Related Experiment Videos
Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B
1Department of Biochemistry, Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Abstract:
Protein kinase B lies "downstream" of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans, is rapidly phosphorylated by human protein kinase Balpha (PKBalpha) at Thr-24, Ser-256, and Ser-319 in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans.
Insights
FKHR is phosphorylated by protein kinase B (PKB), a key signaling molecule for growth factors. This suggests FKHR mediates PKB
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Transcription factor regulation
Background:
- Protein kinase B (PKB) is a crucial intracellular mediator for insulin and growth factor signaling.
- FKHR is a human homolog of the C. elegans transcription factor DAF16.
- DAF16 is part of a conserved signaling pathway involving insulin/IGF-1 receptor, PtdIns 3-kinase, and PKB.
Purpose of the Study:
- To investigate if FKHR is a direct substrate of PKB.
- To identify the specific phosphorylation sites on FKHR by PKB.
- To determine if FKHR mediates PKB's effects on gene expression.
Main Methods:
- In vitro phosphorylation assays using purified PKBalpha and FKHR.
- Cotransfection experiments with FKHR, PKB, and PDK1 in 293 cells.
- Stimulation of cells with insulin-like growth factor 1 (IGF-1) and treatment with specific inhibitors (wortmannin, PD 98059, rapamycin).
Main Results:
- FKHR is rapidly phosphorylated by PKBalpha at Thr-24, Ser-256, and Ser-319 in vitro.
- These sites are also phosphorylated in cells upon cotransfection with PKB or PDK1.
- IGF-1 stimulation induces phosphorylation of these sites, dependent on PtdIns 3-kinase activity and not MAPK or mTOR pathways.
- The phosphorylation pattern suggests FKHR is a physiological substrate of PKB.
Conclusions:
- FKHR is a direct and physiological substrate of protein kinase B (PKB).
- PKB-mediated phosphorylation of FKHR may play a role in regulating gene expression.
- The findings support a conserved role for PKB in regulating transcription factors like FKHR/DAF16 across species.