CD45 and RPTPα display different protein tyrosine phosphatase activities in T lymphocytes

Abstract

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPα, RPTPα was expressed in a CD45-, T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPα did not fully restore either proximal or distal TCR-mediated signalling events. RPTPα was unable to reconstitute the phosphorylation of CD3ζ and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPα did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56lck or p59fyn, suggesting that RPTPα does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPα was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56lck as substrates indicated that CD45 was consistently more active than RPTPα, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPα, which provides one reason why RPTPα cannot effectively dephosphorylate p56lck and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.