Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase alpha.
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.
Buist
A
A
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Blanchetot
C
C
Tertoolen
L G
LG
den Hertog
J
J
D016475:3T3 Cells; D000818:Animals; D050719:Crk-Associated Substrate Protein; D005353:Fibronectins / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D051379:Mice; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D013379:Substrate Specificity; D014162:Transfection
C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D005353:Fibronectins; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases