Cdc42GAP, reactive oxygen species, and the vimentin network

Am J Physiol Cell Physiol. 2009 Aug;297(2):C299-309. doi: 10.1152/ajpcell.00037.2009. Epub 2009 Jun 3.

Abstract

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cells, Cultured
  • Dogs
  • Enzyme Activation
  • GTPase-Activating Proteins / genetics
  • GTPase-Activating Proteins / metabolism*
  • Hydrogen Peroxide / metabolism
  • Muscle Contraction / physiology
  • Myocytes, Smooth Muscle / cytology
  • Myocytes, Smooth Muscle / metabolism
  • Oxidants / metabolism
  • Phosphorylation
  • Reactive Oxygen Species / antagonists & inhibitors
  • Reactive Oxygen Species / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Serotonin / metabolism
  • Vimentin / genetics
  • Vimentin / metabolism*
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*
  • p21-Activated Kinases / metabolism
  • rac1 GTP-Binding Protein / metabolism
  • rho GTP-Binding Proteins / metabolism

Substances

  • GTPase-Activating Proteins
  • Oxidants
  • Reactive Oxygen Species
  • Recombinant Proteins
  • Vimentin
  • Serotonin
  • Hydrogen Peroxide
  • p21-Activated Kinases
  • cdc42 GTP-Binding Protein
  • rac1 GTP-Binding Protein
  • rho GTP-Binding Proteins