BIOCHEMICAL-CHARACTERIZATION OF A HUMAN BAND 4.1-RELATED PROTEIN-TYROSINE-PHOSPHATASE, PTPH1

PTPH1 is a human protein-tyrosine phosphatase with homology to the ban d 4.1 superfamily of cytoskeleton-associated proteins, Here, we report the purification and biochemical characterization of this enzyme from baculovirus-infected insect cells. The purified protein exhibited an apparent M(r) of 120,000 on SDS gels. The native enzyme dephosphorylat ed both myelin basic protein (MBP) and reduced, carboxamidomethylated, and maleylated lysozyme (RCML) but was over 5-fold more active on MBP . The K-m values for the two substrates were similar (1.45 mu M for MB P and 1.6 mu M for RCML). Phosphorylation of PTPH1 by protein kinase C in vitro resulted in a decrease in K-m but had no effect on V-max. Re moval of the NH2-terminal band 4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated dephosphorylation of RCML but inhibited i ts activity toward MBP, The dephosphorylation of RCML by full-length P TPH1 was enhanced up to B-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M Nacl, whereas trypsinized preparations of P TPH1 containing the isolated catalytic domain were unaffected. These r esults suggest that in addition to a potential role in controlling sub cellular localization, the NH2-terminal band 4.1 homology domain of PT PH1 may exert a direct effect on catalytic function.