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Association of p21ras with phosphatidylinositol 3-kinase
A Sjölander1, K Yamamoto, B E Huber
1Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Abstract:
In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3-kinase activity.
Insights
Ras proteins (p21ras) are linked to cancer. This study shows phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity is involved in p21ras signal transduction, potentially mediating cancer cell changes.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- Ras genes encode 21-kDa GTP-binding proteins (p21ras) implicated in cell growth, differentiation, and neoplastic transformation.
- The precise signal transduction pathways of p21ras remain largely undefined.
- Phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a known component in growth factor signaling pathways.
Purpose of the Study:
- To investigate the role of PtdIns 3-kinase activity in the signal transduction of Ha-ras-transformed epithelial cells.
- To determine if p21ras directly associates with PtdIns 3-kinase activity.
Main Methods:
- Utilized a Ha-ras-transformed epithelial cell line.
- Stimulated cells with insulin or insulin-like growth factor I.
- Performed immunoprecipitation using anti-phosphotyrosine, anti-receptor antibodies, and the anti-Ras monoclonal antibody Y13-259.
- Assessed PtdIns 3-kinase activity in immunoprecipitates.
Main Results:
- Demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor immunoprecipitates upon insulin/IGF-I stimulation.
- Showed that PtdIns 3-kinase activity could be immunoprecipitated by the anti-Ras antibody Y13-259.
- Confirmed the specificity of the p21ras-PtdIns 3-kinase association using antigen peptide neutralization and by observing no activity when the ras gene was inactive.
Conclusions:
- PtdIns 3-kinase activity is a crucial component in the p21ras signal transduction cascade.
- This activity may mediate cytoskeletal alterations and aberrant growth observed in ras-transformed cells.
- Suggests a direct link between p21ras signaling and PtdIns 3-kinase in cellular transformation.