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.
Chen
Ying
Y
Department of Anatomy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.
Chen
Chia-Huei
CH
Tung
Po-Yuan
PY
Huang
Shih-Horng
SH
Wang
Seu-Mei
SM
D022005:Adherens Junctions / Q000378:metabolism*; D015820:Cadherins / Q000737:chemistry*; D051177:Catenins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000737:chemistry; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry; D019061:src-Family Kinases / Q000378:metabolism*; D000095002:Delta Catenin
D015820:Cadherins; D051177:Catenins; D004791:Enzyme Inhibitors; D025801:Ubiquitin; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D046988:Proteasome Endopeptidase Complex; D000095002:Delta Catenin; C000722347:CTNND1 protein, human