Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 9 January 2003 ; accepted in final form 4 March 2003
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ABSTRACT |
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Rac; Cdc42; actin; ezrin; adenosine 5'-triphosphate; guanosine 5'-triphosphate
Ischemia is a pathophysiological state that is characterized in part by poor tissue perfusion and subsequent anoxia with inhibition of oxidative phosphorylation caused by substrate depletion (13). Although ischemia is a complex multifactorial process, many of its consequences result from, and can be recapitulated by, depletion of the normal intracellular concentration of ATP (3). Ischemic injury to the epithelial cells of the kidney proximal tubule is a factor in the initiation and progression of acute renal failure and typifies many of the features of ischemic injury in other cell types and organs. The normal architecture of the actin cytoskeleton is an early casualty of ischemic injury and ATP depletion, and this disruption directly results in impaired cell-cell and cell-substrate adhesion, loss of tight junction barrier function (3, 17), and mixing of apical and basolateral transporters (30), with resultant impaired organ physiology and structure. There is a loss of actin from the apical microvilli, terminal web, cortical junctional complexes, and stress fibers. This results in membrane blebbing, internalization, and loss of apical microvilli (15, 40). The loss of actin and villin from microvilli is coincident with abnormal perinuclear accumulation of F-actin (42) and an increase in the ratio of F- to G-actin in the cell.
The complexity and pervasiveness of the cytoskeletal alterations that are precipitated by ischemic injury indicate that they are probably the result of the combined effect of many different pathways. Perturbations of normal signaling pathways are probably a major factor in this aspect of injury, and in view of their critical role in cytoskeletal regulation, Rho GTPases are obvious candidate molecules. Evidence for the role of RhoA during ATP depletion in kidney epithelium comes from experiments using the constitutively active Rho mutant RhoV14. When RhoV14 is microinjected into tissue culture cells derived from renal proximal tubule epithelium before ATP depletion, cortical actin and stress fibers are maintained during subsequent depletion (33). Conversely, when the bacterial toxin C3 transferase, which specifically inactivates Rho, is injected, stress fibers are not reassembled during recovery from ATP depletion. RhoV14 also protects tight junctions against disassembly during ATP depletion (17), whereas the corresponding constitutively active mutant of Rac, RacV12, protects adherens junctions (16).
Additional evidence for altered activity of Rho GTPases during ischemia or nucleotide triphosphate depletion is furnished by altered regulation of cytoskeletal proteins known to be downstream targets of Rho GTPase signaling. For example, ezrin is a protein link between the plasma membrane and the actin cytoskeleton (1) that is regulated by RhoA. In normal proximal tubule epithelial cells ezrin is localized in microvilli and at sites of cell-cell contact (19, 35). Ezrin binds to the cytoplasmic domain of the cell surface glycoprotein CD44 (36), and the carboxy terminus of ezrin binds to actin (37). The interaction of ezrin and CD44 is of high affinity only in the presence of phosphatidylinositol 4,5-bisphosphate (21). RhoA is thought to regulate this interaction through its effector, phosphatidylinositol 4-phosphate 5-kinase (29). Another effector of RhoA, Rho kinase (ROK), phosphorylates ezrin in vitro (28). The dephosphorylation of ezrin is an early event during ischemia in renal epithelium (8). As a result of dephosphorylation, ezrin dissociates from the cytoskeleton and can be extracted with nonionic detergents.
The intracellular concentration of GTP is tightly coupled to that of ATP, and the two decrease in tandem with ischemia or chemical anoxia (10). Given the evidence for involvement of Rho GTPase signaling and the paucity of data concerning the effect of intracellular GTP levels on Rho protein signaling mechanisms in vivo, we used the ability of the GTPase binding domains from Rho effectors to discriminate between the GTP- and GDP-bound forms of the GTPase (24) to measure the relative amount of active and inactive RhoA, Rac1, and Cdc42 during ATP depletion induced by treatment with the mitochondrial poison antimycin A combined with substrate depletion. We measured ATP and GTP levels in parallel with these assays to establish the degree of correlation between the activation state of the GTPase and nucleotide levels in the cell. We also followed the localization of actin stress fibers and the detergent solubility of ezrin during nucleotide triphosphate depletion and recovery. Finally, we measured the activity of RhoA, Rac1, and Cdc42 during nucleotide depletion and recovery in the presence of constitutively active RhoAL63.
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MATERIALS AND METHODS |
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Cell culture. LLC-PK10 porcine proximal tubule cells were grown with KP medium [DMEM-Ham's F-12 medium (1:1) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 14 mM NaHCO3, and 12.5 mM HEPES] with 10% fetal bovine serum in a humidified chamber (95% air-5% CO2) at 37°C. Cells were grown in collagen coated T-25 flasks (Techno Plastic Products, Trasadingen, Switzerland). Cells were grown for 2 days after confluence and rinsed with warm PBS before use. To model ischemia, cells were incubated with depleted DMEM (medium without amino acids, glucose, serum, and antibiotics) and 100 nM antimycin A. If recovery of these cells was required, they were rinsed with warm PBS and incubated with supplemented KP medium. For some experiments cells were rinsed twice and incubated with serum-free normal growth medium to control for the effect of removing growth factors. We analyzed the effect of ATP depletion in quiescent cells by incubating cells in serum-free medium for 24 h before the experiment.
Nucleotide triphosphate assay. Cell cultures were rinsed three times with ice-cold PBS and scraped with 300 µl of ice-cold acetonitrile followed by 700 µl of ice-cold water (2) The extract was centrifuged at 16,000 g for 10 min at 4°C. The supernatant was gassed with N2 while on ice for 30 min to evaporate the acetonitrile. The residue was solubilized with 1 N NaOH, and the protein concentration was determined by Coomassie blue assay (Pierce Chemical, Rockford, IL). To separate nucleotide triphosphates, samples were diluted 1:1 and 100 µl was injected onto a 4-µm Nova-Pak C18 HPLC cartridge (100 x 8 mm ID) equipped with a radial compression chamber (Waters, Milford, MA). The elution buffer consisted of 20% acetonitrile, 10 mM ammonium phosphate, and 2 mM PIC Reagent A ion-pairing reagent (Waters). The column was run isocratically at 2 ml/min (18) on a Hewlett-Packard Chemstation model 1100 with ultraviolet detection at 254 nm (Hewlett-Packard, Wilmington, DE). Internal standards showed that nucleotide recovery was >90%.
Visualization of stress fibers. Cells were plated on collagen-coated coverslips in 35-mm petri dishes. Cells were treated with antimycin A as described in Cell culture. After the depletion time course, coverslips were fixed in PBS-5% formaldehyde, permeabilized with PBS plus 0.2% Triton X-100, and incubated with 0.66 µM rhodamine phalloidin (Molecular Probes, Eugene, OR). Confocal images were collected with a Zeiss Axiovert LSM510.
GTPase activity assay. The ROK Rho binding domain (ROK-BD, amino
acids 9411,075) and the p-21 associated kinase (Pak) binding domain
(Pak-BD, amino acids 67150) cloned into pGEX vectors were transformed
into Escherichia coli strain BL21. pGEX-ROK-BD was a kind gift from
Dr. Kozo Kaibuchi (Nagoya University, Nagoya, Japan; Ref.
27), and pGEX-Pak-BD was a
kind gift from Dr. Gary Bokoch (Scripps Research Institute, La Jolla, CA;
4). Production of the fusion
protein was initiated with isopropyl -D-thiogalactopyranoside
(IPTG). The fusion protein was isolated by binding to
S-hexylglutathione-agarose as described in the manufacturer's
instructions. The glutathione S-transferase (GST) fusion proteins
bound to glutathione agarose (GST-ROK-BD and GST-Pak-BD) were stored in
aliquots in liquid nitrogen for no more than 1 mo. The amount of fusion
protein required to capture all of the available GTPase was determined
empirically.
Affinity isolation was carried out as described previously
(5) with modifications. After
treatment with antimycin A cells were rinsed with warm PBS. Three hundred
fifty microliters of buffer A [25 mM Tris (pH 7.5 at 4°C), 150 mM
K acetate, 5 mM EDTA, 5 mM EGTA, 10 mM dithiothreitol (DTT), 1% Triton X-100,
60 mM n-octyl--D-glucopyranoside, 50 µM butylated
hydroxytoluene (BHT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM
benzamidine, 2 µg/ml aprotinin (Roche Molecular Biochemicals, Indianapolis,
IN), 1 µg/ml pepstatin A (Roche Molecular Biochemicals), 50 µg/ml
leupeptin (Peptides International, Osaka, Japan), and 40 µg/ml bestatin
(Roche Molecular Biochemicals)] were added to the T-25 flasks and incubated on
ice for 10 min with periodic manual rocking to evenly distribute the
extraction buffer. The buffer was removed and centrifuged briefly at 15,000
g. Twenty-five microliters of detergent extract were mixed with
thirty microliters of GST-ROK-BD to assay for RhoA activity or fifteen
microliters of GST-Pak-BD to assay for Rac1 or Cdc42 activity and incubated at
4°C for 1 h on a rotator. The GST-binding domain was rinsed three times
with 500 µl of buffer A without detergent or protease inhibitors.
This unbound fraction was precipitated with 10% trichloroacetic acid, rinsed
with 70% ethanol-acetone, dried, and dissolved in SDS sample buffer [50 mM
Tris 7.5, 2% sodium dodecyl sulfate (SDS), 5%
-mercaptoethanol, 6 M
urea, bromophenol blue]. The bound fraction was dried in a vacuum and
solubilized in SDS sample buffer. Bound and unbound fractions for each time
point were run on a 15% SDS-PAGE polyacrylamide gel (BioWhittaker Molecular
Applications, Rockland, ME). This was followed by Western blotting and
immunostaining with 1 µg/ml anti-RhoA antibody (26C4; Santa Cruz
Biotechnology, Santa Cruz, CA), 2 µg/ml anti-Rac1 antibody (Santa Cruz
Biotechnology), or 2 µg/ml anti-Cdc42 antibody (Santa Cruz Biotechnology)
and horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson
Immunoresearch, West Grove, PA). Blots were visualized with enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) and Biomax
ML film (Kodak, Rochester, NY). To determine the ratio of bound to unbound
GTPase, film images were digitized and the optical density of each band was
quantified with a Fluor-S MultiImager with Quantity One 4.1 software (Bio-Rad
Laboratories, Hercules, CA).
For assay validation experiments the detergent lysate was incubated with 1
mM GDP or 1 mM GTPS or without additional nucleotide for 10 min at
30°C. As a control for nonspecific binding to GST a sample of lysate was
incubated with glutathione-agarose-GST.
To ensure the validity of using a ratio of bound to unbound GTPase to express activity, the levels of the GTPases were measured by densitometry of immunoblots of whole cell extracts and normalized to the total amount of protein in LLC-PK10 cell lysates. When expressed as GTPase per microgram of total protein for each of three time points, 0 and 90 min of nucleotide triphosphate depletion and 90 min followed by 60 min of repletion, there was no significant change in GTPase (RhoA, Rac1, and Cdc42) concentration during the time course of the experiment (data not shown).
Ezrin solubility assay. The solubility of ezrin during depletion and recovery was determined with the lysates from the RhoA activity assay. The amount of ezrin in the detergent-insoluble fraction left after the lysate was made was determined by adding SDS sample buffer to the T flask containing the detergent-insoluble adherent cell layer and heating it to 80°C for 2 min. The viscous solution was scraped out, sonicated to reduce viscosity, and run with the detergent-soluble fractions on a 12% PAGE gel. Immunoblotting was performed with an anti-ezrin polyclonal antibody (kind gift of Jing Chen, Indiana State University, Terre Haute, IN; Ref. 9). Visualization and quantification were as described in GTPase activity assay.
To demonstrate the effect of the RhoA signaling pathway on ezrin detergent solubility, 10 µM of the ROK inhibitor Y27632 (Upstate Biotechnology, Lake Placid, NY; Ref. 11) was incubated with cells during recovery from depletion (as above) and the ezrin detergent solubility was determined as described above.
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RESULTS |
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Stress fiber integrity during depletion and recovery. The actin cytoskeleton is remodeled during ATP depletion (16, 17, 30). Actin stress fibers are regulated by RhoA and found near the basal membrane in LLC-PK10 cells (33). During the time course over which we measured ATP and GTP levels, we followed the integrity of stress fibers by labeling F-actin with rhodamine phalloidin (Fig. 2). Normal stress fibers (Fig. 2; 0 min) began to break down within 5 min after antimycin A treatment started and became progressively more punctate (Fig. 2; 590 min). After repletion of ATP with incubation in normal medium, stress fibers progressively reorganized (Fig. 2; 90/30 and 90/60 min of depletion/repletion).
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Measurement of RhoA activity. A fusion protein (GST-ROK-BD)
consisting of GST and the effector-binding domain of human ROK was used to
selectively capture the GTP-bound form of RhoA with glutathioneagarose resin
(24). Because only the
GTP-bound form of RhoA binds to the GST-ROK-BD, the active and the inactive
proteins could be separated and detected by immunoblotting with anti-RhoA
antibody (Fig. 3). We compared
the active and inactive RhoA fractions in a control cell detergent lysate
(Fig. 3A, lanes
1 and 2) and after 90 min of nucleotide triphosphate depletion
(Fig. 3A, lanes
7 and 8). After 90 min of depletion, the amount of inactive RhoA
increased and the amount of active RhoA decreased. The level of GTP-bound RhoA
decreased substantially when the cell lysate was incubated with a large excess
of exogenous GDP (Fig.
3A, lanes 3 and 4). Glutathione-agarose
was incubated with GST as an additional control. Lanes 5 and
6 in Fig. 3A
show that RhoA did not bind to GST-glutathione-agarose nonspecifically.
Additionally, after 90 min of nucleotide depletion, excess exogenous
GTPS (Fig. 3A,
lanes 9 and 10), but not GDP
(Fig. 3A, lanes
11 and 12), shifted the balance back toward the active form of
RhoA.
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Because removal of the growth factors present in the normal growth medium could affect the activity of RhoA independently of ATP depletion, we tested the effect of removing serum on RhoA activity in control cells not subjected to ATP depletion and the effect of the presence or absence of serum during the repletion phase. As shown in Fig. 3B, the presence or absence of serum during the assay did not affect RhoA activity significantly, although in the absence of serum RhoA activity levels during recovery were slightly lower than those observed with serum present. Serum starvation for 24 h (Fig. 3C) did not significantly alter RhoA activity in these cells, and it did not alter the affect of nucleotide triphosphate depletion.
RhoA activity during nucleotide triphosphate depletion and repletion with normal medium following the time course of stress fiber disruption seen in Fig. 2 is illustrated in Fig. 4A. Control cells (Fig. 4A, lanes 1 and 2) showed that a large fraction of RhoA was active (bound) in a confluent LLC-PK10 cell culture. During the time course of nucleotide triphosphate depletion (Fig. 4A, lanes 312) the amount of active RhoA decreased. On repletion of ATP levels with normal medium the level of active RhoA increased (Fig. 4A, lanes 1316). The average of three experiments expressed as a ratio of bound protein (active) to unbound protein (inactive) is shown in Fig. 4B. A significant loss of RhoA activity was detected after as little as 5 min of nucleotide triphosphate depletion, by which time the activity ratio dropped to 46.2 ± 15.1% of the control value. After 10 min of nucleotide triphosphate depletion the activity ratio dropped to 11.4 ± 8.2% of the control value. No measurable RhoA activity was detected at 60 min of nucleotide triphosphate depletion. On repletion with normal medium, the ratio recovered to 83.9 ± 78.9% of the control value by 30 min.
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Measurement of ezrin detergent solubility. To determine whether alterations in the amount of activated RhoA during ATP depletion and recovery were reflected in the behavior of a known cytoskeletal target of Rho signaling, we followed the detergent solubility of the ERM (ezrin, radixin, moesin) protein ezrin as an indication of its association with the actin cytoskeleton during the time course of nucleotide depletion and repletion (Fig. 5A). The control detergent fractionation of ezrin (0 min) shows that under our conditions much of the protein was recovered in the detergent-soluble fraction but a significant fraction remained in the detergent-insoluble fraction (Fig. 5A, lanes 1 and 2). With nucleotide triphosphate depletion (Fig. 5A, lanes 312) the amount of ezrin in the detergent-insoluble fraction decreased, suggesting that ezrin had dissociated from the cytoskeleton. As the cells recovered from depletion (Fig. 5A, lanes 1316), the amount of detergent-insoluble ezrin increased. An average detergent solubility for ezrin during the time course (n = 3) was expressed as the ratio of detergent insoluble to soluble ezrin. The detergent insolubility of ezrin decreased 60% during nucleotide triphosphate depletion and increased to control level after 30 min of repletion with normal medium (Fig. 5B) and showed an overall time course similar to that of RhoA activity with depletion and repletion (Fig. 4B).
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The serine/threonine kinase Rho kinase (or ROCK) is activated by binding to RhoA GTP and phosphorylates ezrin among other targets (28). To demonstrate the effect of RhoA signaling on ezrin detergent solubility, we incubated the ROK inhibitor Y27632 (11) with LLC-PK10 cells during nucleotide triphosphate depletion and repletion. Figure 5C shows a Western blot of a typical experiment. In control cells a substantial amount of ezrin was associated with the cytoskeleton in the detergent-insoluble fraction (Fig. 5C, lanes 1 and 2). After 90 min of depletion, ezrin dissociated from the cytoskeleton and a decreased amount was found in the detergent-insoluble fraction (Fig. 5C, lanes 3 and 4). However, with 60 min of repletion in normal medium (Fig. 5C, lanes 5 and 6), ezrin was again associated with the cytoskeleton in the insoluble fraction. When Y27632 was included in the incubation medium, ezrin failed to reassociate with the cytoskeleton after 60 min of repletion, suggesting that ROK, and therefore RhoA signaling, is important for the stable association of ezrin with the cytoskeleton.
Activity measurement of Cdc42 and Rac1. The depletion of
nucleotide triphosphates likely affects the activity of the other Rho family
GTPases as well as RhoA itself. We measured the activity of Cdc42 and Rac1
with a similar pull-down assay using a fusion of GST to the effector domain of
Pak (containing the CRIB domain) to selectively bind GTP-bound Rac1 and Cdc42.
To validate the assay, nucleotides were added to detergent extracts of
LLC-PK10 cells to change the activity of the GTPases
(Fig. 6A). Without
exogenously added nucleotides (Fig.
6A, lanes 1 and 2) most of Rac1 and
nearly all of Cdc42 was found in the bound (active) lane. The addition of GDP
increased the amount of GTPase in the unbound (inactive) fraction for both
Cdc42 and Rac1. GST alone (Fig.
6A, lanes 5 and 6) did not pull down
any GTPase. When the cells were first depleted for 90 min with antimycin A,
Cdc42 did not show any significant decrease in activity. The level of active
Rac1 was decreased after 90 min of depletion, but a significant fraction of
GTP-bound Rac1 remained (Fig.
6A, lanes 7 and 8). Incubation with
GTPS increased the amount of GTPase in the active fraction for both
proteins (Fig. 6A,
lanes 9 and 10). Addition of GDP decreased the total amount
of Cdc42 that was recovered, making it impossible to determine the effect of
GDP on the activity of Cdc42 in this in vitro assay. GDP decreased the amount
of active Rac1 (Fig.
6A, lanes 11 and 12). We tested whether
serum deprivation contributed to the effects we observed. Substituting
serum-free medium for normal growth medium, without induction of ATP depletion
(Fig. 6B, lanes
14), did not affect Rac1 or Cdc42 activity, and it did not alter
the effect of ATP depletion (Fig.
6B, lanes 5 and 6). Serum starvation
(24 h) had no appreciable effect on Rac1 activity but did result in a decrease
in Cdc42 activity (Fig.
6C, lanes 1 and 2). However, Cdc42 and
Rac1 maintained high levels of activity after 90 min of ATP depletion, even
with prior serum starvation (Fig.
6C, lanes 9 and 10).
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Because, in contrast to our results with RhoA (Fig. 3, lanes 7 and 8, and Fig. 4), there was no significant decrease in the amount of active Cdc42 and Rac1 with depletion, we sought to demonstrate that the GTPase assay was capable of detecting physiological changes in the activity of Rac1 and Cdc42 in LLC-PK10 cells. Because cadherin-mediated cell-cell adhesion was shown to result in alterations in Rho GTPase activity (32), the activities of RhoA, Rac1, and Cdc42 were assayed with the pull-down assay 24 h after LLC-PK10 cells were seeded at a range of different densities. A representative Western blot is shown in Fig. 7A. RhoA activity increased >36-fold from the lowest to the highest initial cell density (graphed in Fig. 7B), whereas Rac1 and Cdc42 activity decreased by 91.1 ± 6.5% and 85.2 ± 14.7%, respectively. These experiments showed that our assay conditions were sufficiently sensitive to have detected physiological changes in the activity of all three GTPases during ATP depletion and recovery.
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A comparison of the changes in the activity of RhoA, Rac1, and Cdc42 during 90 min of nucleotide triphosphate depletion and depletion, followed by 60 min of repletion in normal medium, is shown in Fig. 8A. Under normal culture conditions (t = 0) in postconfluent cultures all three GTPases were active (bound fraction). Whereas RhoA was completely inactive after 90 min of depletion (unbound fraction), Rac1 was still active and Cdc42 activity had changed negligibly. A more complete time course of nucleotide triphosphate depletion and repletion for Rac1 activity revealed that Rac1 activity dropped after initiation of depletion to a minimum at 30 min but rebounded to higher levels after 60 and 90 min (Fig. 8B). With repletion Rac1 activity increased to control levels after 30 min.
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The increase in Rac1 activity after an initial decrease during depletion raised the possibility that Rac1, RhoA, or Cdc42 activity may fluctuate over a longer time course. However, a time course extended to 240 min of nucleotide triphosphate depletion showed that the activity of each of the three GTPases was relatively constant over longer time periods. RhoA activity remained near zero, Rac1 activity remained steady during 90240 min of depletion, and Cdc42 remained almost entirely in the active state (Fig. 9).
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Effect of activated RhoA during depletion. Considerable evidence exists for cross talk between the activities of different Rho family GTPases (22). Because RhoA activity decreases so rapidly with nucleotide depletion, we considered that this might affect the activity of Rac1 or Cdc42. To test this possibility, the constitutively active RhoA mutant RhoAL63 was transfected into LLC-PK10 cells. Subsequently, nucleotide triphosphate depletion was induced with antimycin A and the activities of RhoA, Rac1, and Cdc42 were measured after 90 min of depletion and after depletion followed by 60 min of repletion. No effect was found on the activity of endogenous RhoA or Cdc42 (data not shown). However, the ratio of active to inactive Rac1 decreased 32.1% during repletion in the presence of RhoAL63 compared with nontransfected controls. However, this change did not reach statistical significance (P = 0.189; n = 3).
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DISCUSSION |
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In contrast to the effect of ATP depletion on RhoA, the activation state of Rac1 and Cdc42 did not directly correlate with the level of ATP or GTP in the cell. We confirmed that our assay was sensitive to physiological changes in Rac1 and Cdc42 activity by measuring large decreases in activity as cell density increased to confluence. It is interesting to note that the changes we observed in RhoA, Rac1, and Cdc42 activity were in the opposite direction from those observed in similar experiments on cell density in another renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells (32). This discrepancy is not necessarily surprising, because other work has shown that signaling pathways involving Rho GTPases differ between MDCK and LLC-PK cells (16a). It should be noted that the assays we used here are valid as an indication of relative changes in GTPase activity that result from the manipulations we used to alter ATP levels or cadherin engagement but cannot be used as an indication of whether the basal level of GTPase activity in these cells is high or low compared with studies in other cells by other investigators, because of the sensitivity of the assay to buffer conditions and other variables in the protocol used.
Our assay showed that Cdc42 activity did not decrease during ATP depletion, whereas the level of active Rac1 initially declined but later recovered somewhat, even as ATP and GTP levels were still falling. Disruption of actin cytoskeletal organization is not, therefore, attributable to a net decrease in the cellular level of Cdc42 activity and cannot be related in a simple way to the level of Rac1 activity. It may be that the activity of Cdc42 and Rac1 is still an important factor in cytoskeletal disruption, but this disruption results instead from an imbalance in the level of activity of these Rho GTPases. In the absence of detectable RhoA activity, continued Cdc42 and Rac1 activity might indeed be one of the causes of aberrant actin polymerization into F-actin aggregates in the center of the cell that characterizes ATP depletion in renal proximal tubule cells (31), as well as in other cell types such as fibroblasts and endothelial cells (14, 20). Actin monomers released by the destruction of normal cytoskeletal assemblies after RhoA inactivation, and from the thymosin-sequestered pool, could be induced to polymerize at nuclei provided by the activity of Rac1 and Cdc42 effectors such as cortactin (38, 41).
The partial recovery of Rac1 activity at later times during ATP depletion, when ATP and GTP levels are still falling, is surprising. Because in many cell types RhoA activity antagonizes that of Rac1 (23, 39), we considered that the nearly complete inactivation of RhoA could explain the partial recovery of Rac1. However, the constitutively active RhoA mutant RhoAL63 had no effect in Rac1 activity under ATP-depleted conditions, so the signaling relays that normally operate between the two GTPases appear to be disrupted by ATP depletion but are again operable during repletion.
Under the conditions used here for ATP depletion, cellular ATP levels
decrease to 1% of the level in control cells, whereas the levels of GTP
decrease to a minimum of 5.8% of control levels. Assuming that the normal
concentrations of ATP and GTP in our LLC-PK10 cells are comparable
to those in other cell types
(26), this corresponds to a
decrease in ATP concentration from 25 mM to 2050 µM and a
decrease in GTP concentration from
300 µM to
17 µM. These GTP
concentrations still significantly exceed the affinity constants of the
GTPases for GTP. RhoA, Rac1, and Cdc42 GTPases' affinities for GTP are
comparable (KD
0.2 µM), as are their slightly
lower affinities for GDP (KD
0.50.6 µM)
(43). Exchange factors (GEFs)
act by increasing the dissociation rate constant for bound nucleotide, so that
in normal cells the cytoplasmic excess of GTP over GDP ensures that when GEFs
bind the result is formation of the GTP-bound form of the protein. Under
depleted conditions, however, GDP is in excess over GTP (at least over the
short time scales used here) and the activity of exchange factors could be
expected to result in inactivation of Rho proteins, because the relative
affinities for GTP and GDP do not bias the exchange reaction sufficiently to
prevent this. This may well be what happens to RhoA because the rapid kinetics
of RhoA inactivation that we observe would be hard to account for by the
intrinsic dissociation or hydrolysis rates and must therefore result from the
action of GEFs or GAPs. A role for regulatory proteins also seems to be
required in view of the very different behavior of RhoA, Rac1, and Cdc42. If
alterations in Rho protein activity were simply the result of their
equilibration to the ambient GTP-to-GDP ratio in the cytoplasm one would
expect to see the same effect of ATP depletion on all three GTPases, because
their nucleotide affinities, dissociation rates, and hydrolysis rates are
comparable. The fact that the effect of ATP depletion is so different for each
protein argues that depletion selectively impacts the regulation of upstream
pathways that control the activities of the GEF and GAP proteins. These
upstream pathways may still directly sense the GTP-to-GDP or ATP-to-ADP ratio
but could transduce the effects of alterations in these ratios differentially
to each Rho family member. Because it is generally accepted that GAP proteins
are more promiscuous and GEF proteins more specific, it is reasonable to
suppose that the differences in the effects of ATP depletion on RhoA, Rac1,
and Cdc42 result from different effects on specific GEF proteins.
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ACKNOWLEDGMENTS |
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We thank Jing Chen, Gary Bokoch, and Kozo Kaibuchi for reagents; Cathy Cox, Zoya Plotkin, and Matt Loesch for technical assistance; and Bruce Molitoris, Tim Sutton, and Jim Marrs for valuable discussion.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Au JL, Su MH,
and Weintjes MG. Extraction of intracellular nucleosides and nucleotides
with acetonitrile. Clin Chem
35: 4851,
1989.
3. Bacallao R,
Garfinkel A, Monke S, Zampighi G, and Mandel LJ. ATP depletion: a novel
method to study junctional properties in epithelial tissues. I. Rearrangement
of the actin cytoskeleton. J Cell Sci
107: 33013313,
1994.
4. Benard V, Bohl
BP, and Bokock GM. Characterization of Rac and Cdc42 activation in
chemoattractant-stimulated human neutrophils using a novel assay for active
GTPases. J Biol Chem 274:
1319813204, 1999.
5. Benard V and Bokoch GM. Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol 345: 349359, 2002.[ISI][Medline]
6. Bishop AL and Hall A. Rho GTPases and their effector proteins. Biochem J 348: 241255, 2000.[ISI][Medline]
7. Bokoch GM. Regulation of cell function by Rho family GTPases. Immunol Res 21: 139148, 2000.[ISI][Medline]
8. Chen J, Cohn JA, and Mandel LJ. Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. Proc Natl Acad Sci USA 92: 74957499, 1995.[Abstract]
9. Chen J, Doctor
BR, and Mandel LJ. Cytoskeletal dissociation of ezrin during renal anoxia:
role in microvillar injury. Am J Physiol Cell Physiol
267: C784C795,
1994.
10. Dagher PC.
Modeling ischemia in vitro: selective depletion of adenine and guanine
nucleotide pools. Am J Physiol Cell Physiol
279: C1270C1277,
2000.
11. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95105, 2000.[ISI][Medline]
12. De Vendittis E, Zahn R, and Fasano O. Regeneration of the GTP-bound from the GDP-bound form of human and yeast ras protein by nucleotide exchange. Stimulatory effect of organic and inorganic polyphosphates. Eur J Biochem 161: 473478, 1986.[Abstract]
13. Edelstein CL, Ling H, and Schrier RW. The nature of renal cell injury. Kidney Int 51: 13411351, 1997.[ISI][Medline]
14. Glascott PA Jr, McSorley KM, Mittal B, Sanger JM, and Sanger JW. Stress fiber reformation after ATP depletion. Cell Motil Cytoskeleton 8: 118129, 1987.[ISI][Medline]
15. Glaumann B, Glaumann H, Brerezesky IK, and Trump BF. Studies on the pathogenesis of ischemic cell injury. II. Morphological changes of the pars convoluta (P1 and P2) of the proximal tubule of the rat kidney made ischemic in vivo. Virchows Arch 19: 281302, 1975.
16. Gopalakrishnan S, Dunn KW, and Marrs JA. Rac1, but not RhoA,
signaling protects epithelial adherens junction assembly during ATP depletion.
Am J Physiol Cell Physiol 283:
C261C272, 2002.
16. Gopalakrishnan S, Hallett MA, Atkinson SJ, and Marrs JA.
Differential regulation of junctional complex assembly in renal epithelial
cells. Am J Physiol Cell Physiol
285: C102C111,
2003.
17. Gopalakrishnan S, Raman N, Atkinson SJ, and Marrs JA. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol Cell Physiol 275: C798C809, 1998.[Abstract]
18. Grune T, Seims W, Gerber G, Tikhonov YV, Pimenov AM, and Toguzov RT. Changes in nucleotide patterns in liver, muscle and blood during the growth of Ehrlich ascites cells: application of the reversed-phase and ion-pair reversed-phase high-performance liquid chromatography with radial compression column. J Chromatogr 563: 5361, 1991.[Medline]
19. Hanzel D, Reggio H, Bretscher A, Forte JG, and Mangeat P. The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli. EMBO J 10: 23632373, 1991.[Abstract]
20. Hinshaw DB,
Burger JM, Miller MT, Adams JA, Beals TF, and Omann GM. ATP depletion
induces an increase in the assembly of a labile pool of polymerized actin in
endothelial cells. Am J Physiol Cell Physiol
264: C1171C1179,
1993.
21. Hirao M, Sato N, Kondo T, Yonemura S, Monden M, Sasaki T, Takai Y, Tsukita S, and Tsukita S. Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J Cell Biol 135: 3751, 1996.[Abstract]
22. Kjoller L and Hall A. Signaling to Rho GTPases. Exp Cell Res 253: 166179, 1999.[ISI][Medline]
23. Kozma R, Sarner S, Ahmed S, and Lim L. Rho family GTPases and neuronal growth cone remodeling: relationship between increased complexity induced by Cdc42Hs, Rac1 and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol Cell Biol 17: 12011211, 1997.[Abstract]
24. Kraenburg O,
Poland M, van Horck FPG, Drechsel D, Hall A, and Moolenaar WH. Activation
of RhoA by lysophosphatidic acid and G12/13 subunits in
neuronal cells: induction of neurite retraction. Mol Biol
Cell 10:
18511857, 1999.
25. Madaule P and Axel R. A novel ras-related gene family. Cell 41: 3140, 1985.[ISI][Medline]
26. Mathews C and Van Holde KE. Biochemistry. Menlo Park, CA: Benjamin/Cummings, 1996, p. 425.
27. Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J 15: 22082216, 1996.[Abstract]
28. Matsui T, Maeda
M, Doi Y, Yonemura S, Amano M, Kaibuchi K, Tsukita S, and Tsukita S. Rho
kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM)
proteins and regulates their head-to-tail association. J Cell
Biol 140:
647657, 1998.
29. Matsui T, Yonemura S, Tsukita S, and Tsukita S. Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases. Curr Biol 9: 12591262, 1999.[ISI][Medline]
30. Molitoris BA, Geerdes A, and McIntosh JR. Dissociation and redistribution of the Na+,K+-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion. J Clin Invest 88: 462469, 1991.[ISI][Medline]
31. Molitoris BA and Nelson WJ. Alterations in the establishment and maintenance of epithelial cell polarity as a basis for disease processes. J Clin Invest 85: 39, 1990.[ISI][Medline]
32. Noren NK,
Niessen CM, Gumbiner BM, and Burridge K. Cadherin engagement regulates Rho
family GTPases. J Biol Chem
276: 3330533308,
2001.
33. Raman N and
Atkinson SJ. Rho controls actin cytoskeletal assembly in renal epithelial
cells during ATP depletion and recovery. Am J Physiol Cell
Physiol 276:
C1312C1324, 1999.
34. Sutton TA, Mang
HE, and Atkinson SJ. Rho-kinase regulates myosin II activation in MDCK
cells during recovery after ATP depletion. Am J Physiol Renal
Physiol 281:
F810F818, 2001.
35. Takeuchi K, Sato N, Kasahara H, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 125: 13711384, 1994.[Abstract]
36. Tsukita S, Oishi K, Sato N, Sargara J, Kawai A, and Tsukita S. ERM family members as molecular linkers between the cell surface glycoprotein CD44 and the actin-based cytoskeleton. J Cell Biol 126: 391401, 1994.[Abstract]
37. Turunen O, Wahlstrom T, and Vaheri A. Ezrin has a COOH-terminal actin binding site that is conserved in the ezrin protein family. J Cell Biol 126: 14451453, 1994.[Abstract]
38. Uruno T, Liu Zhang P J, Fan Y, Egile C, Li R, Mueller SC, and Zhan X. Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat Cell Biol 3: 259266, 2001.[ISI][Medline]
39. Van Leeuwen FN,
Kain HET, van der Kammen RA, Michiels F, Kranenburg OW, and Collard JG.
The guanine nucleotide exchange factor Tiam1 affects neuronal morphology:
opposing roles for the small GTPases Rac and Rho. J Cell
Biol 139:
797807, 1997.
40. Venkatachalam MA, Jones DB, Rennke HG, Sandstrom D, and Patel Y. Mechanisms of proximal tubule brush border loss and regeneration following mild renal ischemia. Lab Invest 45: 355365, 1981.[ISI][Medline]
41. Weaver AM, Karginov AV, Kinley AW, Weed SA, Li Y, Parsons JT, and Cooper JA. Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation. Curr Biol 11: 370374, 2001.[ISI][Medline]
42. White P, Doctor
RB, Dahl RH, and Chen J. Coincident microvillar actin bundle disruption
and perinuclear sequestration in anoxic proximal tubule. Am J
Physiol Renal Physiol 278:
F886F893, 2000.
43. Zhang B, Zhang
Y, Wang Z, and Zheng Y. The role of Mg2+ cofactor in
the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family
GTP-binding proteins. J Biol Chem
275: 2529925307,
2000.