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Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.