CD45 AND RPTP-ALPHA DISPLAY DIFFERENT PROTEIN-TYROSINE-PHOSPHATASE ACTIVITIES IN T-LYMPHOCYTES

To examine the substrate specificity and function of two receptor prot ein tyrosine phosphatases, CD45 and RPTP alpha, RPTP alpha was express ed in a CD45(-), T-cell receptor (TCR)(+), BW5147 T-lymphoma cell. Hig h levels of expression of RPTP alpha did not fully restore either prox imal or distal TCR-mediated signalling events. RPTP alpha was unable t o reconstitute the phosphorylation of CD3 zeta and did not increase th e expression of the activation marker, CD69, on stimulation with TCR/C D3. RPTP alpha did not significantly alter the phosphorylation state o r kinase activity of two CD45 substrates, p56(lck) or p59(fyn), sugges ting that RPTP alpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTP alpha was approx. one-seventh to one-ten th as active as CD45 when tested against artificial substrates. This d ifference in activity was also observed in vitro with purified recombi nant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTP alpha, having both higher V-max and lower K-m values. Thus CD45 is intrinsically a much more act ive phosphatase than RPTP alpha, which provides one reason why RPTP al pha cannot effectively dephosphorylate p56(lck) and substitute for CD4 5 in T-cells. This work establishes that these two related protein tyr osine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase act ivity.