Studies on the biological functions of protein tyrosine phosphatase PEST in cellular signaling

Identification of physiological substrates of protein tyrosine phosphatases is a key step in understanding the function of these enzymes. We have generated fibroblast cell lines from PTP-PEST −/− embryos (in which the PTP-PEST gene was targeted by homologous recombinaison in ES cells) in order to identify potential substrates with the premise that specific substrates of this enzyme would exist in a hyperphosphorylated state. Analysis of the profile of the phosphotyrosine proteins in the PTP-PEST −/− cells revealed the presence of hyperphosphorylated proteins of 180, 130 and 97 kDa when compared to control cells. The p130 was identified as p130Cas which is constitutively hyperphosphorylated in cells lacking PTP-PEST. In addition, p130Cas was also isolated by a substrate trapping mutant of PTP-PEST in the PTP-PEST −/− cell lysates. Furthermore, the first proline rich sequence of PTP-PEST interacted with the p130Cas family (also including Hef1 and Sin) via their SH3 domain in vitro. In this part of the work, I have combined genetic and biochemical strategies to allow the identification of PTP-PEST substrates.; In the second part of this thesis, I have studied the association of PTP-PEST with another focal adhesion protein, paxillin. I demonstrated that a proline rich segment of PTP-PEST (Pro 2) is essential for this interaction. Conversely, LIM3 and LIM4 domains were found both necessary for binding of PTP-PEST both in vitro and in vivo. Finally, using a “substrate trapping” approach, I showed that unlike p130Cas, paxillin is not a substrate for PTP-PEST. Interestingly, the focal adhesion targeting of paxillin is mediated by LIM 3. Thus we propose that PTP-PEST, by a competition with the unidentified ligand of paxillin in the focal adhesion complex, could contribute to the removal of paxillin from the adhesion sites and consequently promote focal adhesion turnover.; In the last part of my work, I tested the hypothesis that PTP-PEST could interact with PSTPIP. Our results show that the association between the two proteins is mediated by the CTH domain of PTP-PEST and the coiled-coil domain of PSTPIP. I tested several pathways that could regulate the tyrosine phosphorylation of PSTPIP and found that EGF and PDGF are potent stimulators in murine fibroblasts. Furthermore, I provided evidence that tyrosine 344 of PSTPIP is a docking site for SH2-containing tyrosine kinases. In this respect, I also demonstrated that PTP-PEST can dephosphorylate PSTPIP at tyrosine 344 thereby regulating the association between SH2 domain containing proteins and PSTPIP.