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Characterization of protein tyrosine phosphatase E Melhado, Ian Granville

Abstract

Protein tyrosine phosphatases have been shown to play an important role in immune cell regulation. This study focused upon identifying PTPases that were regulated during inflammation. Using PCR mediated differential hybridization PTPε was identified as a candidate PTPase mRNA upregulated by pro-inflammatory stimuli. Northern blotting analysis confirmed that the message for this PTPase was induced by a limited number of stimuli in cells of the monocyte/macrophage lineage. With the development of highly specific polyclonal antibodies, monocyte/macrophage derived PTPs was observed as a 72/74 kDa doublet. p72/74PTPε was found to possess PTPase activity in vitro, predominantly within the cytosol, and its expression was observed to be regulated during cellular differentiation. Further characterization of PTPε indicated that the PTPase was phosphorylated and tyrosine phosphorylation could be induced in vivo. Tyrosine phosphorylation was found to induce the association of the small adapter protein GRB2 with PTPε in pervanadate treated cells. These observations and others pointed to a novel isoform of the previously described transmembrane PTPε PTPase. An in vitro analysis of recombinant PTPε was undertaken to characterize the PTPase activity of the enzyme. This analysis revealed that the PTPase had a pH optimum of 5.6. PTPε exhibited some substrate specificity especially when compared to other recombinant PTPases such as CD45 and PTPα. From the preceding analysis and the fact that the closely related PTPα appears to show specificity for pp60c'src, the src family kinases were the signaling proteins initially investigated as potential in vivo substrates of PTPε. By utilizing cDNAs encoding both transmembrane and non-transmembrane isforms of PTPε, PTPases were introduced into mammalian cells. In the case of transmembrane PTPε, various mutants were characterized in vivo. These studies revealed that plOO/11OPTPε is a highly glycosylated membrane protein, found predominantly at cell-cell junctions. While plOO/11OPTPε was capable of associating with and modulating the phosphotyrosine content and kinase activity of the src family kinase members pp60c" s r c and pp59*" p72/74PTPε was not. This phenomenon may be explained by the observation that the localization of p72/74PTPε was to the nucleus and not the plasma membrane where src family kinases are found. Finally, the PTPε homologue was reported to contain a SH3 binding site, however, this site was not conserved in PTPε. A proline residue was introduced at position Fl 12 and resulted in the reconstitution of an SH3 domain-binding site thus turning PTPε into a PTPα like molecule. PTPεF112P exhibited altered substrate specificity showing increased activity towards src family kinases and reduced activity towards the in vivo substrate Crkll.

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