorylation of all studied TJ proteins was observed during ATP
depletion, followed by rephosphorylation during ATP repletion;
rephosphorylation of occludin was inhibited by genistein. Furthermore,
during the ATP repletion phase, tyrosine phosphorylation of Triton
X-100-insoluble occludin, which is localized at the junction, as well
as ZO-2, p130/ZO-3 (though not ZO-1), and other proteins was evident;
this tyrosine phosphorylation was completely inhibited by genistein.
This indicates that tyrosine kinase activity is necessary for TJ
reassembly during ATP repletion and suggests an important role for the
tyrosine phosphorylation of occludin, ZO-2, p130/ZO-3, and possibly
other proteins in the processes involved in TJ (re)formation.
occludin; zonula occludens 1; cytoskeleton; tyrosine phosphorylation; ATP depletion
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