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Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a
1Developmental Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
In the nematode Caenorhabditis elegans, mutations of the insulin/insulin-like growth factor-1 receptor homologue Daf-2 gene cause developmental arrest at the dauer stage. The effect of Daf-2 mutations is counteracted by mutations in the Daf-16 gene, suggesting that Daf-16 is required for signaling by Daf-2. Daf-16 encodes a forkhead transcription factor. Based on sequence similarity, the FKHR genes are the likeliest mammalian Daf-16 homologues. FKHR proteins contain potential sites for phosphorylation by the serine/threonine kinase Akt. Because Akt is phosphorylated in response to insulin and has been implicated in a variety of insulin effects, we investigated whether insulin affects phosphorylation of FKHR. Insulin stimulated phosphorylation of endogenous FKHR and of a recombinant c-Myc/FKHR fusion protein transiently expressed in murine SV40-transformed hepatocytes. The effect of insulin was inhibited by wortmannin treatment, suggesting that PI 3-kinase activity is required for FKHR phosphorylation. Mutation of serine 253, located in a consensus Akt phosphorylation site at the carboxyl-terminal end of the forkhead domain, abolished the effect of insulin on FKHR phosphorylation. In contrast, mutation of two additional Akt phosphorylation sites, at amino acids threonine 24 or serine 316, did not abolish insulin-induced phosphorylation. These data indicate that FKHR may represent a distal effector of insulin action.
Insights
Mutations in the Daf-2 gene cause developmental arrest in C. elegans, but Daf-16 gene mutations counteract this effect. FKHR phosphorylation by insulin suggests it is a key player in insulin signaling pathways.
Area of Science:
- Molecular Biology
- Genetics
- Cell Signaling
Background:
- Mutations in the insulin/insulin-like growth factor-1 receptor homologue Daf-2 gene in C. elegans lead to developmental arrest.
- The Daf-16 gene, encoding a forkhead transcription factor, counteracts Daf-2 mutations, indicating its role in Daf-2 signaling.
- FKHR genes are mammalian homologues of Daf-16, and FKHR proteins have potential phosphorylation sites for Akt kinase.
Purpose of the Study:
- To investigate whether insulin affects the phosphorylation of FKHR.
- To determine if PI 3-kinase activity and specific Akt phosphorylation sites are involved in insulin-mediated FKHR phosphorylation.
Main Methods:
- Studied endogenous FKHR and recombinant c-Myc/FKHR fusion protein phosphorylation in murine hepatocytes.
- Utilized wortmannin treatment to assess the role of PI 3-kinase.
- Mutated specific Akt phosphorylation sites (Serine 253, Threonine 24, Serine 316) on FKHR to evaluate their impact on insulin-induced phosphorylation.
Main Results:
- Insulin stimulated the phosphorylation of both endogenous and recombinant FKHR.
- Insulin-induced FKHR phosphorylation was inhibited by wortmannin, indicating PI 3-kinase dependence.
- Mutation of Serine 253 abolished insulin-induced FKHR phosphorylation, while mutations at Threonine 24 and Serine 316 did not.
Conclusions:
- FKHR phosphorylation is regulated by insulin in a PI 3-kinase-dependent manner.
- Serine 253 is a critical site for insulin-induced FKHR phosphorylation.
- FKHR may function as a downstream effector in insulin signaling pathways.