Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

Abstract

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp6fyrc as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)-comprising ca. 20, 45, and 35%, respectively, of the total activity-were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2 , vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F-under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonylprotein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

Insulin-receptor phosphotyrosyl-protein phosphatases

“…These studies did not, however, exclude a contribution of calmodulindependent protein phosphatase to the phosphotyrosylprotein phosphatase activity, because comparative measurements were not made under conditions whereby calmodulin-dependent protein phosphatase was highly active (Brautigan et al, 1981;Foulkes et al, 1983;Chernoff & Li, 1983;Shriner & Brautigan, 1984). Although Foulkes et al (1983) Li, 1983;Shriner & Brautigan, 1984;Okada et al, 1986) and found no more than 36-45 % of the phosphatase in membrane fractions (Martensen, 1982;Nelson & Branton, 1984;Rotenberg & Brautigan, 1987). This difference may be attributable to the use of non-specific artificial substrates in the previous studies, whereas specific receptor substrates have been utilized in the present work.…”

Inhibition of protein tyrosine phosphatase activity by diamide is reversed by epidermal growth factor in fibroblasts

“…Recent evidence suggests that tyrosine kinase phosphorylation could be regulated by a diverse family of enzymes referred to as protein tyrosine phosphatases (PT-Pax) [3,4], whose catalytic activity is dependent on a specific cysteine residue [5]. PTPase activity is found associated with the plasma membrane and/or in the cytosol [6]. Their specific activities are much greater than protein tyrosine kinases .xrd they therefore have thecapacity to regulate the levei of tyrosine phosphorylation of proteins involved in signal transduction pathways in the cell [7].…”

Inhibition of Hepatoma Cell Growth in Vitro by Arylating and Non-arylating K Vitamin Analogs

“…1 and 3). Sodium orthovanadate, the prototype PTPase inhibitor (24,25), has also been shown to interact with the cysteine residue within covalent bond distance (26). Peroxovanadate (a mixture of orthovanadate and hydrogen peroxide) oxidizes the cysteine by forming cysteic acid (26), whereas alendronate forms sulfonic acid with the cysteine (27), and aromatic disulfides (28) and nitric oxide (29) form disulfides and inactivate the enzyme activity.…”