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Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways
1Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, California 94720-3204, USA.
Abstract:
An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded.
Insights
v-Src transformation of chicken embryo fibroblasts relies on two parallel pathways: Ras-MAPK and PI3K-mTOR. Inhibiting either pathway alone is insufficient, but simultaneous blockade effectively prevents transformation, highlighting their combined role.
Area of Science:
- Cell Biology
- Molecular Oncology
- Signal Transduction
Background:
- v-Src mediated transformation of chicken embryo fibroblasts (CEF) is a complex process.
- While active Ras (GTP-bound Ras) is not essential for transformation, suggesting alternative signaling routes.
- The phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway is a potential Ras-independent contributor.
Purpose of the Study:
- To investigate the contribution of the PI3K-mTOR pathway to v-Src transformation.
- To determine the combined effect of inhibiting both Ras-MAPK and PI3K-mTOR pathways on CEF transformation by v-Src.
Main Methods:
- Transformation parameters assessed: morphological alteration, hexose uptake, density inhibition, anchorage-independent growth.
- Ras-MAPK pathway inhibition using dominant-negative HRasN17 or MEK inhibitor PD98059.
- PI3K-mTOR pathway inhibition using PI3K inhibitor LY294002 or mTOR inhibitor rapamycin.
Main Results:
- Inhibition of Ras-MAPK or PI3K-mTOR pathways individually reduced transformation parameters but did not block it.
- Simultaneous inhibition of both Ras-MAPK and PI3K-mTOR pathways effectively blocked v-Src-induced transformation.
- These findings indicate two parallel, cooperating pathways are crucial for transformation.
Conclusions:
- v-Src transformation of CEF is mediated by two parallel signaling pathways: Ras-MAPK and PI3K-mTOR.
- Both pathways are necessary and contribute significantly to the transformation process.
- Targeting both pathways concurrently offers a potential strategy to inhibit v-Src-driven transformation.