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Updated: May 7, 2026

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Published on: March 31, 2012

Extracellular signal-regulated kinase regulates RhoA activation and tumor cell plasticity by inhibiting guanine

Anne von Thun1, Christian Preisinger, Oliver Rath

  • 1The Beatson Institute for Cancer Research, Glasgow, United Kingdom.

Molecular and Cellular Biology
|September 18, 2013
PubMed

Insights

Short-term ERK inhibition boosts RhoA signaling via GEF-H1, independent of Fra-1. This pathway regulates cell motility and invasiveness, identifying GEF-H1 as a key ERK effector.

Area of Science:

  • Cell Biology
  • Molecular Signaling

Background:

  • Extracellular signal-regulated kinase (ERK) pathway dysregulation is implicated in cancer.
  • ERK inhibition in Ras mutant cells typically increases RhoA activity and reduces cell motility, linked to Fra-1.
  • A Fra-1-independent mechanism for RhoA regulation by ERK inhibition was investigated.

Purpose of the Study:

  • To identify novel effectors of ERK signaling that regulate RhoA activity.
  • To elucidate the mechanism by which ERK inhibition impacts RhoA signaling and cell behavior.
  • To investigate the role of guanine nucleotide exchange factor H1 (GEF-H1) in this process.

Main Methods:

  • Mass spectrometry-based proteomics to identify protein interactions.
  • Western blotting and kinase assays to assess protein phosphorylation.
  • Gene knockdown and site-directed mutagenesis (S959A GEF-H1 mutant) to study protein function.
  • Cell motility and invasiveness assays.

Main Results:

  • Proteomics identified GEF-H1 as a mediator of RhoA signaling augmentation upon short-term ERK inhibition.
  • ERK directly binds and phosphorylates GEF-H1 at serine 959 (S959).
  • Phosphorylation of S959 inhibits GEF-H1 activity, decreasing RhoA signaling.
  • GEF-H1 S959 phosphorylation is critical for regulating cell motility and invasiveness.

Conclusions:

  • GEF-H1 is a novel, critical effector of ERK signaling controlling RhoA activity.
  • The ERK-GEF-H1 interaction and S959 phosphorylation regulate cell motility, morphology, and invasiveness.
  • This Fra-1-independent pathway provides new insights into cancer cell migration regulation.

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