Am J Physiol Cell Physiol 285: C129-C138, 2003.
First published March 5, 2003; doi:10.1152/ajpcell.00007.2003
0363-6143/03 $5.00
RECEPTORS AND SIGNAL TRANSDUCTION
Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury
Mark A. Hallett,
Pierre C. Dagher, and
Simon J. Atkinson
Department of Medicine, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Submitted 9 January 2003
; accepted in final form 4 March 2003
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.
Rac; Cdc42; actin; ezrin; adenosine 5'-triphosphate; guanosine 5'-triphosphate
Address for reprint requests and other correspondence: S. J. Atkinson, Indiana
Univ. School of Medicine, Dept. of Medicine-Nephrology, 950 W. Walnut St.,
R2–202, Indianapolis, IN 46202 (E-mail:
satkinso{at}iupui.edu).
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Copyright © 2003 by the American Physiological Society.
