Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury

Mark A. Hallett, Pierre C. Dagher, Simon J. Atkinson

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK10 and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations.

Original languageEnglish (US)
Pages (from-to)C129-C138
JournalAmerican Journal of Physiology - Cell Physiology
Volume285
Issue number1 54-1
StatePublished - Jul 1 2003

Fingerprint

rho GTP-Binding Proteins
Guanosine Triphosphate
Nucleotides
Wounds and Injuries
Adenosine Triphosphate
Actins
Ischemia
Antimycin A
rho-Associated Kinases
Proximal Kidney Tubule
GTP Phosphohydrolases
Glutathione Transferase
GTP-Binding Proteins
Energy Metabolism
triphosphoric acid
Proteins
Cells
Membranes
Recovery
Cell Line

Keywords

  • Actin
  • Adenosine 5′-triphosphate
  • Cdc42
  • Ezrin
  • Guanosine 5′-triphosphate
  • Rac

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury. / Hallett, Mark A.; Dagher, Pierre C.; Atkinson, Simon J.

In: American Journal of Physiology - Cell Physiology, Vol. 285, No. 1 54-1, 01.07.2003, p. C129-C138.

Research output: Contribution to journalArticle

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