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Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and

C M Cahill1, G Tzivion, N Nasrin

  • 1Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA.

The Journal of Biological Chemistry
|January 1, 2001
PubMed

Insights

Insulin signaling regulates the DAF-16 transcription factor through PI 3-kinase/AKT. This pathway impacts DAF-16 DNA binding and activity via 14-3-3 dependent and independent mechanisms.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Genetics

Background:

  • The insulin-like signaling pathway, involving phosphatidylinositol 3-kinase (PI 3-kinase) and AKT, is crucial for regulating cellular processes.
  • In Caenorhabditis elegans, this pathway negatively impacts the activity of the DAF-16 transcription factor.
  • DAF-16 homologs in mammals, FKHR and FKHRL1, are known targets of AKT, influencing their nuclear localization.

Purpose of the Study:

  • To investigate the regulation of C. elegans DAF-16 by the PI 3-kinase/AKT pathway in mammalian cells.
  • To elucidate the mechanisms by which AKT phosphorylation affects DAF-16's DNA binding and transcriptional activity.
  • To identify both 14-3-3 dependent and independent modes of PI 3-kinase signaling on DAF-16 function.

Main Methods:

  • Utilized mammalian cell culture (HepG2 cells) to study DAF-16 function.
  • Employed in vitro phosphorylation assays with AKT and DAF-16.
  • Created DAF-16 mutants lacking AKT/14-3-3 binding sites.
  • Used a GAL4 DNA binding domain/DAF-16 fusion protein to assess transcriptional activity.
  • Applied the PI 3-kinase inhibitor LY294002.

Main Results:

  • AKT directly phosphorylates DAF-16, creating 14-3-3 binding sites and regulating its nuclear/cytoplasmic distribution.
  • AKT-phosphorylated DAF-16 interaction with 14-3-3 inhibits its binding to the insulin response element (IRE) in the IGFBP-1 gene.
  • Insulin signaling to PI 3-kinase/AKT inhibits DAF-16's transcriptional activity via the IRE.
  • Mutations preventing 14-3-3 association abolish this inhibition, while LY294002 enhances DAF-16 activity.
  • Constitutively nuclear DAF-16 mutants lacking 14-3-3 sites also show enhanced activity upon LY294002 treatment, indicating a 14-3-3-independent mechanism.

Conclusions:

  • PI 3-kinase/AKT signaling regulates DAF-16 DNA binding and transcription through at least two distinct mechanisms.
  • One mechanism involves 14-3-3-dependent regulation of DAF-16 localization and DNA binding.
  • A second, 14-3-3-independent mechanism also contributes to PI 3-kinase-mediated regulation of DAF-16 activity.

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