Purification and characterization of a protein tyrosine phosphatase containing SH2 domains.

Abstract

A protein tyrosine phosphatase (PTP) containing two SH2 domains (PTP1C) was purified to near homogeneity from an adenovirus expression system by a two-step chromatographic procedure with a yield of 67%. The purified enzyme behaves as a monomer of 68 kDa on gel filtration and is totally specific for phosphotyrosyl residues. Its optimal pH is around neutrality for protein substrates such as reduced, carboxyamidomethylated, maleylated (RCM)-lysozyme and myelin basic protein but below 5 for low molecular weight compounds such as para-nitrophenyl phosphate (p-NPP) and phosphotyrosine. Furthermore, with the protein substrates, it displays an activity less than 1% of that obtained with other known PTPs but comparable activities toward p-NPP and phosphotyrosine. Its responsiveness toward the usual PTP activators (e.g. spermine) or inhibitors (e.g. vanadate, molybdate, heparin, or Zn2+) varied considerably with the nature of the substrates involved. Limited digestion with trypsin caused the cleavage of a C-terminal segment of the enzyme, giving rise to a 63-kDa fragment; this cleavage resulted in an approximately 20- and 10-fold activation of the enzyme toward RCM-lysozyme and myelin basic protein, respectively.