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Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster
Z Tuhácková1, V Sovová, E Sloncová
1Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Prague. tuhack@img.cas.cz
Abstract:
Increased phosphorylation of the translational repressor protein 4E-BP1 was found in the cell line derived from the tumor induced in Syrian hamster by Rous sarcoma virus (RSV). This was accompanied by its dissociation from the complex with initiation factor eIF4E. The ribosomal S6 protein kinase p70S6k is supposed to be regulated by the same or a closely related rapamycin-sensitive signalling pathway to that which modulates 4E-BP1. Phosphorylation and activity of p70S6k were found to be also increased in RSV-transformed H19 cells that express significantly higher amounts of the Src protein (p60src) relative to the non-transformed hamster fibroblasts NIL-2. The increased activity and phosphorylation of p70S6k were blocked by rapamycin, indicating that the rapamycin-sensitive pathway is involved in its regulation in v-src-transformed hamster fibroblasts. In agreement with this, rapamycin reduced the expression of elongation factor eEF1alpha (whose translation is regulated by a rapamycin-sensitive mechanism thought to involve p70S6k) and did not affect the production of a housekeeping protein, alpha-tubulin, in these cells. Synthesis of Src protein was also inhibited in cells treated with rapamycin. However, treatment of cells with a concentration of rapamycin sufficient to completely inhibit the activity and phosphorylation of p70S6k resulted in only partial de-phosphorylation of 4E-BP1 and its re-association with eIF4E in the transformed cells, indicating that additional rapamycin-insensitive mechanisms/pathways are implicated in the control of 4E-BP1 phosphorylation in RSV-transformed hamster fibroblasts. Over-expression of eIF4E favours cell proliferation and can lead to a transformed phenotype, while over-expression of 4E-BP1 has the opposite effect. The altered signalling to the phosphorylation of 4E-BP1 in RSV-transformed cells, which leads to its dissociation from eIF4E and thus relief of inhibition of eIF4E function, may therefore represent an important regulatory mechanism in malignant cell growth.
Insights
Rous sarcoma virus (RSV) transformation increases 4E-BP1 phosphorylation and eIF4E release in hamster cells, promoting malignant growth. Rapamycin partially reverses this, suggesting additional pathways regulate 4E-BP1 in cancer.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Rous sarcoma virus (RSV) transformation alters cellular signaling pathways.
- The translational repressor protein 4E-BP1 and initiation factor eIF4E play critical roles in protein synthesis and cell proliferation.
- The p70S6 kinase (p70S6k) is implicated in cell growth regulation and is sensitive to rapamycin.
Purpose of the Study:
- To investigate the role of 4E-BP1 phosphorylation and its interaction with eIF4E in RSV-transformed hamster cells.
- To determine the involvement of the rapamycin-sensitive pathway, including p70S6k, in regulating 4E-BP1 in these transformed cells.
- To elucidate the contribution of altered translational control to malignant cell growth.
Main Methods:
- Analysis of 4E-BP1 and p70S6k phosphorylation and activity in RSV-transformed (H19) and non-transformed (NIL-2) hamster cells.
- Treatment with rapamycin to assess the inhibition of signaling pathways.
- Examination of protein-protein interactions between 4E-BP1 and eIF4E using co-immunoprecipitation.
- Quantification of protein expression levels, including Src, eEF1alpha, and alpha-tubulin.
Main Results:
- RSV-transformed cells exhibited increased 4E-BP1 phosphorylation and dissociation from eIF4E, alongside elevated p70S6k phosphorylation and activity.
- Rapamycin treatment inhibited p70S6k activity and phosphorylation, reduced eEF1alpha expression, and partially reversed 4E-BP1 de-phosphorylation and eIF4E re-association.
- Src protein synthesis was also inhibited by rapamycin, but additional rapamycin-insensitive mechanisms were implicated in 4E-BP1 regulation.
Conclusions:
- Altered 4E-BP1 phosphorylation and subsequent eIF4E release in RSV-transformed cells contribute to malignant cell growth by relieving translational inhibition.
- The rapamycin-sensitive pathway, involving p70S6k, plays a role in regulating translation factors in these cells, but is not solely responsible for 4E-BP1 control.
- These findings highlight the complex interplay of signaling pathways in cancer development and suggest potential therapeutic targets for controlling malignant proliferation.