Investigations into the physiological role of the human low molecular weight phosphotyrosyl protein phosphatase

Recombinant human low molecular weight phosphotyrosyl protein phosphatase isoenzymes (HCPTP-A and HCPTP-B) were expressed in high yield and purified to homogeneity from a T7-polymerase Escherichia coli expression system. The purified enzyme was used to generate monoclonal and polyclonal antibodies. Antibody epitopes were determined for two isoenzyme-specific monoclonal antibodies. Polyclonal antibodies were characterized and used in indirect immunofluorescence localization studies. Loss of the enzyme upon fixation of the cells confirmed the soluble nature of the enzyme in vivo and suggested a mostly cytoplasmic localization of the enzyme during basal cellular growth. Kinetic studies were conducted using various phosphopeptide substrates to determine second-order rate constants with each isoenzme. HCPTP-A was more efficient in the hydrolysis of all peptide substrates tested except for one that contained three consecutive phosphorylated tyrosines. Such studies also showed that both isoenzymes were more specific for the hydrolysis of FMN than for phosphopeptides. Site-directed mutagenesis was used to individually mutate the tyrosine residues in HCPTP-A to phenylalanine. These mutants were used to determine the sites of phosphorylation by specific protein tyrosine kinases. Using proteolytic peptide maps, we determined the phosphorylation sites of the enzyme by Src, Syk, Elk, and Flk kinases. The effect of phosphorylation on enzymatic activity was also explored