Related Experiment Video

Updated: May 12, 2026

RhoC GTPase Activation Assay
09:58

RhoC GTPase Activation Assay

Published on: August 23, 2010

RhoA influences the nuclear localization of extracellular signal-regulated kinases to modulate p21Waf/Cip1 expression

Brian S Zuckerbraun1, Richard A Shapiro, Timothy R Billiar

  • 1University of Pittsburgh, Department of Surgery, Pittsburgh, Pa, USA. zuckerbraunbs@msx.upmc.edu

Circulation
|July 23, 2003
PubMed
Abstract

Insights

Small GTPase RhoA influences smooth muscle cell proliferation by affecting the nuclear localization of extracellular signal-regulated kinases (ERKs). RhoA regulation of the cytoskeleton impacts ERK

Area of Science:

  • Cell Biology
  • Molecular Signaling
  • Smooth Muscle Physiology

Background:

  • Mitogen-activated protein kinases (extracellular signal-regulated kinases, ERKs) regulate smooth muscle cell (SMC) cell-cycle progression.
  • The small GTPase RhoA modulates ERK activity, influencing SMC proliferation, but the precise mechanism remains unclear.
  • RhoA's role in actin cytoskeleton regulation and its convergence on signaling pathways are key to understanding ERK modulation.

Purpose of the Study:

  • To investigate how RhoA regulates ERK downstream signaling and SMC proliferation.
  • To determine if RhoA affects ERK signaling through cytoskeletal changes and nuclear localization.
  • To elucidate the mechanistic link between RhoA, the cytoskeleton, and ERK-mediated SMC proliferation.

Main Methods:

  • SMCs were treated with Clostridia botulinum C3 exoenzyme to inhibit RhoA.
  • ERK phosphorylation and nuclear localization were assessed.
  • p21Waf1/Cip1 transcription and protein levels were measured.
  • Immunostaining was used to visualize phosphorylated ERK and the actin cytoskeleton.
  • Leptomycin B was used to inhibit nuclear export and induce ERK nuclear accumulation.

Main Results:

  • RhoA inhibition decreased SMC proliferation and increased p21Waf1/Cip1 levels, effects partially reversed by inhibiting ERK phosphorylation.
  • Inactivation of RhoA increased nuclear localization of phosphorylated ERK without altering overall ERK phosphorylation levels.
  • Phosphorylated ERK was found to associate with the actin cytoskeleton, which was disrupted by C3 exoenzyme.
  • Inhibition of nuclear export (Leptomycin B) also increased p21Waf1/Cip1 levels.

Conclusions:

  • RhoA inhibition enhances the nuclear levels of phosphorylated ERK.
  • Inhibition of RhoA or nuclear export leads to increased p21Waf1/Cip1 expression and reduced SMC proliferation, with partial dependence on ERK.
  • RhoA's regulation of the actin cytoskeleton likely dictates ERK's subcellular localization and subsequent impact on SMC proliferation.

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