Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST.
PTP-PEST is a ubiquitously expressed, cytosolic, mammalian protein tyrosine phosphatase (PTP) which exhibits high specific activity in vitro. We have investigated the substrate specificity of PTP-PEST by a novel substrate-trapping approach in combination with in vitro dephosphorylation experiments. We initially identified a prominent 130-kDa tyrosine-phosphorylated protein in pervanadate-treated HeLa cell lysates which was preferentially dephosphorylated by PTP-PEST in vitro. In order to identify this potential substrate, mutant (substrate-trapping) forms of PTP-PEST were generated which lack catalytic activity but retain the ability to bind substrates. These mutant proteins associated in stable complexes exclusively with the same 130-kDa protein, which was identified as p130(cas) by immunoblotting. This exclusive association was observed in lysates from several cell lines and in transfected COS cells, but was not observed with other members of the PTP family, strongly suggesting that p130(cas) represents a major physiologically relevant substrate for PTP-PEST. Our studies suggest potential roles for PTP-PEST in regulation of p130(cas) function. These functions include mitogen- and cell adhesion-induced signalling events and probable roles in transformation by various oncogenes. These results provide the first demonstration of a PTP having an inherently restricted substrate specificity in vitro and in vivo. The methods used to identify p130(cas) as a specific substrate for PTP-PEST are potentially applicable to any PTP and should therefore prove useful in determining the physiological substrates of other members of the PTP family.
Pubmed ID: 8887669 RIS Download
Animals | COS Cells | Crk-Associated Substrate Protein | Cytosol | Enzyme Inhibitors | HeLa Cells | Humans | Kinetics | Mammals | Mutagenesis, Site-Directed | Phosphoproteins | Point Mutation | Protein Tyrosine Phosphatase, Non-Receptor Type 12 | Protein Tyrosine Phosphatases | Proteins | Recombinant Proteins | Retinoblastoma Protein | Retinoblastoma-Like Protein p130 | Substrate Specificity | Transfection | Tumor Cells, Cultured | Vanadates