Kinetic analysis of two closely related receptor-like protein-tyrosine- phosphatases, PTPα and PTPε

Abstract

Among transmembrane protein-tyrosine-phosphatases, the membrane distal catalytic domain (D2) of protein-tyrosine-phosphatase α (PTPα) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the catalytic properties of PTPα D2 in association with D1, kinetic parameters of activity were established for PTPα D1D2 proteins containing 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 absence of inactive D1, and the presence or absence of inactive D2 affected the velocity but not the K(m) of D1 catalysis. While D1 appears to be the major catalytic contributor to PTPα activity, D2 possesses a significantly higher substrate-specific activity relative to wild-type D1D2 than the D2 domains of other protein-tyrosine-phosphatases. Also, PTPα D2 is an active phosphatase with comparable or better efficiency, on the basis of k(cat)/K(m) criteria, to some of the dual specificity phosphatases. Kinetic parameters of a closely related receptor-like protein-tyrosine-phosphatase, PTPε, were determined. PTPε D1 is the major, if not the only, catalytic moiety of PTPε, and has much higher turnover numbers than D1 of PTPα. The PTPε D2 activity is insignificant compared to that of PTPε-D1D2, with lower turnover numbers than FTPα D2. Thus, the intrinsic activity of PTPα D2 is high compared to other D2 domains and, more outstandingly, its activity relative to D1 appears unique. These are also apparent upon in vitro assay of full-length PTPα catalytic mutants expressed in mammalian cells. Together, these results suggest potential catalytic and regulatory roles for PTPα D2, and that PTPα may be an optimal model transmembrane protein-tyrosine- phosphatase for investigating the former within the cell.