Identification of p130(cas) as an in vivo substrate of receptor protein-tyrosine phosphatase alpha

We have employed a substrate trapping strategy to identify physiological su bstrates of the receptor protein-tyrosine phosphatase alpha (RPTP alpha). H ere we report that a substrate-trapping mutant of the RPTP alpha membrane p roximal catalytic domain (D1), RPTP alpha-D1C433S, specifically bound to ty rosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTP alpha (D2) and mutants thereof did not bin d to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylat ed proteins that bound to RPTP alpha-D1-C433S varied between cell lines, bu t 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 w eb, suggesting that p130(cas) is a physiological substrate of RPTP alpha. R PTP alpha dephosphorylated p130(cas) in vitro, and RPTP alpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection e xperiments with activated SrcY529F, p130(cas), and RPTP alpha or inactive, mutant RPTP alpha indicated that RPTP alpha dephosphorylated p130(cas) in v ivo. Tyrosine-phosphorylated epidermal growth factor receptor was not depho sphorylated 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 RPT P alpha in vivo.