A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). One positive clone was isolated and the nucleotide sequence was determined. It contained 1305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues (Mr, 48,400). This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

The amino acid sequence of the cytosolic human placenta protein-tyrosine-phosphatase 1B (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been determined. It consists ofa single chain of321 residues with an N-acetylated N-terminal methionine and an unusually proline-rich C-terminal region. The enzyme is structurally related to the two cytoplasmic domains of both the leukocyte common antigen CD45 and LAR, a CD45-flke molecule with an external segment that resembles a neural cell adhesion molecule. A low molecular weight protein encoded by a cDNA clone from T cells also shows extensive sequence smllarties. The present study defines homologous domains common to this diverse family of PTPases that includes both soluble and receptor-like transmembrane forms. The cysteinyl residues 121 and 215 of PTPase 1B are conserved among all members of the family and are candidates for involvement in catalysis since PTPase 1B is inactivated by thiol modifying reagents. Two segments rich in positively charged residues (residues 33-47 and 227-238) may provide sites of interaction with inhibitory anionic polymers such as heparin or poly(Glu/Tyr).

A human T-cell cDNA encoding a 48-kDa protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) was cloned into a mammalian expression vector and introduced into baby hamster kidney cells, and stable colonies were isolated. The expressed PTPase was found to be associated with the particulate fraction of the cells, where it was essentially inactive in an in vitro assay unless first subjected to limited trypsinization; trypsin treatment generated an active fragment of 33 kDa by the removal of a carboxyl-terminal segment of the full-length enzyme. Gel filtration indicated that the expressed enzyme was associated with a complex of >600 kDa. Introduction of a premature stop codon into the T-cell cDNA at position 1012 resulted in the production of a fully active 37-kDa species that distributed between both the particulate and soluble fractions. The truncated form of the enzyme was readily solubilized by detergents and was eluted within its predicted molecular mass range. These results suggest that the carboxyl-terminal segment is important in determining the localization and regulation of the PTPase. The level of protein-tyrosine phosphorylation observed after 5 min of platelet-derived growth factor stimulation was reduced in cells overexpressing either form of the phosphatase, indicating that both are active in vivo. Overexpressing the truncated enzyme resulted in a growth rate that was approximately 50% of that observed in cells transfected with either the full-length PTPase cDNA or the vector alone.