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Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured
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 pp60src 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-phosphothreonyl-protein 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.
Insights
Researchers identified unique phosphoprotein phosphatases that remove phosphate groups from phosphotyrosine residues. These enzymes are distinct from other phosphatases and may play a role in cell transformation.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Tyrosine phosphorylation is crucial for cell proliferation and cancer development.
- Specific enzymes, phosphoprotein phosphatases, are responsible for dephosphorylating phosphotyrosine residues.
Purpose of the Study:
- To partially purify and characterize phosphoprotein phosphatases specific for phosphotyrosine residues from chicken embryo fibroblasts.
- To investigate the properties of these enzymes and compare them to known phosphatases.
Main Methods:
- Partial purification of phosphoprotein phosphatases from chicken embryo fibroblasts using ion-exchange chromatography and gel filtration.
- Assay of enzyme activity using 32P-labeled immunoglobulin G phosphorylated by pp60src.
- Characterization of enzyme properties including pH optimum and inhibition by various ions and nucleotides.
Main Results:
- Three distinct phosphotyrosyl-protein phosphatase (pTPI, pTPII, pTPIII) species were resolved with apparent molecular weights of 55,000, 50,000, and 95,000.
- All three enzymes exhibited a pH optimum around 7.4 and were inhibited by Zn2+, vanadate, ATP, and ADP.
- These enzymes were unaffected by divalent metal cations, EDTA, and F- at physiological ionic strength.
- A slight increase in phosphotyrosyl-protein phosphatase activity was observed in cells transformed by Rous sarcoma virus.
Conclusions:
- The identified enzymes represent a unique class of phosphoprotein phosphatases distinct from phosphoseryl- and phosphothreonyl-protein phosphatases.
- These enzymes likely play a significant role in the dephosphorylation of phosphotyrosine-containing proteins.
- The observed increase in activity during viral transformation suggests a potential involvement in oncogenesis.