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Ras-mediated phosphorylation of a conserved threonine residue enhances the transactivation activities of c-Ets1 and
B S Yang1, C A Hauser, G Henkel
1Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.
Insights
Ras oncogene signaling specifically targets Ets1 and Ets2 transcription factors. Phosphorylation of a key threonine residue in Ets1/Ets2 is essential for Ras-mediated gene activation in cancer cells.
Area of Science:
- Molecular Biology
- Oncology
- Cell Signaling
Background:
- Ras oncogene products are crucial regulators of gene expression in transformed cells.
- The Ets family of transcription factors plays a role in Ras-mediated gene regulation.
Purpose of the Study:
- To identify specific Ets transcription factors targeted by Ras signaling pathways.
- To elucidate the molecular mechanisms underlying Ras-mediated activation of Ets factors.
Main Methods:
- Transient transfection assays were used to examine the activation of Ras-responsive enhancer (RRE) reporters.
- Reporter constructs containing Ets-AP-1 and Ets-Ets binding sites were employed.
- Phosphoamino acid analysis was performed on radiolabeled Ets2 to assess phosphorylation.
- Site-directed mutagenesis was used to investigate the role of specific threonine residues.
Main Results:
- Ets1 and Ets2, but not other tested Ets factors, significantly activated RRE reporters in the presence of oncogenic Ras.
- Ras and Ets2 co-activated an Ets-Ets binding site reporter, but not the c-fms promoter.
- Mutating a conserved threonine residue in Ets1 (Thr38) and Ets2 (Thr72) abrogated reporter gene activation.
- Ras induced threonine-specific phosphorylation of Ets2, which was dependent on Thr72.
Conclusions:
- Ets1 and Ets2 are specific nuclear targets of Ras signaling pathways.
- Phosphorylation of a conserved threonine residue is a critical step in Ras-mediated activation of Ets1 and Ets2.
- These findings provide insight into the molecular mechanisms of oncogenic Ras signaling.