Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation
Hitomi Matsuzaki1, Hiroaki Daitoku, Mitsutoki Hatta
1Center for Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Abstract:
Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.
Insights
Insulin signaling triggers the degradation of Forkhead transcription factor FKHR (Foxo1) in liver cells. This process involves FKHR phosphorylation, ubiquitination, and subsequent proteasomal breakdown, revealing a novel regulatory mechanism.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Metabolic regulation
Background:
- Forkhead transcription factor FKHR (Foxo1) is crucial for glucose homeostasis, cell cycle, and apoptosis.
- Insulin and growth factors phosphorylate FKHR, leading to cytoplasmic retention and gene expression repression.
Purpose of the Study:
- To investigate the fate of FKHR following insulin stimulation in HepG2 cells.
- To elucidate the molecular mechanisms underlying insulin-induced FKHR regulation.
Main Methods:
- Utilized HepG2 cells for experiments.
- Employed insulin stimulation, proteasome inhibitors, and ubiquitination assays (in vivo and in vitro).
- Investigated the role of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) signaling pathways.
Main Results:
- Insulin treatment reduced endogenous FKHR protein levels in HepG2 cells.
- FKHR undergoes ubiquitination, which is enhanced by insulin.
- Insulin-induced FKHR degradation is mediated by the PI3K/PKB pathway.
- Mutations in PKB phosphorylation sites on FKHR inhibited ubiquitination.
- Efficient FKHR ubiquitination requires both phosphorylation and cytoplasmic retention.
Conclusions:
- Insulin-induced FKHR phosphorylation triggers a multi-step negative regulatory process.
- This regulation involves both nuclear exclusion and ubiquitination-mediated degradation of FKHR.
- Unveiled a novel mechanism for insulin's control over FKHR stability and function.
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