Identification of Src phosphorylation sites in the catenin p120ctn.

Mariner DJ, Anastasiadis P, Keilhack H, Böhmer FD, Wang J, Reynolds AB
J Biol Chem. 2001 276 (30): 28006-13

PMID: 11382764 · DOI:10.1074/jbc.M102443200

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.

MeSH Terms (27)

Animals Binding Sites Blotting, Western Catenins Cell Adhesion Molecules Cell Line COS Cells Cytoplasm DNA Mutational Analysis Electrophoresis, Gel, Two-Dimensional Epidermal Growth Factor Gene Deletion Genes, Dominant Glutathione Transferase Humans Mutagenesis, Site-Directed Mutation Phenylalanine Phosphoproteins Phosphorylation Precipitin Tests Protein Binding Protein Structure, Tertiary Proto-Oncogene Proteins pp60(c-src) Transfection Tyrosine Vanadates

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