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Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter
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Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity

Margret H Bülow1, Torsten R Bülow2, Michael Hoch3

  • 11] Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, 8093 Zurich, Switzerland [2] Life and Medical Sciences (LIMES) Institute, Program Unit Development and Genetics, Laboratory for Molecular Developmental Biology, University of Bonn, 53115 Bonn, Germany.

Scientific Reports
|February 12, 2014
PubMed

Insights

Src signaling regulates insulin pathways by controlling the dFOXO transcription factor in fruit flies. Lowering Src activity moves dFOXO into the nucleus, impacting gene expression and growth.

Area of Science:

  • Molecular Biology
  • Cellular Signaling
  • Genetics

Background:

  • Src family kinases (SFKs) are implicated in mammalian insulin signaling.
  • The precise in vivo physiological link between Src and insulin pathway effectors remains unclear.

Purpose of the Study:

  • To investigate the in vivo physiological link between Src signaling and the insulin pathway effector dFOXO in Drosophila.
  • To determine if dFOXO is a downstream effector of Src signaling.

Main Methods:

  • Utilized Drosophila melanogaster as a model organism.
  • Manipulated Src42A signaling levels through ectopic activation, loss-of-function mutations, and pharmacological inhibition.
  • Assessed dFOXO nuclear localization via microscopy.
  • Quantified the expression of dFOXO target genes (d4EBP, dInR) using RT-qPCR.
  • Employed genetic epistasis analysis by introducing dFOXO loss-of-function mutations into Src42A mutants.

Main Results:

  • Ectopic Src42A activation in larval fat bodies caused dFOXO nuclear exclusion.
  • Reduced Src signaling (mutations or inhibition) led to nuclear dFOXO localization in growing larvae.
  • Lowered Src signaling induced transcription of dFOXO target genes d4EBP and dInR.
  • dFOXO loss-of-function rescued target gene induction and body size reduction in Src42A mutants.
  • Evidence suggests conserved regulation of FOXO by Src in mammalian cells.

Conclusions:

  • dFOXO is a critical downstream effector of Src signaling in vivo.
  • Src signaling modulates dFOXO activity, influencing target gene expression and organismal growth.
  • The regulatory relationship between Src and FOXO is evolutionarily conserved.

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