Phosphatidylinositol-3 kinase is activated in v-src, v-yes, and v-fps transformed chicken embryo fibroblasts
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Y Fukui1, A R Saltiel, H Hanafusa
1Rockefeller University, New York 10021.
Abstract:
PI-3 kinase activity has been shown to associate with p60v-src. We found that immunoprecipitates of p60v-src exhibit an activity that catalyzes the formation of PI-3-P, PI-3,4-P2 and PIP3 from PI, PI-4-P, and PI-4,5-P2, respectively. Transformation of chicken embryo fibroblasts (CEF) by p60v-src of Rous sarcoma virus (RSV) caused elevation of PI-3-P, PI-3,4-P2, and PIP3, suggesting that the PI-3 kinase may be activated in these cells. Similar elevations were seen in cells transformed with the v-yes or v-fps oncogenes, but not with v-ros or v-ras. We have established also a system that allows the binding of PI-3 kinase to purified p60v-src in vitro, reproducing the binding seen in vivo. This assay indicated that more PI-3 kinase activity binds to purified p60v-src in cell lysates from CEF transformed with v-yes or v-fps, suggesting that some modification or over-expression of PI-3 kinase takes place in these cells.
Insights
Phosphatidylinositol 3-kinase (PI-3 kinase) activity is elevated in cells transformed by Rous sarcoma virus (RSV) p60v-src. This suggests PI-3 kinase activation by specific oncogenes like v-yes and v-fps.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Phosphatidylinositol 3-kinase (PI-3 kinase) activity is implicated in cellular signaling pathways.
- The association between PI-3 kinase and the p60v-src protein kinase has been previously suggested.
Purpose of the Study:
- To investigate the catalytic activity of PI-3 kinase associated with p60v-src.
- To determine if PI-3 kinase activity is altered in chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) and other oncogenes.
- To establish an in vitro system to study the binding of PI-3 kinase to p60v-src.
Main Methods:
- Immunoprecipitation of p60v-src to assess associated PI-3 kinase activity.
- Analysis of phosphoinositide levels (PI-3-P, PI-3,4-P2, PIP3) in transformed CEF.
- Development of an in vitro binding assay for PI-3 kinase and purified p60v-src.
Main Results:
- Immunoprecipitates of p60v-src catalyzed the formation of PI-3-P, PI-3,4-P2, and PIP3.
- Transformation of CEF with p60v-src (RSV) led to elevated levels of PI-3-P, PI-3,4-P2, and PIP3.
- Similar elevations were observed in cells transformed by v-yes and v-fps oncogenes, but not v-ros or v-ras.
- The in vitro assay confirmed PI-3 kinase binding to p60v-src and showed increased binding in lysates from v-yes or v-fps transformed cells.
Conclusions:
- p60v-src possesses PI-3 kinase activity, catalyzing the production of key phosphoinositides.
- PI-3 kinase is likely activated in CEF transformed by RSV p60v-src, v-yes, and v-fps oncogenes.
- The enhanced binding of PI-3 kinase to p60v-src in transformed cells suggests PI-3 kinase modification or overexpression.