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Distinct repression of translation by wortmannin and rapamycin
S Pedersen1, J E Celis, J Nielsen
1Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
Abstract:
The role of phosphatidylinositol 3-kinase and FK506-binding protein rapamycin-associated protein (FRAP) in translational control has been examined by treating RD-rhabdomyosarcoma cells with wortmannin and rapamycin and studying the effects on cell-growth, translation initiation, and protein synthesis. Whereas wortmannin and rapamycin exhibit subtle effects on global translation, examination of individual mRNAs in sucrose gradients and of individual proteins in two-dimensional polyacrylamide gels reveals that wortmannin and rapamycin exhibit distinct effects on the translation of individual mRNAs. Wortmannin represses the synthesis of a third of cellular proteins, whereas rapamycin affects a subset of these proteins. Since ribosomal protein S6 was rapidly dephosphorylated following wortmannin and rapamycin treatment, and the phosphorylation status of the eukaryotic initiation factor 4E was unchanged, our data imply that the p70 signalling pathway has at least one branch-point upstream of FRAP leading to an additional route of translational control.
Insights
Wortmannin and rapamycin impact cell growth by distinctively altering protein synthesis, suggesting a complex translational control pathway involving phosphatidylinositol 3-kinase and FRAP signaling.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Phosphatidylinositol 3-kinase (PI3K) and the FK506-binding protein rapamycin-associated protein (FRAP) are key regulators of cellular processes.
- Translational control is crucial for protein synthesis and cellular function.
- Understanding the specific roles of PI3K and FRAP in translation is essential for deciphering cellular signaling pathways.
Purpose of the Study:
- To investigate the distinct roles of wortmannin (a PI3K inhibitor) and rapamycin (an inhibitor targeting FRAP signaling) in regulating protein synthesis and cell growth.
- To elucidate the effects of these inhibitors on global and individual mRNA translation.
- To identify potential branch points in the signaling pathways controlling translation.
Main Methods:
- RD-rhabdomyosarcoma cells were treated with wortmannin and rapamycin.
- Cell growth, translation initiation, and overall protein synthesis were assessed.
- Individual mRNA translation was analyzed using sucrose density gradients.
- Protein synthesis patterns were examined using two-dimensional polyacrylamide gel electrophoresis.
- Phosphorylation status of ribosomal protein S6 and eukaryotic initiation factor 4E was determined.
Main Results:
- Wortmannin and rapamycin showed subtle effects on global translation but distinct impacts on individual mRNA translation.
- Wortmannin repressed the synthesis of approximately one-third of cellular proteins.
- Rapamycin affected a subset of the proteins repressed by wortmannin.
- Ribosomal protein S6 was rapidly dephosphorylated by both treatments.
- The phosphorylation status of eukaryotic initiation factor 4E remained unchanged.
Conclusions:
- The p70 signaling pathway, upstream of FRAP, likely involves at least one additional route for translational control.
- Distinct effects of wortmannin and rapamycin on protein synthesis highlight complex regulatory mechanisms.
- These findings contribute to a deeper understanding of PI3K and FRAP involvement in translational regulation.