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Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion
Graham Rena1, Yvonne L Woods, Alan R Prescott
1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. grena1@biochem.dundee.ac.uk
Abstract:
FKHR is phosphorylated by protein kinase B (PKB) at Thr24, Ser256 and Ser319 in response to growth factors, stimulating the nuclear exit and inactivation of this transcription factor. Here we identify two further residues, Ser322 and Ser325, that become phosphorylated in insulin-like growth factor-1 (IGF-1)-stimulated cells and which are mediated by the phosphatidylinositol 3-kinase-dependent PKB-catalysed phosphorylation of Ser319. Phosphorylation of Ser319 forms a consensus sequence for phosphorylation by CK1, allowing it to phosphorylate Ser322, which in turn primes the CK1-catalysed phosphorylation of Ser325. IGF-1 stimulates the phosphorylation of Thr24, Ser256, Ser319, Ser322 and Ser325 in embryonic stem (ES) cells, but not in PDK1-/- ES cells, providing genetic evidence that PDK1 (the upstream activator of PKB) is required for the phosphorylation of FKHR in mammalian cells. In contrast, the phosphorylation of Ser329 is unaffected by IGF-1 and the phosphorylation of this site is not decreased in PDK1-/- ES cells. The cluster of phosphorylation sites at Ser319, Ser322, Ser325 and Ser329 appears to accelerate nuclear export by controlling the interaction of FKHR with the Ran-containing protein complex that mediates this process.
Insights
Growth factors trigger FKHR phosphorylation by protein kinase B (PKB), promoting nuclear exit. New sites Ser322 and Ser325 phosphorylation are identified, dependent on PDK1, impacting FKHR nuclear export.
Area of Science:
- Cellular biology
- Molecular biology
- Signal transduction
Background:
- Forkhead transcription factors regulate gene expression.
- FKHR (Forkhead in rhabdomyosarcoma) is a transcription factor regulated by growth factor signaling.
- Phosphorylation of FKHR by protein kinase B (PKB) controls its nuclear localization and activity.
Purpose of the Study:
- To identify novel phosphorylation sites on FKHR.
- To elucidate the signaling pathway regulating FKHR phosphorylation in response to insulin-like growth factor-1 (IGF-1).
- To investigate the role of PDK1 in FKHR phosphorylation and nuclear export.
Main Methods:
- Phosphorylation site mapping using mass spectrometry.
- Site-directed mutagenesis to assess the function of specific phosphorylation sites.
- Analysis of FKHR phosphorylation in wild-type and PDK1-deficient embryonic stem (ES) cells.
- Investigation of FKHR interaction with the Ran-importin complex.
Main Results:
- Two novel FKHR phosphorylation sites, Ser322 and Ser325, were identified in IGF-1-stimulated cells.
- Phosphorylation of Ser319 by PKB creates a consensus site for CK1-mediated phosphorylation of Ser322, which then primes Ser325 phosphorylation.
- IGF-1-induced phosphorylation of Thr24, Ser256, Ser319, Ser322, and Ser325 in ES cells requires PDK1.
- Phosphorylation of Ser329 is independent of IGF-1 and PDK1.
- The cluster of phosphorylation sites (Ser319, Ser322, Ser325, Ser329) enhances FKHR nuclear export by modulating its interaction with the Ran-containing complex.
Conclusions:
- PDK1 is essential for IGF-1-stimulated FKHR phosphorylation in mammalian cells.
- A sequential phosphorylation cascade involving PKB and CK1 regulates FKHR.
- FKHR phosphorylation sites play a critical role in controlling its nuclear export and transcriptional activity.