Kinetic analysis of two 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 alpha) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the catalytic properties of PTP alpha D2 in association with D1, kinetic parameters of activity were established for PTP alpha 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 Km of D1 catalysis. While D1 appears to be the major catalytic contributor to PTP alpha activity, D2 possesses 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)/Km criteria, to some of the dual specificity phosphatases. Kinetic parameters of a closely related receptor-like protein-tyrosine-phosphatase, PTP epsilon, were determined. PTP epsilon D1 is the major, if not the only, catalytic moiety of PTP epsilon, and has much higher turnover numbers than D1 of PTP alpha. The PTP epsilon D2 activity is insignificant compared to that of PTP epsilon-D1D2, with lower turnover numbers than PTP alpha D2. Thus, the intrinsic activity of PTP alpha 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 PTP alpha catalytic mutants expressed in mammalian cells. Together. these results suggest 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.

Purification and characterization of a higher-molecular-mass form of protein phosphotyrosine phosphatase (PTP 1B) from placental membranes.

Purification of a major placental membrane protein phosphotyrosine phosphatase (PTP-I) through the use of a nonhydrolysable phosphotyrosine analogue affinity ligand has enabled identification of the enzyme as a single polypeptide of at least 46 kDa. This phosphatase specifically dephosphorylates phosphotyrosine-containing substrates, including the src peptide, the epidermal-growth-factor receptor tyrosine kinase and the non-receptor tyrosine kinase p56lck. The p56lck can be dephosphorylated by PTP-I at two tyrosine residues (Tyr-394 and Tyr-505), which are differentially phosphorylated in vitro and in vivo and have been suggested to modulate kinase activity. The activity of PTP-I towards these substrates indicates a possible function of regulation of cellular tyrosine phosphorylation pathways at the level of growth-factor receptor and/or oncogene/proto-oncogene tyrosine kinases. Kinetic analyses show that PTP-I exhibits a Km value of about 2 microM with either src peptide or reduced, carboxyamidomethylated and maleylated (RCM)-lysozyme as substrate, and is inhibited in a mixed competitive manner by the polyanions heparin and poly(Glu4,Tyr1). Sequencing of PTP-I peptides reveals almost complete identity with sequences within the N-terminal half of the 37 kDa non-receptor tyrosine phosphatase 1B. However, the size and amino acid composition of PTP-I are similar to that of a higher-molecular-mass form of PTP 1B predicted from cDNA cloning. These results suggest that the 37 kDa PTP 1B is a proteolysed form of PTP-I, and provide evidence that a larger form of PTP 1B exists in vivo, at least in association with placental membranes.