Tyrosine-phosphorylation of the 12th armadillo-repeat of β-catenin is associated with cadherin dysfunction in human cancer

Abstract

Abstract. Tyrosine phosphorylation of ß-catenin, an intracytoplasmic cadherin-binding protein, causes disruption of the cadherin-mediated cell adhesion system in cancer cells. We identified that the c-erbB-2 protein activated by TGF· was capable of binding to the 12th armadillo repeat (arm12) of ß-catenin with increased phosphorylation-state level. We produced a monoclonal antibody, 4G7, which recognizes a phosphorylated-tyrosine residue (Tyr-654) located at arm12 of ß-catenin. Tyrosine phosphorylation of the arm12 was detected after stimulation with TGF· in TMK-1. Immunoprecipitation analyses using 4G7, anti-ß-catenin, ·-catenin, the c-erbB-2 gene product and phosphotyrosine antibodies indicated that tyrosine phosphorylation of the arm12 was closely correlated with dissociation between E-cadherin/ ß-catenin and ·-catenin or the c-erbB-2 gene product, but not with dissociation between ß-catenin and E-cadherin. Immunocytochemical staining of TMK-1 cells using the 4G7 antibody revealed that tyrosine phosphorylation of the arm12 was localized at cell-cell contact sites of cancer cells transiently and only after TGF· treatment. Immunohistochemical localization of the 4G7 antibody was observed in cancer cells at the invasive front where they detached from cancer nests. These findings indicated that the tyrosine phosphorylation of arm12 is a key marker of cadherin dysfunction and the 4G7 antibody may be useful in screening for a ß-catenin phosphorylation ligand or tyrosine kinases.