ABSTRACT
To better understand the mechanisms that regulate p120-catenin (p120) and E-cadherin function, we are systematically generating phospho-specific monoclonal antibodies (MAb) to the major p120 phosphorylation sites. p120 has emerged recently as a master regulator of E-cadherin stability and an important modulator of RhoGTPase activities. A number of phosphorylation sites have been identified, but none have as yet been linked to specific regulatory roles. Here, we describe a novel phospho-specific monoclonal antibody to the major PKC-induced p120 phosphorylation site, phospho-serine 879 (pS879). With a few exceptions, p120 MAb pS879 is remarkably specific for the phosphorylated S879 epitope and works effectively in common applications such as Western blot analysis, immunoprecipitation, and immunofluorescence. p120 MAb pS879 should facilitate efforts to identify the role of S879 phosphorylation and to map signaling pathways that modify p120 function through activation of PKC.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Phospho-Specific/biosynthesis , Antibodies, Phospho-Specific/chemistry , Antibody Specificity , Cell Adhesion Molecules/immunology , Phosphoproteins/immunology , Serine/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Phospho-Specific/metabolism , COS Cells , Catenins , Cell Adhesion Molecules/deficiency , Cell Adhesion Molecules/genetics , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Dogs , Epitopes/immunology , HCT116 Cells , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Phosphoproteins/deficiency , Phosphoproteins/genetics , Phosphorylation , Rats , Serine/genetics , Serine/metabolism , Delta CateninABSTRACT
p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity.
