Negative Regulation of the Forkhead Transcription Factor FKHR by Akt

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.