KINETIC-ANALYSIS OF 2 CLOSELY-RELATED RECEPTOR-LIKE PROTEIN-TYROSINE-PHOSPHATASES, PTP-ALPHA AND PTP-EPSILON

Among transmembrane protein-tyrosine-phosphatases, the membrane distal catalytic domain (D2) of protein-tyrosine-phosphatase alpha (PTP alph a) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the cataly tic properties of PTP alpha D2 in association with D1, kinetic paramet ers of activity were established for PTP alpha D1D2 proteins containin g an inactivating point mutation in D1 and/or D2. In this context, D2 activity was unchanged by the presence (N-terminal or C-terminal) or a bsence of inactive D1, and the presence or absence of inactive D2 affe cted the velocity but not the K-m of D1 catalysis. While D1 appears to be the major catalytic contributor to PTP alpha activity, D2 possesse s a significantly higher substrate-specific activity relative to wild- type D1D2 than the D2 domains of other protein-tyrosine-phosphatases. Also, PTP alpha D2 is an active phosphatase with comparable or better efficiency, on the basis of k(cat)/K-m criteria, to some of the dual s pecificity phosphatases. Kinetic parameters of a closely related recep tor-like protein-tyrosine-phosphatase, PTP epsilon, were determined. P TP epsilon D1 is the major, if not the only, catalytic moiety of PTP e psilon, and has much higher turnover numbers than D1 of PTP alpha. The PTP epsilon D2 activity is insignificant compared to that of PTP epsi lon-D1D2, with lower turnover numbers than PTP alpha D2. Thus, the int rinsic activity of PTP alpha D2 is high compared to other D2 domains a nd, more outstandingly, its activity relative to D1 appears unique. Th ese are also apparent upon in vitro assay of full-length PTP alpha cat alytic mutants expressed in mammalian cells. Together, these results s uggest potential catalytic and regulatory roles for PTP alpha D2, and that PTP alpha may be an optimal model transmembrane protein-tyrosine- phosphatase for investigating the former within the cell.