COMBINATION OF GENE TARGETING AND SUBSTRATE TRAPPING TO IDENTIFY SUBSTRATES OF PROTEIN-TYROSINE PHOSPHATASES USING PTP-PEST AS A MODEL

Identification of physiological substrates of protein tyrosine phospha tases is a key step in understanding the function of these enzymes. We have generated fibroblast cell lines having a gene-targeted PTP-PEST in order to identify potential substrates with the premise that specif ic substrates of this enzyme would exist in a hyperphosphorylated stat e. Analysis of the profile of the phosphotyrosine proteins in the PTP- PEST -/- cells revealed the presence of hyperphosphorylated proteins o f 180, 130, and 97 kDa when compared to control cells. The p130 was id entified as p130(Cas), and direct immunoprecipitates of p130(Cas) demo nstrate that this protein is constitutively hyperphosphorylated in cel ls lacking PTP-PEST. In addition, p130(Cas) was also isolated by the s ubstrate-trapping mutant of PTP-PEST in the PTP-PEST -/- cell lysates. Interestingly, we have demonstrated for the first time that PTP-PEST, through its first proline-rich sequence (PPKPPR337)-P-332, interacts with ether members of the p130(Cas) family (Hef1 and Sin) via their SH 3 domain in vitro. This result suggests that Hef1 and Sin could also b e potential substrates of PTP-PEST. In conclusion, we have combined ge netic and biochemical strategies to allow the identification of PTP-PE ST substrates. This experimental approach could potentially be used to identify substrates of other PTPases. Furthermore, the Cas-like molec ules Hef1 and Sin associate via their SH3 domains with a proline-rich motif found on PTP-PEST, suggesting the possibility that PTP-PEST coul d be a general modulator of the Gas family of proteins.