Negative Regulation of the Forkhead Transcription Factor FKHR by Akt*
- 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 ‖Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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.
Footnotes
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↵* This work was supported by the National Institutes of Health (to K.-L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed (E. D. T. or K.-L. G.): Dept. of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109. Fax: 734-763-4581 (for both E. D. T. and K.-L. G.); E-mail: edtang{at}umich.edu (for E. D. T.) or kunliang{at}umich.edu (for K.-L. G.).
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↵2 E. D. Tang and K.-L. Guan, unpublished data.
- Abbreviations:
- PKB
-
protein kinase B
- HA
-
hemagglutinin
- β-gal
-
β-galactosidase
- PAGE
-
polyacrylamide gel electrophoresis
- GST
-
glutathione S-transferase
- HNF-3
-
hepatic nuclear factor 3
- X-gal
-
5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside
- IGFBP-1
-
insulin-like growth factor-binding protein-1
- IRS
-
insulin-responsive sequence
- EMSA
-
electrophoretic mobility shift assay
- MOPS
-
4-morpholinepropanesulfonic acid
- WT
-
wild-type
-
- Received April 5, 1999.
- Revision received April 19, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
