Correspondence to: Shigeo Ohno, Department of Molecular Biology, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236, Japan. Tel:81-045-787-2596 Fax:81-045-785-4140 E-mail:ohnos{at}med.yokohama-cu.ac.jp.
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Abstract |
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We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:36073614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.
Key Words: atypical PKC, tight junction, epithelial cell polarity, PAR-3, PAR-6
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Introduction |
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Protein kinase C comprises a family of serine/threonine kinases with NH2-terminal autoregulatory regions containing unique cysteine-rich Zn-finger motifs (for reviews, see /
and
isoforms whose activities are affected by neither calcium nor diacylglycerol/phorbol esters. Several works including ours have shown that aPKCs are activated in vivo through a pathway involving phosphatidylinositol 3-kinase (
Recently, we identified a mammalian homologue of Caenorhabditis elegans PAR-3, one of the six par gene products indispensable for the establishment of the cell polarity of the one-cell embryo (
In this work, we report the introduction of a dominant-negative mutant of aPKC (aPKCkn) into MDCK II cells using an adenovirus expression vector to directly examine the functional importance of aPKC in epithelial cell polarity. We present evidence that aPKCkn blocks the completion of tight junction formation after calcium switch or during normal cell growth. Impairment of cell surface polarity of aPKCkn-expressing cells is also demonstrated by increased interdomain diffusion of fluorescent membrane lipids and the disrupted asymmetric distribution of Na+,K+-ATPase. On the other hand, we also demonstrate that aPKC associates with not only ASIP/PAR-3, but also with a mammalian homologue of another par-gene product, PAR-6, which colocalizes and functions interdependently with PKC-3 and PAR-3 in the C. elegans embryo (
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Materials and Methods |
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cDNA Cloning of Human PAR-6
A database search of human EST clones in GenBank identified a human EST clone (AA609625) encoding a peptide closely similar to a part of the C. elegans PAR-6 protein. To obtain cDNA clone(s) covering the entire human PAR-6 protein coding region, we performed backscreening of a human kidney cDNA library (CLONTECH Laboratories, Inc.) using this EST clone as a probe, and identified five cDNA clones, each encoding human PAR-6. The longest clone, n32, carries a 1,269-pb insert containing a 1,041-bp open reading frame encoding a protein of 346 amino acid residues with a calculated molecular weight of 37,388.32. Screening of a HeLa cDNA library (CLONTECH Laboratories, Inc.) with the same probe led to the identification of a different class of cDNA clones encoding a human PAR-6 isoform. The longest clone, 16-5-5, contains 1,162 bp and encodes a 276 amino acid residue, corresponding to a part of the protein (see Supplemental Fig. S1).
Northern Blot Analysis
Northern blot analysis was performed following a standard procedure using human multiple tissue Northern blot (CLONTECH Laboratories, Inc.). Radio-labeled cDNA inserts of clone n32 (1,269 bp) and clone 16-5-5 (1,162 bp) were used as probes to detect human PAR-6 and 16-5-5 mRNA, respectively (see Supplemental Fig. S2).
Antibodies
The antibodies used in this study were: rabbit antinPKC(
5), antiaPKC
(
1,
2), and antiASIP(C2-3) polyclonal antibodies, previously raised in our laboratory (
, E-cadherin, and ß-catenin monoclonal antibodies (Transduction Laboratories); rabbit antioccludin and ZO-1 polyclonal antibodies (Zymed Laboratories); rabbit antiaPKC
polyclonal antibody (C-20; Santa Cruz Biotechnology, Inc.); mouse antiT7 monoclonal antibody (Novagen); mouse antiFlag monoclonal antibody (M2; Sigma-Aldrich). Antihuman PAR-6 polyclonal antibodies, GW2, GC2, and N12, were generated in rabbits. GW2AP was raised against glutathione-S-transferase (GST)human PAR-6 and purified on a maltose binding protein (MalE)PAR-6 affinity column. GC2AP was raised against GSTPAR-6 (amino acids 126346) and purified as above. N12AP was raised against MalEPAR-6 (amino acids 1-125) and affinity purified on MalE and MalEPAR-6 columns.
Expression Vectors
Complementary DNAs encoding wild-type mouse aPKC and its kinase-deficient mutant (aPKC
kn) were obtained as described previously (
(lysine 376 replaced by alanine;
(lysine 281 replaced by tryptophan) were generous gifts from Dr.Oka (Yamaguchi University, Yamaguchi, Japan); the vector for LacZ was obtained from the Riken Gene Bank (
30 encodes an ASIP splicing isoform lacking amino acid residues 741743 and 827856. Flag-tagged human PAR-6 expression vectors were constructed on pME18S-flag vector; PAR-6wt encodes a full-length PAR-6 (amino acids 1346), PAR-6
aPKCBD lacks amino acids 1125, corresponding to the aPKC-binding domain, and PAR-6
CRIB/PDZ lacks amino acids 126258, corresponding to CRIB and PDZ domain. aPKC
expression vectors encoding wild type or a mutant lacking amino acids 147 (aPKC
N47) have been described previously (
Cell Culture and Adenovirus Infection
MDCK II cells were grown in DMEM containing 10% FCS, penicillin, and streptomycin on 12-mm round coverslips or 12-mm diameter TranswellTM filters (Corning Coaster Corp.) with a pore size of 0.4 µm. For adenovirus infection, cells were seeded on 24-well plates (1.25 x 105 cells/cm2) 1 d before infection, or on filter inserts (1.5 x 105 cells/cm2) 2 d before infection. To enhance the efficiency of viral infection, cellcell adhesion was disrupted by preincubating cells in low Ca2+ (LC) medium containing 5% FCS and 3 µM Ca2+ (
Immunocytochemistry
Cells grown on coverslips or filters were washed twice with PBS containing 0.9 mM CaCl2 and 0.49 mM MgCl2, and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. Cells were then permeabilized with PBS containing 0.5%(vol/vol) Triton X-100 for 10 min and blocked in PBS containing 10% calf serum for 1 h at room temperature. Only in the case of claudin-1 immunostaining, fixation and permeabilization procedures were performed simultaneously using PBS containing 0.3% paraformaldehyde and 0.1% Triton X-100. Antibody incubations were performed at 37°C for 45 min in buffer containing 10 mM Tris/HCl, pH7.5, 150 mM NaCl, 0.01% (vol/vol) Tween 20, and 0.1% (wt/vol) BSA. The secondary antibodies used were: BODIPY, or Alexa488-conjugated goat antirabbit IgG (Molecular Probes Inc.), Cy3-conjugated goat antimouse IgG, Cy3-conjugated goat antirabbit IgG (Amersham Pharmacia Biotech), and FITC-conjugated goat antimouse IgG antibodies (EY Laboratories). To stain F-actin, rhodamine-phalloidin (Molecular Probes Inc.) was used in place of the secondary antibodies. Coverslips were mounted using Vectashield (Vector Laboratories), and examined under a fluorescence microscope equipped with a confocal system (µRadiance; Bio-Rad Laboratories). For immunohistochemistry, a piece of small intestine from a mouse (12-wk old) was fixed in 2% paraformaldehyde/PBS at 4°C for 30 min, rinsed with 50 mM NH4Cl/PBS, treated with PBS containing 1.0 M sucrose at 4°C for 24 h, and embedded in Tissue Tek OTC compound. The frozen tissue was cut in a cryostat. The sections (7 µm) were mounted on glass slides and stained as described above.
Evaluation of the Barrier Functions of TJ in MDCK Cells
Transepithelial electrical resistance (TER) of MDCK II monolayers grown on filters was measured using an ERS electrical resistance system (Millipore) as described elsewhere (
Fluorescent Lipid Labeling of the Plasma Membrane
BODIPY-FL-C5 sphingomyelin (Molecular Probes, Inc.) and defatted BSA (Sigma-Aldrich) were used to prepare sphingomyelin/BSA complexes (5 nmol/ml) in P-buffer (10 mM Hepes, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, and 145 mM NaCl;
Immunoprecipitation Analysis
COS1 cells were transfected with appropriate cDNA expression plasmids by electroporation (Gene Pulser; Bio-Rad Laboratories). These cells cultured in 10-cm dishes were suspended in 200 µl of lysis buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 1 PMSF, 1.8 µg/ml aprotinin, and 1% Triton X-100. After 30-min incubation on ice, the lysates were clarified by centrifugation at 14,000 rpm for 30 min and incubated with antibodies preabsorbed on Protein G-sepharose (Amersham Pharmacia Biotech) for 1 h at 4°C. After washing four times with lysis buffer, the immunocomplexes were denatured in SDS sample buffer and separated by SDS-PAGE.
Electrophoresis and Western Blot Analysis
MDCK II cell extracts prepared by adding 200 µl of SDS-sample buffer to each well or immunocomplexes described above were subjected to SDS-PAGE in 8% gels. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore), which were then soaked in 5% skimmed milk. Blotted membranes were processed for immunoreactions as described previously (
Yeast Two-Hybrid Analysis
Various deletion mutants of PAR-6 and aPKC, subcloned into pAS2-1C and pGAD424 (CLONTECH Laboratories, Inc.), respectively, were simultaneously transformed into the yeast strain HF7C (CLONTECH Laboratories, Inc.) by a standard method (
Online Supplemental Material
Supplemental Fig. S1: comparison of human PAR-6 with other PAR-6 homologues. Supplemental Fig. S2: Northern blot analysis of PAR-6 mRNA in human tissues. Online supplemental materials can be found at http://www.jcb.org/cgi/content/full/152/6/1183/DC1.
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Results |
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Functional Depletion of aPKC Induces the Mislocalization of ASIP/PAR-3 as Well as ZO-1
It has been shown that a kinase-deficient mutant of aPKC (aPKC
kn), in which a conserved lysine residue in the ATP-binding site is replaced by glutamate, exerts dominant-negative effects on aPKC-dependent TRE (TPA-response element) activation in HepG2 cells, as well as on insulin-stimulated glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes (
mutant in MDCK II cells using an adenovirus-mediated gene transfer approach, and analyzed its effects on the junctional localization of ASIP/PAR-3. As shown in Fig 1, a and c, adenoviral infection of confluent monolayers of MDCK II cells resulted in the heterogenous expression of aPKC
kn and aPKC
wt ranging from an extreme high level giving saturated immunofluorescent signals to the lowest level, with signals slightly higher than background. Infection efficiencies in the present conditions estimated from the immunostaining results of aPKC
wt and aPKC
kn were both
40% (Fig 1, a and c, and Table 1). SDS-PAGE analysis revealed an average fivefold overexpression of both proteins compared with endogenous aPKC
(data not shown). Overexpressed aPKC
kn as well as aPKC
wt distributed diffusely in the cytosol and did not show dominant junctional localization as reported for the endogenous protein (
antibody used here could not detect endogenous aPKC
clearly; see Fig 1 c, top left). This appears to be consistent with the fact that substantial amounts of endogenous aPKC also distribute in the cytosol (
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When aPKCkn was expressed in MDCK II cells maintained under normal calcium conditions, the junctional localization of ASIP/PAR-3 was not impaired even in cells showing extremely high levels of aPKC
kn expression (Fig 1 a, -CS). However, if cells expressing aPKC
kn were subjected to calcium switch to induce a disruptionregeneration process of cellcell adhesion, then the junctional staining of ASIP/PAR-3 was significantly affected (Fig 1 a, +CS). Despite the apparently normal restoration of a confluent monolayer as indicated by phase contrast observations (data not shown), ASIP/PAR-3 did not develop a complete junctional distribution over the entire cell circumference, even 6 h after calcium switch (see Fig 4 a), which is achieved <2 h after calcium switch in control cells. This fragmentary staining of the cellcell boundaries remains unchanged until 20 h after calcium switch (Fig 1 a, +CS), suggesting that the junctional localization of ASIP/PAR-3 is significantly inhibited by aPKC
kn. Interestingly, the junctional localization of ZO-1, a TJ marker, in cells was similarly disturbed in a calcium switch-dependent manner, suggesting that TJ formation itself is severely affected (Fig 1 a, right). Table 1 summarizes the statistical results of ZO-1 mislocalization observed 20 h after calcium switch. While most (>99%) of the cells infected with adenovirus vectors carrying LacZ as well as aPKC
wt display normal ZO-1 staining (Fig 1 c, and Table 1), >60% infected with an aPKC
kn-encoding adenovirus vector exhibit partial or complete mislocalization of ZO-1. The severity of ZO-1 mislocalization correlates well with the level of aPKC
kn expression (Table 1, and Fig 1 b); for example, >50% of cells with a high fluorescent signal (+++) exhibit a complete disappearance of ZO-1, whereas the rate is <11% in cells with a low fluorescent signal (+). On the other hand, the data also indicate that >40% of cells apparently negative for an aPKC
fluorescent signal (-/+) also show fragmentary ZO-1 distribution. Considering the low sensitivity of the antiaPKC
antibody used, and the proportional correlation between fluorescent signal intensity and phenotype severity (Fig 1 b), we infer that the actual infection efficiency is higher than estimated and some cells negative for a fluorescent signal also express levels of aPKC
kn that are undetectable but still sufficient to affect ZO-1 distribution.
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Fig 2 a demonstrates that coinfection with increasing amounts of aPKCwt and aPKC
kn gradually restores the network-like staining of ZO-1 in a dose-dependent manner. In addition, Fig 2 b shows that the same phenotypes were observed after the introduction of aPKC
kn, but not nPKC
kn. Because of the significant sequence similarity between aPKC
and aPKC
, we cannot exclude the possibility that aPKC
kn exerts its effect not only on endogenous aPKC
, but also on aPKC
, and vice versa. In fact, we observed that coinfection with aPKC
wt can rescue the defective phenotype of TJ formation caused by aPKC
kn (data not shown). Therefore, we conclude that the observed mislocalizations of ASIP/PAR-3 and ZO-1 are caused specifically by the dominant negative effects of the aPKC kinase-deficient mutants on endogenous aPKC (
and/or
) activity.
The calcium switch dependence of the effects of aPKCkn suggests that this dominant-negative mutant is effective only when the cells develop junctional structures to establish epithelial cell polarity. In fact, we further observed the similar effects of aPKC
kn when cells form de novo cellcell contacts and develop TJ under normal growth conditions. As shown in Fig 3, where cells were sparsely reseeded immediately after viral infection and cultured for 40 h before immunofluorescent analysis, cells expressing LacZ or aPKC
wt show a normal appearance while aPKC
kn-expressing cells exhibit a flattened shape with prominent lamellipodia (Fig 3 c) with many cellcell boundaries negative for ASIP/PAR-3 and ZO-1 staining (Fig 3, a and b). However, it should be noted that aPKC
kn-expressing cells still form islands and remain close to each other through cellcell adhesions even in the absence of ASIP/PAR-3 or ZO-1 staining at the cellcell boundary (Fig 3 c). These results suggest that the effect of aPKC
kn on TJ formation is not the result of the complete disruption of cellcell adhesions.
Aberrant Localization of TJ Components and F-actin Organization in aPKCkn-expressing MDCK II Cells
Fig 4 shows the results of a detailed immunofluorescent analysis of the confluent monolayer of aPKCkn-expressing cells performed 6 h after calcium switch. Double staining for ZO-1 and ASIP/PAR-3 revealed that these peripheral TJ proteins with three PDZ domains colocalize completely, as indicated by the disrupted junctional staining (Fig 4 a). Further, the membrane accumulation of the major membrane proteins that comprise TJ strands, occludin and claudin-1 (
kn overexpression on these membrane proteins seems to be more severe than the effects on ZO-1 or ASIP/PAR-3 (Fig 4 a, arrowheads). Recent results (
kn-expressing cells show defects in the development of the TJ structure itself. The basolateral localization of E-cadherin appears to be less affected, but the fluorescence intensity is rather weaker at contact regions lacking ZO-1 staining (Fig 4 b). Rhodamine-phalloidin staining revealed that aPKC
kn-expressing cells show defects in the formation of developed cortical F-actin bundles surrounding the epithelial cell circumference (Fig 4 b). Instead, these cells show remarkable retention of the stress fiber-like structures of F-actin. Furthermore, some cells show characteristic large F-actin aggregates whose location corresponds completely to aberrant small ring structures containing all the TJ components examined here (Fig 4, a and b, arrows). These results indicate that aPKC
kn interferes with the development of the epithelia-specific junctional structures such as belt-like adherent junction (AJ) and TJ, which is mediated by cooperative interactions between F-actin and junctional components. Since the aberrant small ring structures of TJ are observed only in cells showing low expression of aPKC
kn (Table 1), these structures might be produced when the suppression of endogenous aPKC activity by aPKC
kn is not complete.
Fig 4c and Fig d, show the results of Western blotting analysis to examine the amounts of several junctional proteins in adenovirally infected MDCK II cells harvested 6 h after calcium switch. Prolonged incubation (20 h) of aPKCkn-expressing cells after calcium switch results in a decrease in the amounts of ASIP/PAR-3 and ZO-1, probably because they fail to be stabilized by being recruited into the junctional complexes (data not shown). However, at least in the early phase of cell polarization when aPKC
kn-expressing cells clearly exhibit defects in TJ formation (Fig 4 a), the amounts of the junctional proteins examined do not decrease substantially (Fig 4c and Fig d). These results suggest that the effects of the overexpression of aPKC
kn do not result from an enhanced degradation of junctional proteins.
Disrupted TJ Barrier Functions in aPKCkn-expressing MDCKII Cells
The defects in TJ formation in aPKCkn-expressing cells was further demonstrated functionally by measuring TER to passive ion flow across a cell monolayer grown on permeable support (Fig 5 a). Consistent with the immunostaining results shown in Fig 1 a, none of the ectopically expressed proteins, including LacZ, nPKC
kn, or aPKC
wt, as well as aPKC
kn, affected TER if the experiments were arranged so as to induce the expression of the ectopic proteins after the completion of TJ biogenesis (Fig 5 a, top, 045 h). This also suggests that the overexpression of these proteins, especially aPKC
kn, does not produce any artificial cytotoxic effects on TJ barrier function. On the other hand, if the cells were subsequently subjected to calcium switch (2-h incubation in LC medium followed by switching to NC medium), only cells expressing aPKC
kn showed a large retardation in TER development (Fig 5 a, top, 4570 h). If the preincubation in low calcium medium was prolonged to 20 h to ensure the dissociation of cellcell attachments before calcium switch, the effect of aPKC
kn on TER development was more significant: similar to the immunostaining experiments shown in Fig 1, the expression of ectopic proteins was induced in the cells cultured in LC medium, and calcium switch was applied 20 h after virus infection (Fig 5 a, bottom). In this case, TER development of aPKC
kn-expressing cells was substantially suppressed until 48 h after calcium switch compared with LacZ- or aPKC
wt-expressing control cells, suggesting that the development of functional TJ is strongly suppressed in aPKC
kn-expressing cells.
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The impairment of TJ barrier function in aPKCkn-expressing cells was also demonstrated by measuring the paracellular diffusion of a nonionic solute (Fig 5 b). In these experiments, the cells were prepared as in Fig 1, and the diffusion of FITC-dextran across MDCK II monolayers was measured 48 h after calcium switch to ensure the development of epithelial cell polarity. As shown in Fig 5 b, cells expressing aPKC
kn, but not LacZ or aPKC
wt, still showed fivefold enhanced diffusion of FITC-conjugated dextran 40 K (average 40 kD) over a 3-h period at 37°C. On the other hand, the diffusion of dextran 500 (average 500 kD) showed only a 1.1-fold enhancement, confirming that the enhancement of dextran 40 diffusion is not due to a cytotoxic effect of aPKC
kn expression. These results indicate that aPKC
activity is required for the development of TJ, which is essential for the barrier function of epithelial cells.
Disrupted Cell Surface Polarity in aPKCkn-expressing MDCK II Cells
TJ have been suggested to contribute to the establishment of epithelial cell surface polarity by acting as fences for the diffusion of lipids in the outer leaflet of the plasma membrane between the apical and basolateral membrane domains (kn-expressing cells using confocal microscopy. In Fig 6 a, the apical membranes were labeled with BODIPY-sphingomyelin for 10 min at 4°C, and left for an additional 60 min on ice. Confocal microscopic analysis of the xz sections of these cells demonstrated that only aPKC
kn-expressing cells show markedly enhanced labeling of the lateral membrane after a 60-min chase, suggesting a reduced diffusion fence between the apical and basolateral membranes.
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On the other hand, it has been demonstrated that several membrane proteins, such as a basolateral membrane marker, Na+, K+-ATPase (NKA), as well as an apical membrane marker, gp135, retain their polarized localizations even in the absence of TJ via their interaction with domain-specific membrane-skeletal structures (kn affects epithelial cell surface polarity not only by inhibiting TJ biogenesis, but also by interfering with these additional mechanisms for cell polarization, we examined the distribution of endogenous NKA and gp135 in aPKC
kn-expressing cells. NKA is restricted to the lateral membrane in control cells expressing LacZ, whereas its polarized localization in cells expressing aPKC
wt is slightly disturbed to produce a leaky distribution in the apical membrane (Fig 6 b). Significantly, cells that express aPKC
kn highly show an almost even distribution of NKA in both the apical and basolateral membrane domains (Fig 6 b, arrowheads), suggesting defects in the machinery required for the maintenance of the polarized distribution of NKA. The apical localization of gp135 was also affected by aPKC
kn overexpression (Fig 6 c): cells expressing aPKC
kn tend to show a reduced level of gp135 on the apical membrane. Instead, increased cytosolic signals are often detected in these cells.
aPKC and ASIP/PAR-3 Form a Protein Complex Containing a Mammalian Homologue of C. elegans PAR-6 in Polarized Epithelial Cells
Together with the evolutionarily conserved interaction between aPKC and ASIP/PAR-3, the above finding that aPKC activity is required for the establishment of epithelial cell polarity strongly supports a notion that the cell polarization machinery composed of aPKC and PAR proteins found in C. elegans may also be conserved in mammalian epithelial cells. Therefore, we next determined to identify and characterize mammalian homologue of C. elegans PAR-6, which interdependently works with PKC-3 and PAR-3 in C. elegans one-cell embryo (
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Importantly, Western blot analysis further revealed that aPCK as well as ASIP/PAR-3 are specifically coimmunoprecipitated with PAR-6 (Fig 7 b), raising a possibility that PAR-6 interacts with aPKC-ASIP/PAR-3 complex. Consistently, yeast two-hybrid analyses shown in Fig 7 c confirmed that the NH2-terminal 125 amino acid residues of PAR-6 interact with aPKC
. Since this NH2-terminal region includes two conserved regions, CR1 and CR2, we tried to narrow the regions required for their interaction further. However, neither amino acids 164, including CR1 but not CR2, nor 34346, including CR2 but not CR1, interacts with aPKC
(Fig 7 c), suggesting that the NH2-terminal 125 amino acid sequence including CR1 and CR2 forms a structural domain (termed aPKCBD) required for proteinprotein interaction. Another series of analyses also revealed that the NH2-terminal residues 22113 of aPKC
are sufficient for the interaction with PAR-6 (Fig 7 d). Again, NH2- or COOH-terminal deletion of this region results in the disappearance of the interaction (Fig 7 d), suggesting that this region, corresponding to the D1 region (diversed region 1) of aPKC
, forms another structural domain for proteinprotein interaction.
aPKC Mediates the Interaction between ASIP/PAR-3 and PAR-6 as a Linker
Previously, we demonstrated that aPKC directly binds to ASIP/PAR-3 through its kinase domain. Therefore, the above results raise the possibility that aPKC
binds both ASIP/PAR-3 and PAR-6 simultaneously and mediates the formation of an aPKC-ASIP/PAR-3PAR-6 ternary complex. To examine this possibility, we next performed a series of immunoprecipitation experiments in COS1 cells (Fig 8). As shown in Fig 8 a, when Flag-tagged PAR-6 was overexpressed and immunoprecipitated with an antiFlag antibody, coexpressed T7-tagged ASIP was coprecipitated together with endogenous aPKC
. Significantly, when T7-tagged ASIP
30, which corresponds to an isoform lacking aPKC-binding region was coexpressed with Flag-tagged PAR-6 instead of wild-type ASIP, endogenous aPKC
but not this ASIP isoform was coimmunoprecipitated with PAR-6, suggesting that ASIP indirectly associates with PAR-6 by way of aPKC
. In fact, a T7-tagged PAR-6 mutant lacking NH2-terminal aPKC
-binding region (
aPKCBD) does not show interactions not only with endogenous aPKC
but also with ASIP (Fig 8 b), although the other PAR-6 mutant (
CRIB/PDZ) lacking the CRIB and PDZ domains but retaining aPKC
-binding region can interact with both proteins. Furthermore, Fig 8 c shows that overexpression of aPKC
enhances the coprecipitation of T7-tagged ASIP with Flag-tagged PAR-6. On the other hand, overexpression of aPKC
N47 that cannot bind to PAR-6 does not show such enhancement, but rather suppresses the coprecipitation of ASIP/PAR-3 with PAR-6. This can be explained as a dominant negative effect of this aPKC
mutant on ASIP, inhibiting the indirect association of ASIP with PAR-6 by way of endogenous aPKC
. Taken together, we conclude that aPKC serves as a linker molecule between PAR-6 and ASIP, and mediates the formation of a ternary protein complex composed of aPKC
, ASIP/PAR-3, and PAR-6 (Fig 8 d).
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Colocalization of PAR-6 as Well as aPKC and ASIP/PAR-3 to the Epithelial Junctional Complex with ZO-1
To evaluate the physiological significance of the physical interaction between aPKC, ASIP/PAR-3, and PAR-6 in epithelial cells, we next examined the intracellular localization of PAR-6 in MDCK cells. As shown in Fig 9 a, the antiPAR-6 antibody, GW2AP, clearly stains the cellcell boundary of confluent MDCK II cells. Since the similar result was obtained with the other independent antibody, GC2AP (Fig 9 b), we concluded that these junctional stainings represent the genuine localization of endogenous PAR-6 in MDCK II cells. Closer inspection of the localization of endogenous PAR-6 by confocal z-sectioning revealed PAR-6 staining at the most apical end of the cellcell contact region with ZO-1 (Fig 9 c). Since, as previously suggested (
and ASIP/PAR-3 also localize to the corresponding region with ZO-1 (Fig 9 c), these results strongly suggest that PAR-6 colocalizes with aPKC
and ASIP/PAR-3 to the apical junctional complex of epithelial cells. To clarify further the localization of PAR-6 in epithelial cells, we next stained mouse intestinal epithelia, a typical tissue containing polarized epithelial cells. Similar to aPKC
and ASIP/PAR-3, PAR-6 also localizes to the most apical end of the junctional complex with ZO-1 (Fig 9 d). In addition, like ASIP/PAR-3, the junctional localization of PAR-6 is severely disturbed in aPKC
kn-expressing cells, showing complete colocalization with ZO-1 (Fig 9 e). Taken together with the physical interactions among these three proteins, the above results provide evidence supporting the idea that PAR-6, aPKC
, and ASIP/PAR-3 asymmetrically localize in the apical junctional complex in polarized epithelial cells as a ternary protein complex.
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Discussion |
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aPKC Kinase Activity Is Required for the Establishment of the Junctional Structures and Epithelial Cell Polarity
Pharmacological studies using PKC activators or inhibitors have indicated the involvement of PKC in TJ biogenesis (, nPKC
,
, and aPKC
,
(Shimizu, M., unpublished results). Therefore, the results obtained so far using these agents should reflect the sum of their effects on several PKCs in MDCK cells, even if it is assumed that their targets are restricted to PKC.
In this paper, we used dominant-negative mutants of aPKC to demonstrate that aPKC or
is required for the assembly of TJs. This conclusion is based on the following results. (a) Cells expressing dominantnegative point mutants of aPKC
or
(aPKCkn), but not the wild types, show an inhibited junctional localization of ZO-1 even 20 h after calcium switch. (b) The effects are specific to dominantnegative mutants of aPKCs and are not observed when such mutants of nPKC
and cPKC
(data not shown) are used. (c) The effects of aPKC
kn are rescued by the cointroduction of aPKC
wt in a dose-dependent manner, suggesting that the effects of aPKC
kn are based on its dominant suppression of endogenous aPKC activity. (d) The junctional localizations of not only ZO-1, but all TJ components tested, are similarly affected by aPKC
kn, indicating that the development of the TJ structure itself is blocked in aPKC
kn-expressing cells. (e) Overexpression of aPKC
kn impairs the restoration of the barrier function of MDCK cells after calcium switch, as measured by TER as well as by the paracellular diffusion of dextran. It should be noted that these effects of aPKC
kn are observed only when the ectopic protein is present during the de novo formation of TJ structures. Generally, the dominantnegative effects of kinase-deficient point mutants are thought to arise from competition with the endogenous kinase for interaction with other molecules required for their proper function. The target molecules can be (a) specific substrates of the kinase, (b) activators, or (c) anchoring partners that determine where the kinase should be activated physiologically and efficiently. At this stage, we cannot conclude in which way the aPKCkn mutants we used work within the cells. However, the present results clearly indicate that regulated aPKC kinase activity is required during the dynamic reconstructing of the junctional structures and epithelial cell polarity.
Together with previous pharmacological studies, the features of the effect of aPKCkn reported here agree with those shown for the effects of the PKC-specific inhibitors, H7 and calphostin ( measured in vitro (90 and 1 µM for
-peptide, respectively;
, and this may be why the effects of these inhibitors on ZO-1 distribution are rather weak compared with the effect of aPKCkn (
Molecular Mechanism by which aPKC Regulates the Junctional Structure Formation
The molecular basis of the involvement of aPKC in TJ biogenesis is not yet clear. Considering the fact that endogenous aPKC as well as ASIP/PAR-3, its specific binding protein with three PDZ domains, localize at TJ in polarized epithelial cells (
Of course, the possibility remains that the region where aPKC mainly functions is not the cellcell junction, but the cytosol. In fact, substantial amounts of endogenous aPKC are detected in the cytosol, although some is concentrated at the tip of the lateral membrane of epithelial cells (kn-expressing MDCK cells. On the other hand, we cannot exclude the possibility that the primary target of aPKC may not be TJ biogenesis itself, but upstream events triggered by initial cellcell attachment. In fact, we observed that aPKC
kn-expressing cells also show decreased E-cadherin accumulation to cellcell borders, and defects in the formation of developed F-actin peripheral bundles remaining stress fiber-like structures instead (Fig 4 b). Therefore, aPKC might regulate the reorganization of the submembranous and/or intracellular cytoskeleton, which is closely linked with the asymmetric development of epithelia-specific junctional structures including TJ, belt-like AJ, and desmosome. This idea may be consistent with the recent reports that aPKC
and
participate in the Ras-mediated reorganization of the F-actin cytoskeleton in NIH3T3 cells, or that aPKC
and
work downstream of Cdc42 to disrupt stress fibers in NIH3T3 cells (
kn-expressing MDCK cells can also be explained by this idea. To address these issues, further studies are needed to identify the physiological substrates of this kinase, whose phosphorylated state and activity change dynamically during the process of epithelial cell polarization.
aPKCPAR System in Mammalian Epithelial Cells
Here, we present data showing that the overexpression of a dominantnegative mutant of aPKC disturbs epithelial cell surface polarity along the apico-basal axis (Fig 6). These results correspond well with our previous finding that the C. elegans one-cell embryo lacking aPKC (PKC-3) shows defects in the establishment of the anteriorposterior polarity (kn. Although we have not succeeded in directly demonstrating the involvement of ASIP/PAR-3 and PAR-6 in aPKC function in the present paper, the above coincidences increasingly support the hypothesis that the aPKC-PAR system acts as evolutionarily conserved cell polarization machinery not only in the C. elegans early embryo but also in mammalian epithelial cells. Importantly, a Drosophila homologue of aPKC was recently suggested to associate with Bazooka (Drosophila homologue of PAR-3) and control the polarity of epithelia as well as neuroblasts (
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Footnotes |
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The online version of this article contains supplemental material.
Drs. Suzuki and Yamanaka contributed equally to this work and should be considered co-first authors.
Dr. Izumi's present address is Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.
1 Abbreviations used in this paper: AJ, adherent junction; aPKC, atypical PKC; ASIP, aPKC-specific interacting protein; LC, low Ca2+; NC, normal Ca2+; NKA, Na+, K+-ATPase; TER, transepithelial electrical resistance; TJ, tight junction.
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Acknowledgements |
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We thank S. Tsukita, M. Itoh, and M. Furuse for providing us with antiZO-1 and anticlaudin-1 antibodies and for helpful discussion, J.D. Nelson for providing antiNa+,K+-ATPase and antigp135 antibodies, and Y. Oka and H. Katagiri for providing adenovirus vectors encoding wild-type and kinase-deficient mutants of PKC and
.
This work was supported by grants from the Japan Society for the Promotion of Science (S. Ohno) and the Ministry of Education, Science, Sports and Culture of Japan (S. Ohno and A. Suzuki).
Submitted: 15 June 2000
Revised: 22 January 2001
Accepted: 24 January 2001
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