CHARACTERIZATION AND KINETIC-ANALYSIS OF THE INTRACELLULAR DOMAIN OF HUMAN-PROTEIN-TYROSINE-PHOSPHATASE-BETA (HPTP-BETA) USING SYNTHETIC PHOSPHOPEPTIDES

The intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) (44 kDa) was expressed in bacteria, purified using epitope 'tagging' immunoaffinity chromatography, and characterized with respect to kinetic profile, substrate specificity and potential modulators of enzyme activity. A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (P-i) released from phosphopeptides by HPTP beta. This assay, modified so as to improve its sensitivity, was adapted to a 96-well microtitre plate format, and provided linear detection between 50 and 1000 pmol of P-i. The cytoplasmic domain of HPTP beta was strongly inhibited by vanadate, molybdate, heparin, poly(Glu, Tyr) (4:1) and zinc ions. In order to explore the substrate preferences of this PTPase, we generated 13-residue synthetic phosphotyrosine-containing peptides that corresponded to sites of physiological tyrosine phosphorylation. HPTP beta demonstrated k(cat) values between 76 and 258 s(-1) using four different phosphopeptides. The substrate preference of HPTP beta was in the order src(Tyr-527) > PDGF-R(Tyr-740) > ERK1(Tyr-204) much greater than CSF-1R(Tyr-708) with K-m values ranging from 140 mu M to greater than 10 mM. The variations in affinity were probably due to differences among the four phosphopeptides compared, particularly with respect to the character of the charged amino acids flanking the phosphotyrosine residue.