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Hormones, Growth Factors, and Cell Signaling
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ATP Depletion Increases Tyrosine Phosphorylation of β-Catenin and Plakoglobin in Renal Tubular Cells

JOHN H. SCHWARTZ, THEODORA SHIH, SARAH A. MENZA and WILFRED LIEBERTHAL
JASN November 1999, 10 (11) 2297-2305;
JOHN H. SCHWARTZ
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THEODORA SHIH
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SARAH A. MENZA
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WILFRED LIEBERTHAL
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Abstract

Abstract. This study examines the hypothesis that the loss of integrity of the junctional complex induced by ATP depletion is related to alterations in tyrosine phosphorylation of the adherens junction proteins β-catenin and plakoglobin. ATP depletion of cultured mouse proximal tubular (MPT) cells induces a marked increase in tyrosine phosphorylation of both β-catenin and plakoglobin. The tyrosine phosphatase inhibitor vanadate has the same effect in ATP-replete (control) monolayers, whereas genistein, a tyrosine kinase inhibitor, reduces phosphorylation of both proteins in ATP-replete monolayers and prevents the hyperphosphorylation of these proteins with ATP depletion. This study also demonstrates that the fall in the transepithelial resistance of MPT monolayers induced by ATP depletion can be reproduced by treatment of ATP-replete monolayers with vanadate, whereas genistein substantially ameliorates the fall in transepithelial resistance induced by ATP depletion. Also, using immunofluorescence microscopy it was demonstrated that ATP depletion results in a marked diminution of E-cadherin staining in the basolateral membrane of MPT cells. Vanadate mimics this effect of ATP depletion, whereas genistein ameliorates the reduction in the intensity of E-cadherin staining induced by ATP depletion. Because it is has been well established that hyperphosphorylation of the catenins leads to dissociation of the adherens junction and to dysfunction of the junctional complex, it is proposed that the increase in tyrosine phosphorylation of catenins observed in MPT cells during ATP depletion contributes to the loss of function of the junctional complex associated with sublethal injury.

Sublethal injury to renal tubular cells, induced by ATP depletion, leads to the rapid loss of functional integrity of the tight junction (1,2), the loss of cell polarity (3,4), and severe impairment of the epithelial permeability barrier (2,3,5). After only a few minutes of ATP depletion, the transepithelial electrical resistance (TER) of renal epithelia falls to very low levels and paracellular permeability increases (2,3,5). These functional changes are completely reversible if the ATP levels are restored before lethal cell injury occurs (1,6). This loss of the renal epithelial permeability barrier associated with sublethal injury is believed to contribute, at least in part, to the “back-leak” of glomerular filtrate, which is believed to be an important contributing factor to the profound loss of GFR associated with acute ischemic renal injury (7, 8, 9, 10).

The molecular events that lead to the rapid loss of function of the junctional complex after ATP depletion remain uncertain. The junctional complex between epithelial cells is comprised anatomically of at least three distinct structures: the zonula occludens (ZO), the adherens junction, and the desmosomes (11,12). In mature epithelia, the ZO and adherens junction both completely circumscribe adjoining epithelial cells at the boundary between apical and basolateral membrane domains and both play a role in maintaining the functional integrity of the junctional complex (11,12). Although the ZO is the component of the junctional complex that represents the barrier to the paracellular flux of molecules and ions across the epithelium (11,12), the adherens junction, which lies immediately basal to the ZO, is necessary not only for the formation of the ZO but also for the maintenance of a functionally normal ZO (11,12).

The adherens junction of renal epithelial cells is comprised of a number of proteins that include cadherin and the catenins. E-cadherin is a transmembrane protein that mediates adhesion of adjacent cells to one another. The extracellular domain of the E-cadherin molecules of adjacent cells bind to one another in a homophilic, calcium-dependent manner (11,13,14). The intracellular domain of E-cadherin is connected to the actin cytoskeleton by a complex of cytosolic proteins called the catenins (13,14). At least three different catenins (α-catenin β-catenin and γ-catenin [plakoglobin]) mediate the attachment of E-cadherin to actin by binding to an intermediary actinbinding protein, α-actinin. There is now substantial evidence that tyrosine phosphorylation of the catenins plays an important role in regulating the formation and functional integrity of the adherens junction (14, 15, 16). The maintenance of an intact and stable adherens junction depends on maintaining the catenins in a dephosphorylated state. Increased tyrosine phosphorylation of β-catenin and/or plakoglobin results in the structural and functional disruption of the adherens junction (16,17).

In these studies, we have examined the novel hypothesis that ATP depletion results in hyperphosphorylation of β-catenin and plakoglobin. We will also determine whether hyperphosphorylation of these cytoskeletal proteins contributes to the functional disturbance of the junctional complex associated with ATP depletion.

Materials and Methods

Cell Culture

The primary culture of mouse proximal tubule (MPT) cells was performed as described previously (18). In brief, collagenase digested fragments were obtained from the renal cortices of mice (Harlan, Sprague Dawley, C57BL6) and placed in serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium that contained 2 mM glutamine, 15 mM Hepes, 5 μg/ml transferrin, 5 μg/ml insulin, 50 nM hydrocortisone, 500 μg/ml penicillin, and 50 μg/ml streptomycin. These cells have been identified previously as proximal tubular epithelial cells (18).

Cells were grown either on permeable filter supports (for epithelial permeability studies), in 6-well dishes (for Western blot analysis), or on glass slides (for immunofluorescence studies). The filter supports used for studies of epithelial permeability were 6.5-mm Transwell filter inserts (Costar, Cambridge, MA) consisting of a polycarbonate membrane with 0.4-μm pores coated with rat tail collagen.

ATP Depletion

ATP depletion was induced by chemical anoxia using sodium cyanide (CN) in the absence of glucose. MPT monolayers were rinsed three times with Krebs-Hensleit buffer (KHB) that contained 1 mM calcium and 1 mM magnesium, pH 7.40, at 37°C to remove residual substrates in the medium. Cells were then incubated for 1.5 h in glucose-free KHB containing 5 mM CN. These conditions have been reported previously by our laboratory to lower ATP content to <5% of the control value (2,18). Control monolayers were incubated in CN-free KHB to which 10 mM glucose was added.

Transcellular Electrical Resistance

TER, a sensitive marker of tight junction integrity, was measured with an epithelial Voltohmeter (EVOM; World Precision Instruments, New Haven, CT) in confluent cell monolayers grown on permeable filters. To control for intrinsic resistance of the filters, measurements of electrical resistance were obtained across cell-free inserts equilibrated in KHB at 37°C. The electrical resistance of the cell-free filter insert was then subtracted from all subsequent measurements. The electrodes were sterilized with 95% ethanol and were washed and equilibrated in sterile phosphate-buffered saline before use. To reduce the variability of the determination, the electrodes were placed to the same depth in the solutions bathing the cultured monolayer with a micromanipulator. Monolayers that did not have an initial resistance of at least 175 Ω · cm2 were not used in this study.

Transcellular Permeability

Another measure of the “gate” function of the tight junction is the barrier to the passage of molecules between epithelial cells. To examine permeability, we measured the unidirectional flux across confluent MPT monolayers grown on permeable supports of lucifer yellow, a fluorescence, fluid-phase marker (MW = 482) (3). At the onset of the experiment, 1 mg/ml lucifer yellow was added to the apical solution. The entire volume of the basolateral compartment (1 ml) was sampled at 30-min intervals and then replaced with fresh medium. Apical solution samples (1 μ1) were also obtained at the same intervals as for the basolateral solution. The concentration of lucifer yellow in each compartment was determined by fluorescence spectrofluorometry at an excitation wavelength of 425 nm and an emission of 535 nm. The emission intensity of a standard curve was linear over the range of samples tested and exhibited an r value of >0.95. Flux rates and membrane permeabilities were determined as described previously (2,6).

Preparation of MPT Lysates for Western Blotting

Confluent MPT cells were washed three times in cold phosphate-buffered saline, scraped from the culture dish, and pelleted by centrifugation at 1000 × g for 10 min. The pellet was suspended in 4 vol of ice-cold homogenizing buffer containing 10 mM Tris-HCl, 150 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium vanadate, 5 mM ethylenediaminetetra-acetic acid, aprotinin (0.5 μg/μl), N-tosyl-L-phenylalanine chloromethyl ketone (2 μg/μl), phenylmethylsulfonyl fluoride (4 mM), DNAse (5 μg/ml), RNAase (5 μg/ml) and 1% Nonidet P-40. The suspended pellet was homogenized by ten 1-s strokes in a Teflon homogenizer. The homogenate was centrifuged for 10 min at 1000 × g at 4°C to remove nuclei and remaining intact cells, and the supernatant was stored at -20°C for Western blotting and immunoprecipitation studies.

Immunoprecipitation

MPT lysates were immunoprecipitated using the anti-peptide mouse monoclonal antibodies to β- or γ-catenin (Becton Dickinson, Bedford, MA) according to the following protocol. The homogenate was diluted to a protein concentration of 100 μg/ml with the homogenizing buffer that also contains 0.5% deoxycholate. Nonimmune serum (2 μl) and a 25% suspension of protein A-Sepharose 4B beads (30 μl) was added to a 900-μl aliquot of the cell lysate. The mixture was incubated at 4°C for 2 h and then centrifuged at 13,000 rpm in a microcentrifuge. The supernatant was incubated with 20 μl of anti-β- or anti-γ-catenin antibody and 50 μl of a protein A-Sepharose 4B bead suspension that had been prereacted with goat anti-mouse IgG (Sigma, St. Louis, MO) for 12 h at 4°C. In preliminary studies, we determined that the quantity of primary antibody used was more than adequate to precipitate all of the catenin present in the sample. The beads were pelleted by centrifugation and washed three times and suspended in sodium dodecyl sulfate sample buffer.

Immunoblot

Whole cell homogenates and immunoprecipitated samples, prepared as described above, were heated at 65 or 100°C for 5 min, respectively, before loading on a 7% polyacrylamide sodium dodecyl sulfate gel and run under reducing conditions as described previously (6). Proteins were then electrophoretically transferred to nitrocellulose filters. After transfer, the filters were washed in 150 mM NaCl, 100 mM Tris-HCl, pH 7.5, and 0.05% Tween 20 (TBST), and blocked for 1 h in TBST containing 5% wt/vol nonfat powdered milk (TBSTM) before incubation with the anti-phosphotyrosine antibody PY20 (Transduction Laboratories, Lexington, KY) at a dilution of 1:1000 in TBSTM at 4°C overnight. The filters were then washed three times with TBST and incubated in secondary horseradish peroxidase-labeled goat anti-mouse, 1:2000 in TBSTM for 2 h at room temperature. After three additional washes with TBST, bound secondary antibody was detected using the enhanced chemiluminescence (ECL) system (Amersham).

Immunofluorescence Studies

MPT cells grown on glass coverslips were fixed in 3.7% paraformaldehyde and permeabilized with 0.2% Triton X-100. Detection of E-cadherin in these cells was performed by indirect immunofluorescence using methods we have described previously (2). The anti-E-cadherin antibody (rat monoclonal antibody, clone DECMA-1; Sigma) was used at a dilution of 1:100. The secondary antibody was labeled with CY-3 (Molecular Probes, Eugene, OR) and used at a dilution of 1:500. The monolayers were photographed using a Nikon epifluorescence microscope, and the same exposure time, optimized for the control, was used for all coverslips to depict differences in staining intensity.

Phosphatase and Kinase Inhibitors

Stock solution of vanadate (100 mM) and the sodium salt of okadaic acid (5 mM) were prepared in KHB. Genistein (1 mM), calyculin A (0.5 mM), H7 (1 mM), and H8 (10 mM) were dissolved as stock solutions in DMSO. The final concentration of DMSO in the incubation media never exceeded 1.0%. All of these inhibitors were obtained from Calbiochem (La Jolla, CA).

Protein Assay

Protein concentrations were determined from a colorimetric dye binding assay (Bio-Rad) and expressed in milligrams per milliliter.

Statistical Analyses

Data are expressed as mean ± SEM. Protocols that required comparisons between three or more groups were compared with ANOVA and Fisher post hoc test. When two groups were compared, analysis was performed with an unpaired, two-tailed t test. A P value of <0.05 was defined as significant.

Results

Transepithelial Resistance

We have documented in previous studies that ATP depletion induced by incubating MPT cell monolayers in a glucose-free medium containing CN results in a rapid fall in TER and a rise in permeability to fluid phase markers (2,6). To evaluate the possible role of changes in tyrosine phosphorylation in the alteration in tight junction function, we first examined the effects of phosphotyrosine protein kinase and phosphotyrosine phosphatase inhibitors on transepithelial resistance. Treatment of control, dextrose-treated (ATP-replete) MPT monolayers with the phosphotyrosine phosphatase inhibitor vanadate at 1 mM, a concentration shown in prior studies to increase tyrosine phosphorylation in intact cells (19), resulted in a decline in the transepithelial resistance from 281.2 ± 25.2 to 170.8 ± 18.2 Ω · cm2 (n = 6; P < 0.05) in 20 min and a further decline to 143 ± 12.2 Ω · cm2 in 60 min (Figure 1). Although the initial rate of decline in TER induced by vanadate was similar to that induced by ATP depletion, the reduction in TER after 60 min was somewhat less in the vanadate-treated group than in the ATP-depleted monolayers (Figure 1). The combination of vanadate and ATP depletion resulted in a more rapid decline in TER than with either maneuver alone, but the minimum value obtained after 2 h was no different from that obtained with ATP depletion alone. Similar functional effects of vanadate were observed when the permeability of the epithelium was measured using a fluid phase marker (lucifer yellow) in parallel experiments. Epithelial permeability to lucifer yellow increased 1 h after the addition of vanadate from a control value of 3.9 ± 0.3 × 10-5 to 24.3 ± 0.2 × 10-5 cm/s (n = 5; P < 0.05). This increase in permeability was less than the increase observed with CN-induced ATP depletion (54.7 ± 0.4 × 10-5 cm/s) (n = 6; P < 0.05) or the combined effects of both vanadate and CN (65.3 ± 0.5 × 10 to 5 cm/s) (n = 5; P < 0.05).

Figure 1.
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Figure 1.

Effect of vanadate on transepithelial resistance of ATP-replete (control) and ATP-depleted (cyanide [CN]-treated) mouse proximal tubular (MPT) monolayers grown on permeable supports. □, control; ⋄, vanadate alone; ▵, CN and vanadate; ○, CN. All values are means ± SEM of six studies. *P < 0.05 comparing vanadate versus control; +P < 0.05 comparing vanadate versus vanadate + CN.

Because vanadate can potentially alter the activity of other enzymes in addition to phosphotyrosine phosphatases, its functional effects cannot be definitively attributable to phosphotyrosine phosphatase inhibition alone. We therefore examined the effect of other phosphatase inhibitors that have a different spectrum of action and specificity (Figure 2). Dephostatin (20 μM), another phosphotyrosine phosphatase inhibitor, reduced TER in ATP-replete MPT monolayers by 61 ± 9% (n = 5; P < 0.05), an effect similar to that of vanadate. In contrast, two different serine/threonine phosphatase inhibitors, okadaic acid (20 nM) and calyculin A (10 nM), had no effect on TER of ATP-replete MPT monolayers (Figure 2).

Figure 2.
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Figure 2.

Comparison of the effect of protein tyrosine phosphatase inhibitors with protein phosphatase 1 and 2 inhibitors on transepithelial resistance of ATP-replete monolayers grown on permeable supports. Values are means ± SEM of six studies obtained 60 min after the addition of either diluent (Control) or 1 mM vanadate, 20 μM dephostatin, 20 nM okadaic acid, or 10 nM calyculin A. *P < 0.05 compared with control.

The phosphotyrosine protein kinase inhibitor genistein (19) at a concentration of 25 μM had no effect on TER in control, ATP-replete MPT monolayers (Figure 3). However, genistein substantially ameliorated the reduction in TER induced by ATP depletion (Figures 3 and 4). As depicted in Figures 3 and 4, the decline in TER after 60 min in genistein-treated ATP-depleted monolayers (32 ± 7%) was significantly less than after ATP depletion alone (69 ± 11%, P < 0.05, n = 6). This protective effect of genistein on the reduction in TER induced by ATP depletion could not be reproduced by other types of kinase inhibitors (20). Neither the protein kinase C inhibitor H7 (25 μM) nor the protein kinase A inhibitor H8 (25 μM) affected the reduction in TER induced by ATP depletion (Figure 4).

Figure 3.
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Figure 3.

Effect of genistein on transepithelial resistance of ATP-replete and ATP-depleted MPT monolayers grown on permeable supports. □, control; ○, genistein alone; ▵, CN and genistein; ⋄, CN alone. All values are means ± SEM of six studies. *P < 0.05 comparing CN and genistein or CN alone with control; †P < 0.05 comparing CN and genistein versus CN alone.

Figure 4.
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Figure 4.

Comparison of the effect of protein tyrosine kinase inhibitor genistein with protein kinase A and protein kinase C inhibitors on ATP depletion-induced reduction of transepithelial resistance MPT monolayers grown on permeable supports. Values are means ± SEM of six studies obtained 60 min after the addition of either diluent (Control) or 5 mM cyanide or a combination of cyanide plus 25 μM genistein or 25 μM H7 or 25 μM H8. *P < 0.05 comparing CN with control; †P < 0.05 comparing vanadate versus vanadate plus genistein.

If the vanadate-induced reduction in TER is primarily dependent on tyrosine hyperphosphorylation and not by other mechanisms, then one would predict that its action should be antagonized by a tyrosine kinase inhibitor. To verify this assertion, we investigated whether the reduction in TER induced by vanadate could be ameliorated by the tyrosine kinase inhibitor genistein. In Figure 5, we demonstrate that 1 mM vanadate reduces TER after 1 h by 149 ± 12 Ω · cm2. However, with the simultaneous addition of both 1 mM vanadate and 25 μM genistein, the reduction in TER was reduced by one-third, to only 49 ± 5 Ω · cm2. Thus, the effect of vanadate must be dependent on enhanced tyrosine phosphorylation.

Figure 5.
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Figure 5.

Reversal in the vanadate-induced reduction of transepithelial resistance by genistein. □, control; ⋄, vanadate alone; ○, genistein alone; ▵, vanadate and genistein. All values are means ± SEM of six studies. *P < 0.05 comparing vanadate with control; †P < 0.05 CN versus CN plus genistein.

Phosphorylation of Proteins in Whole Cell Homogenates of MPT Cells

Based on the above observations, we would predict that ATP depletion induces changes in the function of cell junction via tyrosine phosphorylation of proteins. To evaluate this possibility, we first analyzed whole cell lysates of MPT cells by immunoblot analysis to assess the degree of protein tyrosine phosphorylation and to determine the general effects of ATP depletion, vanadate, and genistein on tyrosine phosphorylation of cell proteins (Figure 6). As expected, lysates of MPT cells incubated with 1 mM vanadate for 1 h demonstrated a generalized increase in the immunodetectable degree of tyrosine phosphorylation (Figure 3, lane 2) compared to control cells (lane 1), whereas 25 μM genistein reduced phosphorylation of all protein bands. (Figure 3, lane 3). Most protein bands from MPT cells treated with CN for 1 h (Figure 3, lane 4) demonstrated a decrease in tyrosine phosphorylation. However, several protein bands (one at 50 kD and one at 160 kD) were hyperphosphorylated compared with control MPT cells. There also was a marked increase in the tyrosine phosphorylation of proteins compared to either control or only vanadate treatment when MPT cells were treated with both CN and vanadate (Figure 3, lane 5). This latter change indicates that neither ATP depletion nor kinase activity is rate-limiting for protein tyrosine phosphorylation. In addition, the combination of genistein and ATP depletion (Figure 3, lane 6) reduced phosphorylation more than any one of these maneuvers alone. These observations are consistent with the hypothesis that ATP depletion is associated with alterations in the activity of both tyrosine kinases and phosphatase with resultant dephosphorylation of some proteins and hyperphosphorylation of others.

Figure 6.
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Figure 6.

Effect of tyrosine kinase inhibitors, tyrosine phosphatase inhibitors, and ATP depletion on tyrosine phosphorylation of proteins in MPT cells. An immunoblot of whole cell homogenates (50 μg protein/lane) from monolayers treated with vehicle (control) (lane 1), vanadate (lane 2), genistein (lane 3), CN (lane 4), CN and vanadate (lane 5), and CN and genistein (lane 6) and probed with an antityrosine phosphate antibody. The blot shown is representative of four experiments.

Phosphorylation of β-Catenin and Plakoglobin

Since an intact zonula adherens (ZA) is required to maintain a functional ZO and phosphorylation of catenins is known to regulate the stability of the ZA (E-cadherin complexes), we examined the effect of ATP depletion on catenin phosphorylation. In these studies, β-catenin and plakoglobin were immunoprecipitated from whole cell homogenates of MPT cells. The immunoprecipitates were then subjected to immunoblot analysis and probed first with an anti-tyrosine phosphate antibody and then with an anti-catenin antibody. The amount of β-catenin and plakoglobin expressed was not measurably changed by ATP depletion or exposure to either vanadate or genistein (Figure 7B). The treatment of ATP-replete (control) monolayers with vanadate (Figure 7, lane 2) enhanced tyrosine phosphorylation of β-catenin (upper band) by 398 ± 42% and plakoglobin (lower band) by 283 ± 23% above control (lane 1). Genistein treatment of ATP-replete monolayers reduced the tyrosine phosphorylation of β-catenin by 30 ± 9% and plakoglobin by 35 ± 6% below control (Figure 7, lane 3). ATP depletion with CN increased phosphorylation of β-catenin by 348 ± 37% and plakoglobin by 355 ± 23% (Figure 7, lane 4). The effect of ATP depletion on phosphorylation of both proteins was comparable to that of vanadate alone. The combination of genistein and CN (Figure 7, lane 5) reduced phosphorylation of β-catenin and plakoglobin by 50 ± 11% and 65% ± 10% below control, respectively. Thus, genistein prevented the hyperphosphorylation of both β-catenin and plakoglobin induced by ATP depletion (Figure 7, lane 4). The combination of vanadate and CN (Figure 7, lane 6) resulted in hyperphosphorylation of both β-catenin (428 ± 33%) and plakoglobin (393 ± 53%), an effect somewhat greater than that induced by vanadate (Figure 7, lane 2) or ATP depletion alone (Figure 4, lane 4).

Figure 7.
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Figure 7.

Phosphotyrosine phosphorylation of β-catenin (92 kD) and plakoglobin (82 kD). Whole cell homogenates from monolayers treated with vehicle (control) (lane 1), vanadate (lane 2), genistein (lane 3), CN (lane 4), CN and genistein (lane 5), and CN and vanadate (lane 6) were immunoprecipitated with an antibody directed against both β-catenin and plakoglobin and then analyzed by Western blot for tyrosine phosphorylation (A) or β-catenin and plakoglobin (B). The blot shown is representative of four experiments.

In addition to determining the effect of ATP depletion on the phosphorylation of ZA proteins, we also examined the effect of ATP depletion on two ZO proteins: occludin and ZO-1. These proteins were immunoprecipitated from whole cell homogenates with either an antibody to ZO-1 or to occludin. The immunoprecipitates obtained (Figure 8) were then subjected to immunoblot analysis, and the blots were probed first with an antibody to either ZO-1 or to occludin and then with an anti-tyrosine phosphate antibody (PY20). Neither the amount of ZO-1 (Figure 8, lanes 1 to 2) and occludin (Figure 8, lanes 3, 4) expressed nor the degree of tyrosine phosphorylation of these proteins was affected by ATP depletion (Figure 8, lanes 2, 4, 6, and 8).

Figure 8.
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Figure 8.

Effect of ATP depletion on expression and tyrosine phosphorylation of occludin (68 kD) and ZO-1 (225 kD). ZO-1 and occludin were immunoprecipitated from whole cell homogenate of control (lanes 1 and 3) and ATP-depleted (lanes 2 and 4) MPT monolayers. The immunoprecipitate was analyzed by Western blot for the amount of proteins immunoprecipitated (A) and for the degree of tyrosine phosphorylation of these proteins (B).

Immunofluorescence of E-Cadherin

Tyrosine phosphorylation of β-catenin and/or plakoglobin has been associated with the withdrawal of E-cadherin complexes from the lateral membrane. Immunohistochemical studies were performed to determine whether the maneuvers that induce tyrosine hyperphosphorylation also induce withdrawal of E-cadherin from the lateral membrane. Immunofluorescence microscopy demonstrated that the distribution of E-cadherin in MPT cells is comparable to that described in other epithelial monolayers (11,16). E-cadherin is present primarily at cell-cell borders and in the basolateral membrane (Figure 9a). One hour after treatment of MPT monolayers with 5 mM CN, there is a marked decline in the intensity of basolateral E-cadherin staining (Figure 9b). Treatment of MPT monolayers with 1 mM vanadate resulted in a generalized diminution in E-cadherin staining in a pattern similar to that seen after CN (Figure 9c). E-cadherin staining in MPT monolayers treated with the combination of CN and genistein was similar to that in control monolayers (Figure 9d), suggesting that genistein ameliorates the effect of CN on the redistribution of E-cadherin. Genistein alone had no effect on E-cadherin staining (data not shown).

Figure 9.
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Figure 9.

Immunohistochemical distribution of E-cadherin in MPT cells subjected to ATP depletion. Photomicrographs of MPT cells grown on glass coverslips stained for E-cadherin with a rat monoclonal antibody for mouse E-cadherin. (a) Control monolayer. ATP-replete MPT cells demonstrate the typical basolateral distribution of E-cadherin with predominant staining at cell-cell borders. (b) CN-treated monolayers. After 1 h exposure to 5 mM CN, there is a marked reduction in the intensity of the E-cadherin staining. (c) Vanadate-treated monolayer. MPT cells treated with 1 mM vanadate for 1 h demonstrate a reduction in the intensity of E-cadherin staining, an effect similar to that induced by CN. (d) Genistein and CN. The CN-induced reduction in E-cadherin staining (b) can be ameliorated by the simultaneous incubation with 1 mM genistein. To facilitate an assessment of the relative degree of E-cadherin staining, the exposure time for each photomicrograph was identical. Magnification: ×400.

Discussion

This study is the first to demonstrate that when MPT monolayers are subjected to ATP depletion, the tyrosine residues of some proteins within the cell are dephosphorylated, while others become hyperphosphorylated. Other studies that have examined the effects of ATP depletion on serine-threonine phosphorylation of renal epithelial cell proteins have reported that chemical anoxia induces dephosphorylation of the cytoskeletal protein ezrin (21), as well as other renal tubular cell proteins (22).

Our study, which focuses entirely on tyrosine phosphorylation, demonstrates that ATP depletion causes complex and variable effects on the multiplicity of kinases and phosphatases that modulate protein tyrosine phosphorylation. Because tyrosine phosphorylation plays an integral role in control of cell-cell and cell matrix adhesion, we examined the hypothesis that the dysregulation of tyrosine phosphorylation contributes to many of the functional abnormalities of the renal epithelium following sublethal injury (1,2). In this study, we have examined the role played by alterations of tyrosine phosphorylation of adherens junction proteins in the loss of functional integrity of the junctional complex that is associated with chemical anoxia (1,2,23).

It is now well established that the barrier function of the junctional complex is due to the unique anatomical characteristics of the ZO (11,12). Although the molecular mechanisms involved in the regulation and maintenance of the permeability barrier are still not well understood, it has been demonstrated that occludin, a protein that forms an integral part of the extracellular domain of the ZO (24,25), contributes directly to the permeability characteristics of the tight junction (22). The function of other proteins associated with the ZO, including the cytoplasmic proteins ZO-1 and ZO-2, is less clear but probably relates to assembly and localization of occludin to the ZO (25).

However, it is also clear that the formation and maintenance of a functionally intact ZO also depends indirectly on the presence of an intact adherens junction. The importance of the adherens junction to tight junction integrity and function has been demonstrated in two ways. First, maintenance of normal TER and epithelial permeability characteristics has been demonstrated to be dependent on the presence of extracellular calcium (11) even though the ZO itself is not structurally dependent on calcium (11,26). Thus, the well-known deleterious effects of low extracellular calcium concentrations on the barrier function of the junctional complex is due to the calcium dependency of E-cadherin, the protein that mediates normal cell-cell adhesion at the adherens junction (11,12,25). Second, the importance of an intact adherens junction in maintaining the functional integrity of the tight junction is also suggested by studies in which neutralizing antibodies to cadherin inhibit normal ZO assembly and function (12). Thus, substantial evidence indicates that the normal function of the tight junction depends indirectly on the presence of an intact adherens junction.

Although the mechanisms governing regulation of the adherens junction remain incompletely defined, recent studies have demonstrated that the binding of catenins to cadherin, and the subsequent anchorage of E-cadherin to the cytoskeleton, is regulated by tyrosine phosphorylation of the catenins (14, 15, 16, 17). Tyrosine phosphorylation of β-catenin and/or plakoglobin results in the structural and functional disruption of the adherens junction (17). On the other hand, the dephosphorylation of these proteins is necessary for the establishment and maintenance of a stable adherens junction (16). A family of phosphatases, the receptor tyrosine phosphatases, is believed to participate in the regulation of β-catenin phosphorylation and in adherens junction assembly and disassembly (27, 28, 29). We therefore examined the possibility that the alterations in tight junction function associated with ATP depletion are the consequence of changes in the degree of tyrosine phosphorylation of the catenins.

Initial studies using tyrosine kinase and phosphatase inhibitors were consistent with our hypothesis that tyrosine phosphorylation was important in the process by which ATP depletion induced changes in tight junction function. The pronounced reduction in TER and increase in permeability of MPT monolayers subjected to the tyrosine phosphatase inhibitors (vanadate and dephostatin) support the idea that the tyrosine phosphatases are involved in regulation of the integrity of the junctional complex (17). The absence of any effect of genistein on TER or permeability in ATP-replete monolayers is not surprising since inhibition of kinases involved in adherens junction integrity would be expected to have either no effect or, if anything, might increase the “tightness” of adherens junction mediated cell-cell adhesion. However, the observation that genistein, but not H7 or H8, ameliorates the fall in TER and monolayer permeability associated with either addition of vanadate or ATP depletion is entirely novel. This finding is in keeping with the role of tyrosine kinases and or phosphatases in the regulation of tight junction function and with our hypothesis that selective tyrosine hyperphosphorylation is induced by ATP depletion.

The immunoblots shown in Figures 7 and 8 demonstrate that interventions like vanadate, genistein, and ATP depletion modulate the degree of tyrosine phosphorylation of the ZA proteins β-catenin and plakoglobin, but have no effect on the tyrosine phosphorylation of ZO proteins. ATP depletion as well as vanadate increased phosphorylation of both β-catenin and plakoglobin. Genistein reduced the degree of tyrosine phosphorylation of both proteins in control monolayers and ameliorated the ATP depletion-induced hyperphosphorylation of these proteins. In contrast, the ZO proteins occludin and ZO-1 had no change in tyrosine phosphorylation induced by ATP depletion. These observations indicate that the target that is tyrosinephosphorylated during ATP depletion is not within the ZO but the ZA. Therefore, it is reasonable to suggest that the change in ZA functions is likely to be secondary, at least in part, to redisruption of E-cadherin complexes by phosphorylation of catenin.

There are numerous tyrosine kinases that can phosphorylate catenins. These include epidermal growth factor, human growth factor, and the Src family of tyrosine kinase (14,30,31). Although there is some precedent for activation of Src kinase activity by hypoxia of endothelial cells (32) and myocytes (33), we have not identified the tyrosine kinases activated during ATP depletion in renal tubular cells. However, the potential protein targets for this kinase are also likely to be numerous and include other proteins in addition to the catenins evaluated in this study. For example, the substrates for Src include multiple proteins that regulate actin stress fibers (tensin, vinculin, cortactin, tallin, and annexin II) (31). Thus, it is also possible that tyrosine phosphorylation of one or more of these proteins in response to ATP depletion contributes not only to loss of epithelial permeability, but also to the changes in the actin cytoskeleton and loss of cell-substrate adhesion associated with sublethal injury (2).

We also demonstrate that CN and vanadate result in loss of E-cadherin staining from the cell-cell junctions and basolateral membrane of MPT monolayers (Figure 9, a through c). Furthermore, genistein ameliorates the effects of CN on E-cadherin staining. These observations suggest that alterations in tyrosine phosphorylation of the catenins ultimately disrupt the localization of E-cadherin to the adherens junction. Since changes in phosphorylation of catenins are known to regulate the assembly and disassembly of the adherens junction (15,16,27,28), and since integrity of the adherens junction is necessary for normal tight junction function, it is reasonable to propose that ATP depletion alters the function of the junctional complex, at least in part, via its effect on the phosphorylation of β-catenin and plakoglobin.

Acknowledgments

Acknowledgment

This work was supported by National Institutes of Health Grant DK385101.

Footnotes

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  • © 1999 American Society of Nephrology

References

  1. ↵
    Canfield P, Geerdes A, Molitoris B: Effect of reversible ATP depletion on tight junction integrity. Am J Physiol261 : F1038-F1045,1991
  2. ↵
    Kroshian VM, Sheridan A, Lieberthal W: Functional and cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells.Am J Physiol 266:F21 -F30, 1994
  3. ↵
    Molitoris BA, Dahl RH, Falk SA: Ischemia-induced loss of epithelial polarity: Role of the tight junction. J Clin Invest84 : 1334-1339,1989
  4. ↵
    Molitoris B, Geerdes A, McIntosh J: Dissociation and redistribution of Na+, K+ -ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion. J Clin Invest 88: 462-469,1991
  5. ↵
    Molitoris BA: Putting the actin cytoskeleton into perspective: Pathophysiology of ischemic alterations. Am J Physiol272 : F430-F433,1997
  6. ↵
    Borkan SC, Wang Y-H, Lieberthal W, Burke PR, Schwartz JH: Heat stress ameliorates ATP depletion-induced sublethal injury in mouse proximal tubule cells. Am J Physiol 272:F347 -F355, 1997
  7. ↵
    Donohoe JF, Venkatachalam MA, Bernard DB, Levinsky NG: Tubular leakage and obstruction in acute ischemic renal failure. Kidney Int 13: 208-222,1978
  8. ↵
    Venkatachalam MA, Bernard DB, Donohoe J, Levinsky NG: Ischemic damage and repair in the rat proximal tubule: Differences among S1, S2 and S3 segments. Kidney Int 14:31 -49, 1978
  9. ↵
    Lieberthal W: Biology of ischemic and toxic renal tubular cell injury: Role of nitric oxide and the inflammatory response. Curr Opin Nephrol Hypertens 7:289 -295, 1998
  10. ↵
    Brady HR, Brenner BM, Lieberthal W: Acute renal failure. In:The Kidney , edited by Brenner B, Philadelphia, Saunders,1996 , pp 1200-1252
  11. ↵
    Gumbiner B: Structure, biochemistry and assembly of epithelial tight junctions. Am J Physiol 253:C749 -C758, 1987
  12. ↵
    Citi S: The molecular organization of tight junctions. J Cell Biol 121:485 -489, 1993
  13. ↵
    Geiger B, Ayalon O: Cadherins. Annu Rev Cell Biol8 : 307-332,1992
  14. ↵
    Aberle H, Schwartz H, Kemler R: Cadherin-catenin complex: Protein interactions and their implications for cadherin function. J Cell Biochem 61: 514-523,1996
  15. ↵
    Balsamo J, Leung T, Ernst H, Zanin M, Hoffman S, Lilien J: Regulated binding of a PTP1B-like phosphatase to N-cadherin: Control of cadherin mediated adhesion by dephosphorylation of β-catenin. J Cell Biol 134:801 -812, 1996
  16. ↵
    Kypta RM, Su H, Reichardt LF: Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex. J Cell Biol 134: 1519-1529,1996
  17. ↵
    Staddon JM, Herrenknecht K, Rubin LL: Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci 108: 609-619,1995
  18. ↵
    Sheridan AM, Schwartz JH, Kroshian VM, Tercyak AM, LaRaia L, Masino S, Lieberthal W: Mouse proximal cells are more susceptible to injury than MDCK cells: Role of differences in lipid metabolism. Am J Physiol265 : F342-F350,1993
  19. ↵
    Feick P, Gilhaus S, Schultz I: Pervanadate stimulates amylase release and protein tyrosine phosphorylation of paxillin and p125FAK in differentiated AR4-2J pancreatic acinar cells. J Biol Chem 273:16366 -16373, 1998
  20. ↵
    Hagiwara M, Inagaki M, Watanabe M, Ito M, Onoda K, Tanaka T, Hidaka H: Selective modulation of calcium-dependent myosin phosphorylation by novel protein kinase inhibitors, isoquinoline-sulfonamide derivatives. Mol Pharm 32: 7-12,1987
  21. ↵
    Chen JJ, Cohn A, Mandel LJ: Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. Proc Natl Acad Sci USA 92: 7495-7499,1995
  22. ↵
    Kobryn C, Mandel L: Decreased protein phosphorylation induced by anoxia in proximal renal tubules. Am J Physiol267 : C1073-C1079,1994
  23. ↵
    Mandel L, Bacallao R, Zampighi G: Uncoupling of the molecular “fence” and paracellular “gate” functions in epithelial tight junctions, Nature 361:552 -555, 1993
  24. ↵
    Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S: Occludin: A novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777 -1788, 1993
  25. ↵
    Gumbiner BM: Cell adhesion: The molecular basis of tissue architecture and morphogenesis. Cell 84:345 -357, 1996
  26. ↵
    Stevenson B, Siciliano J, Mooseker M, Goodenough D: Zonulae occludentes in junctional complex-enriched fractions from mouse liver: Preliminary morphological and biochemical characterization. J Cell Biol 98: 1209-1221,1984
  27. ↵
    Brady-Kalnay S, Tonks N: Protein tyrosine phosphatases as adhesion receptors. Curr Opin Cell Biol 7:650 -657, 1995
  28. ↵
    Tonks N, Neel B: From form to function: Signalling by protein tyrosine phosphatases. Cell 87:365 -368, 1996
  29. ↵
    Denu J, Stuckey J, Saper M, Dixon J: Form and function in protein phosphorylation. Cell 87:361 -364, 1996
  30. ↵
    Balkovetz DF, Pollack AL, Mostov KE: Hepatocyte growth factor alters the polarity of MDCK cell monolayers. J Biol Chem272 : 3471-3477,1997
  31. ↵
    Brown MT, Cooper JC: Regulation, substrates and function of Src.Biochim Biophys Acta 1287:121 -149, 1996
  32. ↵
    Tsokias L, Zhou XM, Foster D, Brugge JS, Sukhatme VP: Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature 375:577 -581, 1995
  33. ↵
    Seko Y, Tobe K, Takahashi N, Kaburagi Y, Kadowaki T, Yazaki Y: Hypoxia and hypoxia/regeneration activate Src family tyrosine kinases and p21ras in cultured rat cardiac myocytes. Biochem Biophys Res Commun 226: 520-535,1996
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Journal of the American Society of Nephrology: 10 (11)
Journal of the American Society of Nephrology
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ATP Depletion Increases Tyrosine Phosphorylation of β-Catenin and Plakoglobin in Renal Tubular Cells
JOHN H. SCHWARTZ, THEODORA SHIH, SARAH A. MENZA, WILFRED LIEBERTHAL
JASN Nov 1999, 10 (11) 2297-2305;

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ATP Depletion Increases Tyrosine Phosphorylation of β-Catenin and Plakoglobin in Renal Tubular Cells
JOHN H. SCHWARTZ, THEODORA SHIH, SARAH A. MENZA, WILFRED LIEBERTHAL
JASN Nov 1999, 10 (11) 2297-2305;
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