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Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B

G Rena1, S Guo, S C Cichy

  • 1Department of Biochemistry, Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

The Journal of Biological Chemistry
|June 8, 1999
PubMed

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.

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