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

We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase alpha (RPTP alpha). Here we report that a substrate-trapping mutant of the RPTP alpha membrane proximal catalytic domain (D1), RPTP alpha-D1C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTP alpha (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTP alpha-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 RPTP alpha-D1-C433S as web, suggesting that p130(cas) is a physiological substrate of RPTP alpha. RPTP alpha dephosphorylated p130(cas) in vitro, and RPTP alpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130(cas), and RPTP alpha or inactive, mutant RPTP alpha indicated that RPTP alpha dephosphorylated p130(cas) in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTP alpha under these conditions, suggesting that p130(cas) is a specific substrate of RPTP alpha in living cells. In conclusion, our results provide evidence that p130(cas) is a physiological substrate of RPTP alpha in vivo.