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The oncogenic protein p60v-src has competence activity but does not activate phosphatidylinositol turnover or protein
1Department of Biophysics, University of Rochester Medical Center, NY 14642.
Abstract:
A Balb/c 3T3 cell-line infected with the temperature-sensitive Rous sarcoma virus LA90 has been used to investigate the mitogenic activity of p60v-src and the possible role of phosphatidylinositol turnover and protein kinase C activation in this process. Activation of the p60v-src tyrosine kinase alone was only weakly mitogenic to serum-deprived cells. However, transient activation of the kinase dramatically increased the mitogenic response to epidermal growth factor. Mitogenicity was not additive with phorbol esters. Down-regulation of protein kinase C by chronic administration of phorbol ester did not block the mitogenic effect of p60v-src. Following short-term activation of p60v-src, analysis of protein phosphorylations by giant two-dimensional electrophoresis revealed only very minor changes in any of the 1000 or so detectable phosphoproteins, whereas the activation of protein kinase C by a sub-optimal dose of phorbol ester significantly increased the phosphorylation of at least 10 proteins. Short-term activation of p60v-src did not cause any detectable increase in phosphatidylinositol turnover, or in diacylglycerol level. These results indicate that p60v-src can act, like platelet-derived growth factor, to induce competence in Balb/c 3T3 fibroblasts. They also suggest that even though p60v-src may activate phosphatidylinositol turnover in some other cell-lines, this effect is probably indirect and is not essential for induction of the transformed phenotype or for p60v-src-dependent mitogenesis.
Insights
The Rous sarcoma virus p60v-src tyrosine kinase weakly stimulates cell growth alone but enhances responses to growth factors. Its mitogenic activity is independent of phosphatidylinositol turnover and protein kinase C activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Virology
Background:
- The Rous sarcoma virus (RSV) encodes p60v-src, a tyrosine kinase implicated in cell transformation and mitogenesis.
- Understanding the signaling pathways activated by p60v-src is crucial for deciphering its role in cell proliferation.
Purpose of the Study:
- To investigate the mitogenic activity of p60v-src in Balb/c 3T3 fibroblasts.
- To determine the role of phosphatidylinositol turnover and protein kinase C (PKC) activation in p60v-src-mediated mitogenesis.
Main Methods:
- Utilized a temperature-sensitive RSV LA90-infected Balb/c 3T3 cell line.
- Assessed mitogenic responses to p60v-src activation, epidermal growth factor, and phorbol esters.
- Analyzed protein phosphorylation patterns using two-dimensional electrophoresis.
- Measured phosphatidylinositol turnover and diacylglycerol levels.
Main Results:
- p60v-src activation alone showed weak mitogenicity; however, it significantly potentiated the mitogenic response to epidermal growth factor.
- Mitogenicity was not additive with phorbol esters, and down-regulation of PKC did not inhibit p60v-src's mitogenic effect.
- Short-term p60v-src activation caused minimal changes in phosphoprotein profiles, unlike PKC activation which altered at least 10 proteins.
- No detectable increase in phosphatidylinositol turnover or diacylglycerol levels was observed following p60v-src activation.
Conclusions:
- p60v-src induces competence in fibroblasts, similar to platelet-derived growth factor.
- Phosphatidylinositol turnover and PKC activation are not essential for p60v-src-dependent mitogenesis or transformation, suggesting indirect roles in other cell types.