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Negative regulation of the forkhead transcription factor FKHR by Akt
1Department of Biological Chemistry, University of Michigan Medical School Ann Arbor, Michigan 48109, USA. edtang@unich.edu
Abstract:
The FKHR gene was first identified from its disruption by the t(2;13) chromosomal translocation seen in the pediatric tumor alveolar rhabdomyosarcoma. It encodes for a member of the forkhead family of transcription factors. Recently, a homolog of FKHR in the nematode Caenorhabditis elegans was identified called DAF-16, which is a downstream target of two Akt homologs in an insulin-related signaling pathway. We have examined the possible role of Akt in the regulation of FKHR. We find that FKHR can bind in vitro to the insulin-responsive sequence (IRS) in the insulin-like growth factor-binding protein 1 promoter and can activate transcription from a reporter plasmid containing multiple copies of the IRS. Expression of active but not inactive Akt can suppress FKHR-mediated transcriptional activation. Akt can phosphorylate FKHR in vitro on three phosphoacceptor sites, at least a subset of which can also be phosphorylated by Akt in vivo. Importantly, mutation of these three sites to alanine residues enhances the transcriptional activity of FKHR and renders it resistant to inhibition by Akt. Expression of an Akt-resistant mutant of FKHR causes apoptosis in 293T cells in a manner dependent on DNA binding. These results suggest that FKHR may be a direct nuclear regulatory target for Akt in both metabolic and cell survival pathways.
Insights
The forkhead transcription factor FKHR is regulated by Akt, a key player in insulin signaling. Akt phosphorylation of FKHR impacts its activity, suggesting FKHR is a nuclear target for Akt in metabolic and cell survival pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The FKHR gene is implicated in alveolar rhabdomyosarcoma via chromosomal translocation.
- FKHR is a transcription factor and a homolog of C. elegans DAF-16, a target in insulin signaling.
- Akt is a kinase involved in insulin-related signaling pathways.
Purpose of the Study:
- To investigate the regulatory role of Akt in FKHR activity.
- To determine if Akt directly interacts with and modifies FKHR function.
- To explore the implications of FKHR regulation by Akt in cellular processes.
Main Methods:
- In vitro binding assays to assess FKHR-DNA interaction.
- Reporter gene assays to measure FKHR transcriptional activity.
- In vitro and in vivo phosphorylation studies using Akt and FKHR.
- Site-directed mutagenesis to analyze phosphorylation site function.
Main Results:
- FKHR binds to the insulin-responsive sequence (IRS) and activates transcription.
- Active Akt suppresses FKHR-mediated transcription.
- Akt phosphorylates FKHR at specific sites, which can be mutated to confer Akt resistance.
- Akt-resistant FKHR mutants induce apoptosis in 293T cells.
Conclusions:
- FKHR is a direct nuclear target of Akt.
- Akt-mediated phosphorylation of FKHR plays a role in regulating its transcriptional activity.
- FKHR and Akt signaling are involved in both metabolic regulation and cell survival pathways.