Identification of cadherin tyrosine residues that are phosphorylated and mediate Shc association

Previously, we reported association of the adaptor protein Shc through its SH2 domain with the cytoplasmic domain of the adhesion molecule cadherin (X u et al. [1997] J. Biol. Chem. 272:13463-13466). This association was depen dent on tyrosine phosphorylation of cadherin and could be modulated by extr acellular Ca2+ and epidermal growth factor in intact cells. There are six t yrosine residues in the cytoplasmic domain of cadherin. To define the tyros ine residue(s) that mediate Shc recognition, site-directed mutagenesis was employed to alter Tyr851 and/or Tyr883 in cadherin, which both conform to a predicted Shc SH2 domain recognition sequence, Mutation of either Tyr851 o r Tyr883, but mostly the latter, decreased Src phosphorylation of cadherin and the binding of Shc to cadherin, as determined by Sepharose bead binding and gel overlay assays. Of the two tyrosine residues, Tyr883 is the major Src phosphorylation and Shc binding site. However, the double mutant (Tyr85 1, 883 Phe) exhibited less Shc association than the single Tyr883 Phe mutan t, suggesting a role for Tyr851 also. In addition, the binding of Shc to th e cadherin cytoplasmic domain was competitively inhibited by tyrosine phosp horylated peptides containing either Tyr851 or Tyr883, but not by the corre sponding non-phosphorylated peptides. Mutation of Tyr851 and/or Tyr883 did not alter the capacity of the cytoplasmic domain of cadherin to bind beta-c atenin in vitro. However, Shc binding to cadherin did negatively influence beta-catenin binding to the same molecule. J. Cell. Biochem. 75:264-271, 19 99. (C) 1999 Wiley-Liss, Inc.