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Characterization of the Rous sarcoma virus transforming gene product
The Journal of Biological Chemistry
|May 25, 1983
Summary
This study quantifies in vitro phosphorylation of Rous sarcoma virus pp60src protein using ATP or GTP. Results show distinct phosphorylation sites and kinetics, offering insights into viral oncogene regulation.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The Rous sarcoma virus transforming gene product, pp60src, is a key oncogene.
- Understanding pp60src phosphorylation is crucial for deciphering viral oncogenesis.
Purpose of the Study:
- To quantitatively analyze in vitro phosphorylation of pp60src using ATP and GTP as phosphate donors.
- To compare the kinetic parameters (Km, Vmax) and substrate specificities of ATP and GTP in pp60src phosphorylation.
- To investigate the structural consequences of phosphorylation at different sites using various phosphate donors.
Main Methods:
- In vitro kinase assays using purified pp60src and radiolabeled ATP or GTP.
- Kinetic analysis to determine Km and Vmax values.
- Inhibition studies using various nucleotides to assess substrate specificity.
- Immunoprecipitation with anti-p60src antibody to confirm product identity.
- Structural analysis of phosphorylated sites (tyrosine residues) using mass spectrometry.
- Thermolability studies to assess the stability of phosphorylated pp60src.
Main Results:
- Similar Km values but different Vmax values were observed for pp60src phosphorylation by ATP and GTP.
- ATP-mediated phosphorylation showed higher specificity, with ADP and dATP being significant inhibitors.
- GTP-mediated phosphorylation exhibited lower specificity, with several nucleotides showing inhibitory effects.
- Structural analysis revealed GTP primarily phosphorylates tyrosine in the COOH-terminal region, while ATP phosphorylates additional tyrosines in the NH2-terminal region.
- Phosphorylation by both ATP and GTP resulted in similar thermolability profiles for pp60src.
Conclusions:
- ATP and GTP exhibit distinct kinetic and structural outcomes when phosphorylating pp60src in vitro.
- The phosphorylation site preference differs between ATP and GTP, suggesting differential regulation of pp60src activity.
- These findings provide quantitative insights into the enzymatic properties of pp60src and its interaction with different phosphate donors.
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