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.
Highlights
The FKHR gene was first identified from its disruption by the t(2;13) chromosomal translocation seen in the pediatric tumor alveolar rhabdomyosarcoma
The FKHR gene was first identified by virtue of its fusion to the PAX3 gene in the t(2;13) translocation found in the pediatric soft tissue tumor alveolar rhabdomyosarcoma [11]
In EMSAs, we found that when expressed as a glutathione S-transferase (GST) fusion protein, an N-terminal fragment of FKHR containing the forkhead DNA-binding motif could bind to a probe containing the IGFBP-1 insulin-responsive sequence (IRS) (Fig. 1A)
Summary
From the ‡Department of Biological Chemistry and ¶Department of Pathology and Comprehensive Cancer Center, **Institute of Gerontology, University of Michigan Medical School Ann Arbor, Michigan 48109 and the ʈDepartment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104. 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. The FKHR gene was first identified by virtue of its fusion to the PAX3 gene in the t(2;13) translocation found in the pediatric soft tissue tumor alveolar rhabdomyosarcoma [11] This gene encodes for a protein containing a DNA-binding motif shared with members of the HNF-3/forkhead family of transcription factors [12,13,14]. We have identified three Akt phosphorylation sites in FKHR and have data suggesting that phosphorylation on all or a subset of these sites contributes to the down-regulation of FKHR’s ability to activate transcription and to induce apoptosis. Because its activity can be inhibited by Akt phosphorylation, FKHR is a potential Akt nuclear target in metabolic and survival pathways
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