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A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity
Abstract:
We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.
Insights
The ATP-binding site mutation in pp60v-src (RSV-SF2) abolished its kinase activity, preventing cell transformation and tumor formation. This highlights the essential role of phosphotransferase activity in neoplastic transformation.
Area of Science:
- Molecular Biology
- Oncogenesis
- Protein Kinase Research
Background:
- pp60v-src is a protein tyrosine kinase implicated in cellular transformation and oncogenesis.
- The ATP-binding site is crucial for kinase activity, but its precise role in pp60v-src-mediated transformation requires further elucidation.
Purpose of the Study:
- To investigate the role of the ATP-binding site in pp60v-src's kinase activity and transforming potential.
- To characterize a specific mutant, RSV-SF2, with a lysine-to-methionine substitution at position 295 within the ATP-binding site.
Main Methods:
- Construction and characterization of the RSV-SF2 mutant of pp60v-src.
- Analysis of mutant protein stability, cellular localization, and kinase activity in vitro and in infected rat cells.
- Assessment of cellular morphology, tumor formation, and phosphotyrosine levels in cells expressing the mutant.
Main Results:
- The RSV-SF2 mutant exhibited similar half-life and cellular localization to wild-type pp60v-src.
- RSV-SF2 lacked detectable kinase activity and failed to induce cellular transformation or tumor formation.
- Mutant pp60v-src could not be phosphorylated on serine or tyrosine, unlike the wild-type protein.
Conclusions:
- The phosphotransferase activity of pp60v-src is indispensable for neoplastic transformation.
- ATP binding to pp60v-src likely induces an allosteric conformational change necessary for serine phosphorylation.
- Targeting the ATP-binding site offers a potential strategy for inhibiting oncogenic kinase activity.