Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase alpha

J Biol Chem. 2000 Jul 7;275(27):20754-61. doi: 10.1074/jbc.M001626200.

Abstract

We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase alpha (RPTPalpha). Here we report that a substrate-trapping mutant of the RPTPalpha membrane proximal catalytic domain (D1), RPTPalpha-D1-C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTPalpha (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTPalpha-D1-C433S varied between cell lines, but a protein of approximately 130 kDa was pulled down from every cell line. This protein was identified as p130(cas). Tyrosine-phosphorylated p130(cas) from fibronectin-stimulated NIH3T3 cells bound to RPTPalpha-D1-C433S as well, suggesting that p130(cas) is a physiological substrate of RPTPalpha. RPTPalpha dephosphorylated p130(cas) in vitro, and RPTPalpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130(cas), and RPTPalpha or inactive, mutant RPTPalpha indicated that RPTPalpha dephosphorylated p130(cas) in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTPalpha under these conditions, suggesting that p130(cas) is a specific substrate of RPTPalpha in living cells. In conclusion, our results provide evidence that p130(cas) is a physiological substrate of RPTPalpha in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Crk-Associated Substrate Protein
  • Fibronectins / pharmacology
  • Fluorescent Antibody Technique
  • Mice
  • Mutation
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphotyrosine / metabolism
  • Protein Binding / genetics
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / metabolism*
  • Proteins*
  • Retinoblastoma-Like Protein p130
  • Substrate Specificity
  • Transfection

Substances

  • Bcar1 protein, mouse
  • Crk-Associated Substrate Protein
  • Fibronectins
  • Phosphoproteins
  • Proteins
  • Retinoblastoma-Like Protein p130
  • Phosphotyrosine
  • Protein Tyrosine Phosphatases