Am J Physiol Cell Physiol Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 292: C1094-C1102, 2007. First published August 23, 2006; doi:10.1152/ajpcell.00099.2006
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
292/3/C1094    most recent
00099.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gopalakrishnan, S.
Right arrow Articles by Marrs, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gopalakrishnan, S.
Right arrow Articles by Marrs, J. A.

EXTRACELLULAR MATRIX, CELL INTERACTIONS

aPKC-PAR complex dysfunction and tight junction disassembly in renal epithelial cells during ATP depletion

Shobha Gopalakrishnan, Mark A. Hallett, Simon J. Atkinson, and James A. Marrs

Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana

Submitted 3 March 2006 ; accepted in final form 16 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Renal ischemia and in vitro ATP depletion result in disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity. Tight junction formation is regulated by evolutionarily conserved complexes, including that of atypical protein kinase C (aPKC), Par3, and Par6. The aPKC signaling complex is activated by Rac and regulated by protein phosphorylation and associations with other tight junction regulatory proteins, for example, mLgl. In this study, we examined the role of aPKC signaling complex during ATP depletion and recovery in Madin-Darby canine kidney cells. ATP depletion reduced Rac GTPase activity and induced Par3, aPKC{zeta}, and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP depletion. Zonula occludens (ZO)-1 and Par3 phosphorylation was reduced and association of aPKC{zeta} with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted Madin-Darby canine kidney cells. ATP depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor, which explains how aPKC{zeta} and Rac activities were suppressed. Experimental inhibition of aPKC{zeta} during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery.

ischemia; atypical PKC{zeta}; Par3; zonula occludens-1; mLgl-1

TIGHT JUNCTIONS, the most apical junctional complex, regulate epithelial cell permeability by controlling the passage of molecules through the paracellular space. Tight junctions also contribute to epithelial polarity by restricting the diffusion of proteins and lipids between the apical and basal-lateral plasma membrane domains (6, 27, 32). Tight junctions are composed of the transmembrane proteins occludin, claudin, and junctional adhesion molecule (11, 12, 29). These membrane proteins associate with a cytoplasmic plaque composed of a variety of peripheral membrane proteins including the MAGUK family proteins zonula occludens (ZO)-1, -2, and -3 (18, 23, 44) that help to link tight junction integral membrane proteins to the underlying actin cytoskeleton and provide a scaffold for intracellular signaling protein complexes.

Tight junction assembly is regulated by an evolutionarily conserved protein complex that includes atypical protein kinase C (aPKC) and two homologs of Caenorhabditis elegans partitioning defective gene products, Par3 and Par6 (reviewed in Ref. 41). Successful assembly and activation of the aPKC-Par3-Par6 complex is a critical step in tight junction formation and epithelial polarization (26, 46). Par6 interacts directly with activated forms of Cdc42 and Rac, but not Rho GTPase (24, 36), and this association regulates the serine/threonine kinase activity of aPKC (39, 53). Par3 and mLgl (a member of the evolutionarily conserved tight junction regulatory Scribble complex) have been identified as kinase targets of aPKC (34, 38). aPKC forms independent complexes with mLgl and Par3 during tight junction assembly and epithelial polarization (52).

Ischemic injury is the major cause of acute renal failure in patients with various forms of renal disease and is associated with epithelial cell injury caused by depletion of cellular ATP levels (47). A commonly used, powerful, and reversible model of epithelial cell injury during renal ischemia is ATP depletion of cultured epithelial cells using inhibitors of oxidative phosphorylation. This model recapitulates many pathological effects on epithelial cells associated with renal ischemia in animal models and human patients (10), including disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity.

How ischemia disrupts signaling pathways that regulate tight junctions is unclear. ATP depletion is accompanied by a concomitant reduction in cellular GTP levels (9) and Rho GTPases were shown to be differentially sensitive to nucleotide depletion in LLC-PK cells (17). Activation of Rho and Rac GTPase signaling protects tight junctions from disassembly during ATP depletion (Ref. 15 and S. Gopalakrishnan and J. A. Marrs, unpublished observations). As mentioned previously, Rac GTPase associates with the aPKC-Par6 complex (24, 36) and activates aPKC (39). In the present study, we examined the aPKC signaling pathway in the ATP-depletion model of ischemic epithelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells and reagents. Madin-Darby canine kidney (MDCK) type II cells were maintained in DMEM (GIBCO-BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine (GIBCO Invitrogen, Grand Island, NY). aPKC{zeta} pseudosubstrate (PS) inhibitor was purchased from EMD Biosciences (La Jolla, CA). Other chemicals were purchased from Sigma (St. Louis, MO) or Midwest Scientific (St. Louis, MO).

Antibodies against ZO-1, occludin, Par3, phosphothreonine, and phosphoserine were from Zymed (San Francisco, CA); monoclonal antibodies against Rac1 and Cdc42, and polyclonal antibody against phosphorylated aPKC{zeta} were from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal aPKC{zeta} antibody was from Upstate (Charlottesville, VA); Par3 antibody was a gift from Ian Macara (University of Virginia, Charlottesville, VA), and mLgl-1 antibody was a gift from Patrick Brennwald (Cornell University, Ithaca, NY). Fluorophore-conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Horseradish peroxidase-conjugated goat anti-rabbit antibody was from Amersham-Pharmacia (Piscataway, NJ).

ATP depletion and repletion. MDCK cultures were rinsed in prewarmed depletion medium and ATP depleted for 60 min by incubating cells with depletion medium containing 0.1 µM antimycin A (5). For ATP repletion, depletion medium was replaced with DMEM in the presence or absence of 25 µM aPKC{zeta} pseudosubstrate inhibitor.

Calcium switch assay. The calcium switch assay was previously developed by Cereijido and colleagues (13). MDCK cells were washed once in low-calcium medium (DMEM with 5 µM CaCl2) and incubated with low calcium medium plus 2.5% FBS extensively dialyzed against Tris saline for 3 h to disassemble junctions (48). Cells were then processed as described in Immunoprecipitation and immunoblotting.

Immunofluorescence and image analysis. MDCK cells (2.5 million) were plated on 6-well polycarbonate filter supports (Corning-Costar, Kennebunk, ME) and cultured for 5–7 days (for two-photon microscopy) or cells were plated on coverslips and grown for 3 days. Cells were ATP depleted for 45 or 60 min with 0.1 µM antimycin A or left untreated in the control filters and coverslips. Cells were then fixed in methanol-acetone (1:1) or PBS containing 4% paraformaldehyde for 10 min at room temperature. Cells were washed in PBS and then permeabilized in PBS containing 0.5% Triton X-100 for 5 min at room temperature. Cells were then blocked in PBS containing 0.2% BSA and 2% goat serum (blocking solution) for 30 min at room temperature, followed by incubation with primary antibody diluted in blocking solution for 45 min at room temperature. Cells were washed in PBS containing 0.2% BSA and incubated with secondary antibodies diluted in blocking solution for 45 min at room temperature. Coverslips were washed and mounted in PBS containing 50% glycerol, 0.1% sodium azide, and 100 mg/ml 1,4-diazabicyclo(2.2.2)octane.

For two-photon microscopy, samples were viewed with a confocal/two-photon system (model MRC 1024; Bio-Rad, Hercules, CA) fitted to a Nikon Eclipse inverted microscope (Melville, NY) with a x60 water immersion, 1.2 numerical aperture objective. Data sets were collected as a z-series of ~100 two-photon images with a spacing of 0.1 µm. Acquired images were first processed for background subtraction by applying a 3 x 3 low-pass filter with Metamorph version 4.1.7 image-processing software. The digital contrast of the stacks was manipulated, and a 256 x 256 subsection from each stack was chosen for further analysis. Data stacks were rendered in three dimensions with volume-visualization software (Voxx) (8). Transparency of the volume was manipulated by varying image opacity coefficients with Voxx. Data collection and image analysis for all control and ATP-depleted cells were manipulated identically.

For laser scanning confocal microscopy, individual planes through the entire cell volume were collected at 0.5 µm intervals. A projection image was generated by summing the z-series images through the entire cell volume into one 16-bit image.

Immunoprecipitation and immunoblotting. Control and ATP-depleted cultures were rinsed in ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (0.15 M NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 0.05 M Tris·HCl, pH 8.0) for ZO-1, and in Triton X-100-containing lysis buffer for aPKC and Par3 (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 1 mM PMSF, 1.8 µg/ml aprotinin, 1% Triton X-100, 0.1% deoxycholate, and 0.1% SDS) (22) for 15 min on ice. Monolayers were scraped, and lysates were collected. Extracts were cleared by centrifugation at 15,000 g for 5 min at 4°C. Primary antibody was added to supernatants and tubes were rotated at 4°C for 1 h. Immune complexes were collected with protein A-Sepharose beads (Amersham Pharmacia, Piscataway, NJ), and washed three times in lysis buffers. Beads were resuspended in SDS-PAGE sample buffer, and separated on 7.5% SDS polyacrylamide gels. For analysis of total protein levels of ZO-1 and occludin (Fig. 1B), control and ATP-depleted MDCK cells were extracted in SDS-containing buffer (1% SDS, 10 mM Tris·HCl, pH 7.5, and 2 mM EDTA) at 100°C. Cells were scraped, and lysates were heated at 100°C for 5–10 min and sonicated. Samples were clarified by centrifugation. Protein assays on supernatants were performed with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Thirty micrograms of protein were separated by SDS-PAGE. Samples separated by SDS-PAGE were transferred to nitrocellulose filters (Bio-Rad), blocked in Tris-buffered saline containing Tween 20 (TBST; 10 mM Tris·HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20) containing 3% BSA and 5% nonfat dry milk (ZO-1 and occludin), and in TBST containing 3% BSA for aPKC{zeta}, Par3, mLgl-1, and phosphoserine/threonine. Filters were incubated with primary antibody diluted in blocking solution for 1 h at room temperature. Filters were washed in TBST for 1 h. Filters were then incubated with species-matched horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia) diluted in blocking solution. Filters were again washed in TBST, and signal was detected by enhanced chemiluminescence (ECL kit; Amersham Pharmacia) and exposed to film (Bio-Max ML; Eastman Kodak, Rochester, NY).


Figure 1
View larger version (64K):
[in this window]
[in a new window]

  		Fig. 1. Distribution of tight junction components in control Madin-Darby canine kidney (MDCK) cells and after ATP depletion. A: MDCK cells were processed for immunofluorescence with antibodies specific for occludin or zonula occludens-1 (ZO-1). Stacks of ~100 x-y image planes were collected through the volume of the cell monolayers with a two-photon microscope, projected into a single plane and rendered in three dimensions with Voxx (see MATERIALS AND METHODS). Left, control untreated MDCK cells. Right, MDCK cells that were ATP depleted for 60 min. All images were rotated or tilted –5, 9, and 6 degrees on the x, y, and z-axes, respectively. B: control MDCK cells (C), MDCK cells that were ATP depleted for 60 min (60) were extracted with SDS-containing buffer. Equal amounts of protein were separated by SDS-PAGE and immunoblotted with antibodies against occludin and ZO-1.

 
Rac and Cdc42-GTPase pull-down assays. Cells were rinsed once in PBS, and lysed in buffer A (25 mM Tris·HCl, pH 7.5, 150 mM K acetate, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10% glycerol, 1% Triton X-100, 60 mM n-octyl beta-D-glucopyranoside, and [1 mM PMSF, 1 mM benzamidine, 1 µg/ml pepstatin A, 40 µg/ml bestatin, 5 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µM butyrated hydroxy toluene]) for 10 min on ice. Lysates were cleared by centrifugation for 5 s at 15,000 g. Twenty-five microliters of lysate were then incubated with 15 µl of GST p21-associated kinase (Pak) binding domain (GST PAK-BD) for 1 h at 4°C with rotation. Beads were washed three times in buffer B (25 mM Tris·HCl, pH 7.5, 150 mM K acetate, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, and 10% glycerol). The unbound fractions from each wash were pooled and TCA precipitated. Beads were dried using a Speedvac (Savant Instruments, Holbrook, NY). Beads and TCA precipitates were resuspended in 50 µl x 2 SDS sample buffer (100 mM Tris·HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 6 M urea) and heated to 70°C for 5 min. Equal volumes from each fraction were analyzed on a 15% SDS Polyacrylamide gel, transferred to Immobilon PSQ (Millipore, Bedford, MA) and analyzed by Western blotting with an anti-Rac1 or anti-Cdc42 antibody. Immunoblots were quantified using a Fluor-S MultiImager (Bio-Rad), and the results presented as a ratio of active (bound) to inactive (unbound) Rac1-GTPase.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ATP depletion disrupts tight junction assembly in MDCK cells. Previous studies (2, 49) have shown that ATP depletion causes redistribution of ZO-1 from cell-cell junctions. To study the effects of ATP depletion on tight junction proteins at a higher resolution, distributions of occludin and ZO-1 were examined in three-dimensional reconstructions of immunofluorescence images collected by two-photon microscopy. Data sets were processed with Voxx, a three-dimensional rendering software (8).

MDCK cells were ATP depleted for 60 min, or left untreated in control cells. Cells were processed by immunofluorescence using antibodies against occludin and ZO-1. In control cells both occludin and ZO-1 were concentrated at the apical region of the lateral membrane (Fig. 1A). Distinct breaks were observed in the lateral membrane staining for occludin at 60-min ATP depletion, suggesting that tight junction integrity was disrupted. The fluorescence intensity of ZO-1 at the tight junction was also severely reduced following 60 min of ATP depletion. However, immunoblotting experiments showed that the amounts of the two proteins were unchanged in control and ATP-depleted cells (Fig. 1B), showing that there was redistribution of occludin and ZO-1 without significant changes in ZO-1 or occludin levels by 60 min of ATP depletion (15, 49).

Evidence that ATP depletion downregulates the aPKC signaling pathway in MDCK cells. aPKC and Par3 localize to tight junctions in epithelial cells (22), and stable association of Par3 with junctions requires aPKC activity (45). mLgl, the other known substrate of aPKC, associates with the lateral membrane, and phosphorylation of mLgl by aPKC is required to restrict the protein to the lateral membrane and exclude it from the apical membrane (33, 52). We examined the distribution of aPKC{zeta}, Par3, and mLgl-1 in MDCK cells following ATP depletion and recovery. ATP depletion caused ATP levels to drop rapidly to ~5% of control within 15 min following antimycin A treatment, and ATP levels recover to ~50% of control levels 60 min after cells are returned to normal growth medium (15). Cells were ATP depleted for 45 min, ATP depleted for 45 min and allowed to recover for 45 min, or untreated in controls, and cell monolayers were processed for indirect immunofluorescence. A z-series of x-y images through the entire cell volume of the monolayer was collected with a laser scanning confocal microscope and combined into a single projection image to avoid missing fluorescent signal. ATP depletion caused a redistribution of aPKC{zeta}, Par3, and mLgl-1 from cell junctions (Fig. 2). Wash out of antimycin A and allowing recovery of ATP levels led to reassembly and redistribution of all three proteins back into cell junctions (Fig. 2).


Figure 2
View larger version (139K):
[in this window]
[in a new window]

  		Fig. 2. Effect of ATP depletion on the distribution of Par3, aPKC{zeta}, and mLgl-1 in MDCK cells. MDCK cells were ATP depleted for 45 min, ATP depleted for 45 min and allowed to recover for 45 min, or left untreated in controls. Cells were fixed and processed for immunofluorescence with antibodies to detect Par3, aPKC{zeta}, or mLgl-1. Each image shown is 46.3 µm2. Data shown are representative of 3 independent experiments.

 
We examined the activities of Rac and Cdc42 GTPases in control and ATP-depleted MDCK cells with a pull-down assay using a fusion protein of GST and the effector domain of Pak to selectively bind GTP-bound Rac1 and Cdc42 (3). Activity of Rac was reduced after 60 min of ATP depletion (Fig. 3, A and B). After 60 min of ATP repletion, the activity of Rac GTPase recovered to nearly that of control level (Fig. 3, A and B). Cdc42 remained active during ATP depletion and recovery (data not shown).


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 3. Effect of ATP depletion and recovery on Rac-GTPase activity in MDCK cells. A: MDCK cells were ATP depleted for 60 min (60), ATP depleted for 60 min and allowed to recover for 60 min (60/60), or left untreated in control (C). Detergent lysates were incubated with GST-Pak-BD and separated into bound (B; GTP bound) or unbound (U; GDP bound) GTPase fractions. Proteins bound to GST-Pak-BD were analyzed by immunoblotting with an anti-Rac antibody. Densitometric quantitation of immunoblots was performed. B: graph of 3 experiments as represented in A. The amount of Rac1 in each fraction was quantified by densitometry of Western blots and expressed as means ± SD ratio of bound Rac to unbound Rac in each sample.

 
The Rac-specific exchange factor Tiam-1 has recently been shown to associate with Par3 and regulate activation of Rac GTPase and tight junction assembly (7, 31). We examined the association of Par3 with Tiam-1 in MDCK cells during ATP-depletion and subsequent repletion by immunoprecipitation of Par3 and immunoblotting the immunoprecipitate with Tiam-1 antibody. ATP depletion resulted in an increased association of Tiam-1 with Par3 (Fig. 4), indicating that Tiam-1 was sequestered from its association with Rac, thus preventing GEF activity. Association of Par3 with Tiam-1 was not observed in MDCK cells that were switched to low calcium medium, indicating that this effect is specific to ATP depletion and is not a general response to junction disassembly (see online supplemental Fig. S1A).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 4. Effect of ATP depletion and recovery on the association of Par3 with Tiam-1. MDCK cells were ATP depleted for 60 min, ATP depleted for 60 min and allowed to recover for 60 min, or left untreated in control. Par3 was immunoprecipitated from control (C), ATP-depleted cells (60), and cells that were allowed to recover from ATP depletion (60/60), separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against Tiam-1 or Par3. Data shown are representative of 3 independent experiments.

 
Many protein kinases are inactivated during ATP depletion, resulting in decreased phosphorylation of proteins in epithelial cells (25). Phosphorylation of aPKC{zeta} on Thr410 is essential for aPKC{zeta} activation (43). We examined aPKC{zeta} phosphorylation in MDCK cells by immunoprecipitating total aPKC{zeta} and immunoblotting the immunoprecipitate using a Thr410 phosphorylation-specific aPKC{zeta} antibody (Fig. 5A). ATP depletion reduced Thr410 phosphorylation levels of aPKC{zeta} (Fig. 5B), suggesting that aPKC{zeta} was inhibited by ATP depletion. In addition, its activity could be reduced due to reduced ATP levels during ATP depletion.


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 5. Effect of ATP depletion and recovery on Thr410 phosphorylation of aPKC{zeta} in MDCK cells. A: MDCK cells were ATP-depleted for 60 min, ATP depleted for 60 min and allowed to recover for 60 min, or left untreated in control. aPKC{zeta} was immunoprecipitated from control (C), ATP-depleted cells (60), and cells that were allowed to recover from ATP depletion (60/60), separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against phospho-aPKC{zeta} or aPKC{zeta}. B: graph of 3 experiments as represented in A.

 
Dephosphorylation of ZO-1 and occludin, which are phosphoproteins (1, 51), during ATP depletion could contribute to tight junction disassembly. We have previously shown that occludin is rapidly dephosphorylated during ATP depletion in MDCK cells (15). Phosphorylation of Par3 on Ser827 by aPKC{zeta} is critical for tight junction assembly, and Ser827 phosphorylated Par3 is the predominant form found in mature tight junctions (20). We analyzed the phosphoserine/threonine content of ZO-1 and Par3 in MDCK cells subjected to ATP depletion. Cells were ATP depleted for 60 min or ATP depleted and allowed to recover for 60 min. ZO-1 or Par3 was immunoprecipitated from cell extracts, and the phosphoserine/threonine content was examined by immunoblotting these immunoprecipitates using anti-phosphoserine and anti-phosphothreonine antibodies. ZO-1 phosphoserine/threonine content was significantly reduced in ATP-depleted cells compared with control cells (Fig. 6). Both the 180 and 150 kDa forms of Par3 were detected in MDCK cells (22). The 180 kDa form of Par3 was serine/threonine phosphorylated in control cells, and a significant reduction in Par3 serine/threonine phosphorylation was observed following 60 min of ATP depletion (Fig. 6). After 60 min of ATP repletion, the phosphorylation levels of both ZO-1 and Par3 recovered to nearly that of control levels. Par3 phosphorylation was reduced slightly in MDCK cells switched to low-calcium medium, whereas ZO-1 phosphorylation was not affected (see online supplemental Fig. S2), indicating effects of ATP depletion were not replicated merely by inducing tight junction disassembly.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 6. Effect of ATP depletion and recovery on phosphoserine/threonine content in ZO-1 and Par3. MDCK cells were ATP depleted for 60 min, ATP depleted for 60 min and allowed to recover for 60 min, or left untreated in control. ZO-1 and Par3 were immunoprecipitated from control (C), ATP-depleted cells (60), and cells that were allowed to recover from ATP depletion (60/60), separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against phosphoserine/threonine, ZO-1, or Par3. *ZO-1, **ZO-2, and ***Par3. Data shown are representative of 3 independent experiments.

 
ATP depletion alters protein-protein interactions. The serine/threonine kinase activity of aPKC is necessary for tight junction formation. Several aPKC targets have been identified (19). Par3 and mLgl were identified as kinase targets of the aPKC-Par6 complex, and Par3 and mLgl interactions with aPKC are regulated by aPKC mediated phosphorylation (34, 38). We examined the association between aPKC{zeta} and these substrates in MDCK cells during ATP depletion and recovery. aPKC{zeta} was immunoprecipitated from cell extracts and immune complexes were probed with antibodies against Par3 and mLgl-1. ATP depletion increased the amount of Par3 and mLgl-1 that coimmunoprecipitated with aPKC{zeta} (Fig. 7). Perhaps these interactions are stabilized because aPKC{zeta} binds but cannot phosphorylate its substrates due to reduced Thr410 phopshorylation and reduced levels of ATP. As a control, treatment of MDCK cells with low calcium medium did not alter the interaction of aPKC{zeta} with mLgl-1 (see online supplemental Fig. S1B).


Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 7. Effect of ATP depletion and recovery on the association of aPKC{zeta} with Par3 and mLgl-1. MDCK cells were ATP depleted for 60 min, ATP depleted for 60 min and allowed to recover for 60 min, or left untreated in control. aPKC{zeta} was immunoprecipitated from control (C), ATP-depleted cells (60), and cells that were allowed to recover from ATP depletion (60/60), separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against Par3, mLgl-1 or aPKC{zeta}. *Par3. Data shown are representative of 3 independent experiments.

 
Inhibition of aPKC{zeta} during recovery from ATP depletion interferes with tight junction assembly. Successful assembly and activation of the aPKC-Par3-Par6 complex is critical for tight junction formation and epithelial polarization. Overexpression of a kinase negative aPKC mutant blocks the formation of tight junctions in a calcium switch assay (46). To better understand the role of aPKC{zeta} in tight junction assembly in ischemic injury, we tested the effect of a pseudosubstrate inhibitor of aPKC during recovery from ATP depletion. MDCK cells were established on coverslips for 3 days and ATP depleted for 45 min. ATP-depleted MDCK cells were allowed to recover in the presence of a cell-permeable, myristoylated pseudosubstrate inhibitor of aPKC{zeta}, which directly inhibits aPKC autophosphorylation and transactivation (43), or allowed to recover without pseudosubstrate inhibitor in controls. Distribution of ZO-1 and Par3 was examined by immunofluorescence. Pseudosubstrate inhibitor treatment interfered with the reassembly of ZO-1 and Par3 at cell junctions during recovery from ATP depletion (Fig. 8), indicating that aPKC activity is important for tight junction reassembly during recovery from cell injury.


Figure 8
View larger version (78K):
[in this window]
[in a new window]

  		Fig. 8. Inhibition of aPKC{zeta} during recovery from ATP depletion interferes with reassembly of ZO-1 and Par3. MDCK cells were ATP depleted for 45 min, ATP depleted for 45 min, and allowed to recover for 45 min in the presence or absence of 25 µM aPKC{zeta} pseudosubstrate (PS) inhibitor, or left untreated in controls. Cells were fixed and processed for immunofluorescence with antibodies to detect ZO-1 or Par3. Each image shown is 46.3 µm2. Data shown are representative of 3 independent experiments.

 
To examine the independent effects of aPKC inhibition on tight junction disassembly, confluent MDCK cells were treated with aPKC{zeta} pseudosubstrate inhibitor for 45 or 90 min. Pseudosubstrate treatment of confluent MDCK cells did not dramatically affect the junctional distribution of Par3 (see online supplemental Fig. S3), supporting the conclusion that the pseudosubstrate inhibitor specifically inhibits tight junction reassembly during recovery from ATP depletion. Together, the above results indicate that aPKC signaling pathway participates in tight junction recovery after ischemia.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ATP depletion of renal epithelial cells is a faithful model for in vivo renal ischemia that recapitulates many aspects of cell injury in the organ, including tight junction disassembly and dysfunction. In this study, we hypothesized that ATP depletion disrupts tight junctions by interfering with phosphorylation and protein-protein interactions of key components in the aPKC signaling pathway. Our previous studies showed that occludin is rapidly dephosphorylated during ATP depletion in MDCK cells (15). This is consistent with the loss of occludin from cell-cell junctions because phosphorylation of occludin directly correlates with its assembly into tight junctions (42). ZO-1 was also redistributed from cell-cell junctions, as observed in previous studies (2, 49).

Cdc42 and Rac activation induces aPKC activity by regulating the assembly and activity of the aPKC-Par complex. In turn, activation of aPKC-Par complex stimulates tight junction assembly. Here, we found that ATP depletion of MDCK cells downregulated Rac GTPase activity, and we present evidence that sequestration of the Rac exchange factor Tiam-1 by forming a stable complex with Par3 contributes to the inhibition of Rac activity in ATP-depleted MDCK cells. Association of Par3 with Tiam-1 was not stabilized in MDCK cells treated with low-calcium medium to disrupt tight junctions, indicating that effects of ATP depletion are specific and not a general response to junction disassembly. Interestingly, Cdc42 remained active even after 60 min of ATP depletion (data not shown). Similar differential sensitivity of Rac and Cdc42-GTPases to ATP depletion was also observed in LLC-PK cells (17). Inhibition of aPKC activity, even in the presence of active Cdc42, suggests the possibility that active Cdc42 may be spatially separated from aPKC-Par6 in ATP-depleted MDCK cells and is unable to activate aPKC.

Activity of aPKC is dependent on phosphorylation events. Phosphorylation of aPKC on Thr410 is a prerequisite for its complete activation (43). ATP depletion caused a decrease in Thr410 phosphorylation of aPKC{zeta}. Par3 and mLgl have been identified as substrates for aPKC (34, 38). aPKC was also shown to phopshorylate occludin, claudin, and ZO-1 in vitro (37). We found that the phosphoserine/threonine content of ZO-1 was reduced following ATP depletion, but not after incubation in low-calcium medium. However, in previous studies (21), a decrease in phosphorylation of ZO-1 was observed in MDCK cells that were treated with low-calcium medium for 16 h. Par3 phosphorylation was also significantly reduced in ATP-depleted MDCK cells, and this correlated with a loss of Par3 from cell-cell junctions during ATP depletion. A decrease in Par3 phosphorylation was also observed in MDCK cells incubated in low-calcium medium, consistent with the finding that Par3 phosphorylated at Ser827 accumulates in assembled tight junctions (20).

ATP depletion also caused redistribution of mLgl-1, another substrate of aPKC, from cell junctions to a diffuse cytosolic distribution. Distribution of mLgl-1 at the basolateral membrane is dependent on its phosphorylation (33). ATP depletion may induce dephosphorylation of mLgl-1, similar to that of Par3, but we were unable to detect any serine phosphorylation of mLgl-1 in control MDCK cells using phosphoserine antibody. A previous report (52) that a phosphomimicking mutant of mLgl failed to bind aPKC{lambda} supports the idea that mLgl-1 is dephosporylated during ATP depletion, since we found that there was increased association of mLgl-1 with aPKC{zeta} in ATP-depleted cells compared with control cells. Furthermore, Yamanaka et al. (52), have shown that in MDCK cells expressing a kinase-deficient mutant, aPKC{lambda} kn, mLgl colocalized with Par6 at the apical region of the cell and suppressed tight junction formation. It is possible that in ATP-depleted MDCK cells, mLgl-1 sequesters aPKC{zeta} in an inactive complex. ATP-depletion also enhanced the association between aPKC{zeta} and Par3. Again, this is consistent with the observation that a nonphosphorylatable mutant of Par3, S827A, formed a stable complex with aPKC, and suppressed tight junction formation, whereas S827E (a phosphomimicking mutant) was unable to associate with aPKC (34).

Inhibition of aPKC{zeta} with a pseudosubstrate inhibitor during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions strongly implicating that aPKC activity participates in tight junction assembly dynamics during cell injury and recovery. This is consistent with studies that have shown that expression of a kinase-deficient aPKC mutant inhibited assembly of ZO-1 and Par3 at tight junctions in calcium switch experiments (46).

Our results have led us to propose a working model for tight junction disassembly in ATP-depleted cells (Fig. 9). In normal cells, Rac GTPase is activated by Tiam-1. Rac-GTP activates aPKC{zeta}, which phosphorylates mLgl-1. Phosphorylated mLgl-1 dissociates from aPKC{zeta} and distributes to the lateral membrane. aPKC{zeta} interacts with and phosphorylates Par3, which then associates with tight junctions. How does ATP depletion lead to tight junction disruption? In ATP-depleted cells, various kinase activities are reduced due to reduced ATP substrate concentrations. Phosphatases remain active, which dephosphorylate junctional Par3 and other tight junction proteins. Nonjunctional Par3 associates with Tiam-1 and inhibits Rac activation. Rac-GDP fails to activate aPKC{zeta}. This, coupled with low cellular ATP levels, interferes with the ability of aPKC{zeta} to phosphorylate its substrates thereby affecting aPKC protein interactions, because the association of aPKC with Par3 and mLgl is regulated by phosphorylation. In the absence of aPKC{zeta}-mediated phosphorylation, Par3 and mLgl-1 form "inactive" stable complexes with aPKC{zeta} in ATP-depleted cells, and fail to distribute to cell-cell junctions. This prevents Par3 and mLgl-1 from participating in normal junction assembly processes, which may include associations with other as-yet unidentified binding partners at the junctions. Furthermore, ATP depletion could interfere with phosphorylation of additional proteins that may be required for tight junction stabilization. These could include proteins of the Crumbs-Pals1-PATJ polarity complex, which also participates in tight junction formation (40).


Figure 9
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 9. A model for ATP depletion-induced tight junction disassembly. The aPKC-Par complex is regulated by specific protein interactions, and this complex and aPKC signaling regulate tight junction assembly and other cellular processes (19). Interactions between subsets of these proteins were examined in a model of renal injury. In control cells, (1) Tiam-1 activates Rac GTPase. Rac-GTP associates with the aPKC{zeta}-Par6 complex and activates aPKC{zeta}. (2) aPKC{zeta} associates with and phosphorylates mLgl-1 and Par3. (3) Phosphorylated mLgl-1 and Par3 are released from the complex, and associate with the lateral membrane (LM) and tight junction (TJ), respectively. The aPKC{zeta}-Par6 complex may associate with and phosphorylate other tight junction proteins, such as the Crumbs polarity complex proteins that participate in tight junction assembly. In ATP-depleted cells, Tiam-1 associates with Par3 and is unable to activate Rac GTPase. As a consequence, aPKC{zeta} is not activated. aPKC{zeta} binds mLgl-1 and Par3, but is unable to phosphorylate them. The lack of phosphorylation stabilizes the association of mLgl-1 and Par3 with aPKC{zeta}, thereby preventing their release and localization to the lateral membrane and tight junction, respectively. ATP depletion also interferes with phosphorylation of other tight junction proteins, such as occluding-causing tight junction disassembly. Association of the Crumbs polarity complex with the aPKC{zeta}-Par complex may be altered during ATP depletion and proteins such as Pals1 may also serve as aPKC{zeta} substrates during tight junction formation.

 
Adherens junctions are also disrupted during ATP-depletion by unknown mechanisms that lead to E-cadherin internalization (4, 14, 28). Cadherin mediated adhesion provides both positional information and signaling cues (Rac activation) for the assembly of tight junctions (16, 30, 35, 50), and therefore, adherens junction disassembly may also contribute to tight junction disruption during ATP depletion.

Taken together, the disruption of tight junctions during ATP depletion could involve the synergistic dysfunction of signaling pathways involving aPKC, Crumbs polarity complex, and E-cadherin. A more detailed understanding of these signaling pathways will help identify novel therapeutic targets for protecting renal epithelial cells from ischemic cellular damage.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54518 and DK-53465 (to J. A. Marrs).


   ACKNOWLEDGMENTS
 
We thank Dr. Ian Macara (University of Virginia, Charlottesville) for providing the Par3 antibody and Dr. Patrick Brennwald (Cornell University, Ithaca, NY) for providing the mLgl-1 antibody. We are grateful to Dr. Pierre Dagher for a critical reading of the manuscript. The studies were conducted at the Indiana University Center for Biological Microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Marrs, Dept. of Medicine, Div. of Nephrology, Indiana Univ. Medical Center, R2-223, 950 W. Walnut St., Indianapolis, IN 46202-5116 (e-mail: jmarrs{at}iupui.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Anderson JM, Stevenson BR, Jesaitis LA, Goodenough DA, Mooseker MS. Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J Cell Biol 106: 1141–1149, 1988.[Abstract/Free Full Text]

2. Bacallao R, Garfinkel A, Monke S, Zampighi G, Mandel LJ. ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci 107: 3301–3313, 1994.[Abstract]

3. Benard V, Bohl BP, Bokoch GM. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 274: 13198–13204, 1999.[Abstract/Free Full Text]

4. Bush KT, Tsukamoto T, Nigam SK. Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia. Am J Physiol Renal Physiol 278: F847–F852, 2000.[Abstract/Free Full Text]

5. Canfield PE, Geerdes AM, Molitoris BA. Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F1038–F1045, 1991.[Abstract/Free Full Text]

6. Cereijido M, Valdes J, Shoshani L, Contreras RG. Role of tight junctions in establishing and maintaining cell polarity. Annu Rev Physiol 60: 161–177, 1998.[CrossRef][Web of Science][Medline]

7. Chen X, Macara IG. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat Cell Biol 7: 262–269, 2005.[CrossRef][Web of Science][Medline]

8. Clendenon JL, Phillips CL, Sandoval RM, Fang S, Dunn KW. Voxx: a PC-based, near real-time volume rendering system for biological microscopy. Am J Physiol Cell Physiol 282: C213–C218, 2002.[Abstract/Free Full Text]

9. Dagher PC. Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools. Am J Physiol Cell Physiol 279: C1270–C1277, 2000.[Abstract/Free Full Text]

10. Fish EM, Molitoris BA. Extracellular acidosis minimizes actin cytoskeletal alterations during ATP depletion. Am J Physiol Renal Fluid Electrolyte Physiol 267: F566–F572, 1994.[Abstract/Free Full Text]

11. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 1539–1550, 1998.[Abstract/Free Full Text]

12. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123: 1777–1788, 1993.[Abstract/Free Full Text]

13. Gonzalez-Mariscal L, Chavez de Ramirez B, Cereijido M. Tight junction formation in cultured epithelial cells (MDCK). J Membr Biol 86: 113–125, 1985.[CrossRef][Web of Science][Medline]

14. Gopalakrishnan S, Dunn KW, Marrs JA. Rac1, but not RhoA, signaling protects epithelial adherens junction assembly during ATP depletion. Am J Physiol Cell Physiol 283: C261–C272, 2002.[Abstract/Free Full Text]

15. Gopalakrishnan S, Raman N, Atkinson SJ, Marrs JA. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol Cell Physiol 275: C798–C809, 1998.[Abstract/Free Full Text]

16. Gumbiner B, Stevenson B, Grimaldi A. The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J Cell Biol 107: 1575–1587, 1988.[Abstract/Free Full Text]

17. Hallett MA, Dagher PC, Atkinson SJ. Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury. Am J Physiol Cell Physiol 285: C129–C138, 2003.[Abstract/Free Full Text]

18. Haskins J, Gu L, Wittchen ES, Hibbard J, Stevenson BR. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol 141: 199–208, 1998.[Abstract/Free Full Text]

19. Hirai T, Chida K. Protein kinase C{zeta} (PKC{zeta}): activation mechanisms and cellular functions. J Biochem (Tokyo) 133: 1–7, 2003.[Abstract/Free Full Text]

20. Hirose T, Izumi Y, Nagashima Y, Tamai-Nagai Y, Kurihara H, Sakai T, Suzuki Y, Yamanaka T, Suzuki A, Mizuno K, Ohno S. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J Cell Sci 115: 2485–2495, 2002.[Abstract/Free Full Text]

21. Howarth AG, Singer KL, Stevenson BR. Analysis of the distribution and phosphorylation state of ZO-1 in MDCK and nonepithelial cells. J Membr Biol 137: 261–270, 1994.[Web of Science][Medline]

22. Izumi Y, Hirose T, Tamai Y, Hirai S, Nagashima Y, Fujimoto T, Tabuse Y, Kemphues KJ, Ohno S. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol 143: 95–106, 1998.[Abstract/Free Full Text]

23. Jesaitis LA, Goodenough DA. Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J Cell Biol 124: 949–961, 1994.[Abstract/Free Full Text]

24. Joberty G, Petersen C, Gao L, Macara IG. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol 2: 531–539, 2000.[CrossRef][Web of Science][Medline]

25. Kobryn CE, Mandel LJ. Decreased protein phosphorylation induced by anoxia in proximal renal tubules. Am J Physiol Cell Physiol 267: C1073–C1079, 1994.[Abstract/Free Full Text]

26. Lin D, Edwards AS, Fawcett JP, Mbamalu G, Scott JD, Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat Cell Biol 2: 540–547, 2000.[CrossRef][Web of Science][Medline]

27. Madara JL. Regulation of the movement of solutes across tight junctions. Annu Rev Physiol 60: 143–159, 1998.[CrossRef][Web of Science][Medline]

28. Mandel LJ, Doctor RB, Bacallao R. ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K+-ATPase and E-cadherin. J Cell Sci 107: 3315–3324, 1994.[Abstract]

29. Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol 142: 117–127, 1998.[Abstract/Free Full Text]

30. Matter K, Balda MS. Signalling to and from tight junctions. Nat Rev Mol Cell Biol 4: 225–236, 2003.[CrossRef][Web of Science][Medline]

31. Mertens AE, Rygiel TP, Olivo C, van der Kammen R, Collard JG. The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J Cell Biol 170: 1029–1037, 2005.[Abstract/Free Full Text]

32. Mitic LL, Anderson JM. Molecular architecture of tight junctions. Annu Rev Physiol 60: 121–142, 1998.[CrossRef][Web of Science][Medline]

33. Musch A, Cohen D, Yeaman C, Nelson WJ, Rodriguez-Boulan E, Brennwald PJ. Mammalian homolog of Drosophila tumor suppressor lethal (2) giant larvae interacts with basolateral exocytic machinery in Madin-Darby canine kidney cells. Mol Biol Cell 13: 158–168, 2002.[Abstract/Free Full Text]

34. Nagai-Tamai Y, Mizuno K, Hirose T, Suzuki A, Ohno S. Regulated protein-protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells. Genes Cells 7: 1161–1171, 2002.[Abstract]

35. Nelson WJ. Adaptation of core mechanisms to generate cell polarity. Nature 422: 766–774, 2003.[CrossRef][Medline]

36. Noda Y, Takeya R, Ohno S, Naito S, Ito T, Sumimoto H. Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C. Genes Cells 6: 107–119, 2001.[Abstract]

37. Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CL 3rd, Sontag E. Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol 158: 967–978, 2002.[Abstract/Free Full Text]

38. Plant PJ, Fawcett JP, Lin DC, Holdorf AD, Binns K, Kulkarni S, Pawson T. A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl. Nat Cell Biol 5: 301–308, 2003.[CrossRef][Web of Science][Medline]

39. Qiu RG, Abo A, Martin SG. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKC{zeta} signaling and cell transformation. Curr Biol 10: 697–707, 2000.[CrossRef][Web of Science][Medline]

40. Roh MH, Fan S, Liu CJ, Margolis B. The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J Cell Sci 116: 2895–2906, 2003.[Abstract/Free Full Text]

41. Roh MH, Margolis B. Composition and function of PDZ protein complexes during cell polarization. Am J Physiol Renal Physiol 285: F377–F387, 2003.[Abstract/Free Full Text]

42. Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol 137: 1393–1401, 1997.[Abstract/Free Full Text]

43. Standaert ML, Bandyopadhyay G, Perez L, Price D, Galloway L, Poklepovic A, Sajan MP, Cenni V, Sirri A, Moscat J, Toker A, Farese RV. Insulin activates protein kinases C-{zeta} and C-{lambda} by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes. J Biol Chem 274: 25308–25316, 1999.[Abstract/Free Full Text]

44. Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103: 755–766, 1986.[Abstract/Free Full Text]

45. Suzuki A, Ishiyama C, Hashiba K, Shimizu M, Ebnet K, Ohno S. aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J Cell Sci 115: 3565–3573, 2002.[Abstract/Free Full Text]

46. Suzuki A, Yamanaka T, Hirose T, Manabe N, Mizuno K, Shimizu M, Akimoto K, Izumi Y, Ohnishi T, Ohno S. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol 152: 1183–1196, 2001.[Abstract/Free Full Text]

47. Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N Engl J Med 334: 1448–1460, 1996.[Free Full Text]

48. Troxell ML, Chen YT, Cobb N, Nelson WJ, Marrs JA. Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression. Am J Physiol Cell Physiol 276: C404–C418, 1999.[Abstract/Free Full Text]

49. Tsukamoto T, Nigam SK. Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly. J Biol Chem 272: 16133–16139, 1997.[Abstract/Free Full Text]

50. Watabe M, Nagafuchi A, Tsukita S, Takeichi M. Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line. J Cell Biol 127: 247–256, 1994.[Abstract/Free Full Text]

51. Wong V. Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am J Physiol Cell Physiol 273: C1859–C1867, 1997.[Abstract/Free Full Text]

52. Yamanaka T, Horikoshi Y, Sugiyama Y, Ishiyama C, Suzuki A, Hirose T, Iwamatsu A, Shinohara A, Ohno S. Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity. Curr Biol 13: 734–743, 2003.[CrossRef][Web of Science][Medline]

53. Yamanaka T, Horikoshi Y, Suzuki A, Sugiyama Y, Kitamura K, Maniwa R, Nagai Y, Yamashita A, Hirose T, Ishikawa H, Ohno S. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells 6: 721–731, 2001.[Abstract]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
V. A. Swystun, B. Renaux, F. Moreau, S. Wen, M. A. Peplowski, M. D. Hollenberg, and W. K. MacNaughton
Serine proteases decrease intestinal epithelial ion permeability by activation of protein kinase C{zeta}
Am J Physiol Gastrointest Liver Physiol, July 1, 2009; 297(1): G60 - G70.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
C. Chabu and C. Q. Doe
Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression
Development, August 15, 2008; 135(16): 2739 - 2746.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
292/3/C1094    most recent
00099.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gopalakrishnan, S.
Right arrow Articles by Marrs, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gopalakrishnan, S.
Right arrow Articles by Marrs, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.