An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases-dependent modification of E-cadherin.

We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c-Src-dependent tyrosine phosphorylation of beta-catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6-induced modification of E-cadherin. In control cells cultured at pH 7.4, E-cadherin staining ...
was linear and continuous at cell-cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E-cadherin staining, consistent with the decreased levels of E-cadherin in membrane fractions. pH 6.6 treatment activated c-Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E-cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6-induced tyrosine phosphorylation of E-cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E-cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6-induced detachment of E-cadherin from AJs was blocked by pretreatment with MG132 or NH(4)Cl, indicating the involvement of ubiquitin-proteasomal/lysosomal degradation of E-cadherin. An early loss of p120ctn prior to E-cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E-cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E-cadherin and p120ctn and a weakening of the association of E-cadherin with p120ctn and contributing to the instability of E-cadherin at AJs.
Mesh Terms:
Adherens Junctions, Cadherins, Catenins, Cell Membrane, Enzyme Inhibitors, Gene Expression Regulation, Hep G2 Cells, Humans, Hydrogen-Ion Concentration, Lysosomes, Phosphorylation, Proteasome Endopeptidase Complex, Proto-Oncogene Proteins c-fyn, Ubiquitin, src-Family Kinases
J. Cell. Biochem.
Date: Nov. 01, 2009
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