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 aPKCλ (aPKCλkn), 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 (Akimoto et al. 1996
; Kotani et al. 1998
). As a first step to evaluate the role of aPKC in epithelial cell polarity, we overexpressed this aPKCλ 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 , a and c, adenoviral infection of confluent monolayers of MDCK II cells resulted in the heterogenous expression of aPKCλkn and aPKCλwt 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 aPKCλwt and aPKCλkn were both ~40% (, a and c, and ). SDS-PAGE analysis revealed an average fivefold overexpression of both proteins compared with endogenous aPKCλ (data not shown). Overexpressed aPKCλkn as well as aPKCλwt distributed diffusely in the cytosol and did not show dominant junctional localization as reported for the endogenous protein (Dodane and Kachar 1996
; Izumi et al. 1998
), suggesting that few membrane-anchoring sites for ectopic aPKC remain (note that the anti–aPKCλ antibody used here could not detect endogenous aPKCλ clearly; see c, top left). This appears to be consistent with the fact that substantial amounts of endogenous aPKC also distribute in the cytosol (Izumi et al. 1998
Figure 1 Overexpression of the kinase-deficient mutant of aPKCλ (aPKCkn) in confluent MDCK monolayers disrupts the junctional localization of ASIP and ZO-1 only when the cells are subjected to calcium switch. (a) Confluent MDCK cells seeded on cover slips (more ...)
Correlation between the Expression Level of Ectopic Proteins and the Severity of ZO-1 Mislocalization
When aPKCλkn 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 aPKCλkn expression ( a, −CS). However, if cells expressing aPKCλkn were subjected to calcium switch to induce a disruption–regeneration process of cell–cell adhesion, then the junctional staining of ASIP/PAR-3 was significantly affected ( 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 a), which is achieved <2 h after calcium switch in control cells. This fragmentary staining of the cell–cell boundaries remains unchanged until 20 h after calcium switch ( a, +CS), suggesting that the junctional localization of ASIP/PAR-3 is significantly inhibited by aPKCλkn. 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 ( a, right). 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 aPKCλwt display normal ZO-1 staining ( c, and ), >60% infected with an aPKCλkn-encoding adenovirus vector exhibit partial or complete mislocalization of ZO-1. The severity of ZO-1 mislocalization correlates well with the level of aPKCλkn expression (, and 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 aPKCλ fluorescent signal (−/+) also show fragmentary ZO-1 distribution. Considering the low sensitivity of the anti–aPKCλ antibody used, and the proportional correlation between fluorescent signal intensity and phenotype severity ( b), we infer that the actual infection efficiency is higher than estimated and some cells negative for a fluorescent signal also express levels of aPKCλkn that are undetectable but still sufficient to affect ZO-1 distribution.
Figure 4 Distribution of various junctional components and F-actin in aPKCλkn-expressing cells. Confluent monolayers of aPKCλkn-expressing MDCK II cells were subjected to calcium switch. 6 h later, ZO-1 distribution was compared with that of other (more ...)
a demonstrates that coinfection with increasing amounts of aPKCλwt and aPKCλkn gradually restores the network-like staining of ZO-1 in a dose-dependent manner. In addition, b shows that the same phenotypes were observed after the introduction of aPKCζkn, but not nPKCδkn. Because of the significant sequence similarity between aPKCλ and aPKCζ, we cannot exclude the possibility that aPKCλkn exerts its effect not only on endogenous aPKCλ, but also on aPKCζ, and vice versa. In fact, we observed that coinfection with aPKCζwt can rescue the defective phenotype of TJ formation caused by aPKCλkn (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 (λ and/or ζ) activity.
Figure 2 aPKCkn specifically disturbs the junctional localization of ZO-1 by its dominant negative effect on endogenous aPKC activity. (a) MDCK II cells coinfected with adenovirus vectors encoding aPKCλwt and aPKCλkn were subjected to calcium switch (more ...)
The calcium switch dependence of the effects of aPKCλkn 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 aPKCλkn when cells form de novo cell–cell contacts and develop TJ under normal growth conditions. As shown in , where cells were sparsely reseeded immediately after viral infection and cultured for 40 h before immunofluorescent analysis, cells expressing LacZ or aPKCλwt show a normal appearance while aPKCλkn-expressing cells exhibit a flattened shape with prominent lamellipodia ( c) with many cell–cell boundaries negative for ASIP/PAR-3 and ZO-1 staining (, a and b). However, it should be noted that aPKCλkn-expressing cells still form islands and remain close to each other through cell–cell adhesions even in the absence of ASIP/PAR-3 or ZO-1 staining at the cell–cell boundary ( c). These results suggest that the effect of aPKCλkn on TJ formation is not the result of the complete disruption of cell–cell adhesions.
Figure 3 The overexpression of aPKCkn also disrupts the junctional localization of ASIP and ZO-1 in MDCK II cells under normal growth conditions. MDCK II cells infected with the adenovirus vectors indicated were reseeded sparsely (1.7 × 104 cells/cm2) (more ...)
Aberrant Localization of TJ Components and F-actin Organization in aPKCλkn-expressing MDCK II Cells
shows the results of a detailed immunofluorescent analysis of the confluent monolayer of aPKCλkn-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 ( a). Further, the membrane accumulation of the major membrane proteins that comprise TJ strands, occludin and claudin-1 (Furuse et al. 1993
, Furuse et al. 1998b
), is also inhibited. Since immature junctional structures that show positive staining for ZO-1 but not for occludin or claudin-1 are observed, the effects of aPKCλkn overexpression on these membrane proteins seems to be more severe than the effects on ZO-1 or ASIP/PAR-3 ( a, arrowheads). Recent results (Gow et al. 1999
) have shown that claudin-11–deficient mice lack TJ strands in the myelin sheaths of oligodendrocytes and Sertoli cells in which this claudin subtype is primarily expressed (Morita et al. 1999
). Thus, the above results suggest that aPKC λkn-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 ( b). Rhodamine-phalloidin staining revealed that aPKCλkn-expressing cells show defects in the formation of developed cortical F-actin bundles surrounding the epithelial cell circumference ( 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 (, a and b, arrows). These results indicate that aPKCλkn 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 aPKCλkn (), these structures might be produced when the suppression of endogenous aPKC activity by aPKCλkn is not complete.
and , 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 aPKCλkn-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 aPKCλkn-expressing cells clearly exhibit defects in TJ formation ( a), the amounts of the junctional proteins examined do not decrease substantially ( and ). These results suggest that the effects of the overexpression of aPKCλkn do not result from an enhanced degradation of junctional proteins.
Disrupted TJ Barrier Functions in aPKCλkn-expressing MDCKII Cells
The defects in TJ formation in aPKCλkn-expressing cells was further demonstrated functionally by measuring TER to passive ion flow across a cell monolayer grown on permeable support ( a). Consistent with the immunostaining results shown in a, none of the ectopically expressed proteins, including LacZ, nPKCδkn, or aPKCλwt, as well as aPKCλkn, affected TER if the experiments were arranged so as to induce the expression of the ectopic proteins after the completion of TJ biogenesis ( a, top, 0–45 h). This also suggests that the overexpression of these proteins, especially aPKCλkn, 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 aPKCλkn showed a large retardation in TER development ( a, top, 45–70 h). If the preincubation in low calcium medium was prolonged to 20 h to ensure the dissociation of cell–cell attachments before calcium switch, the effect of aPKCλkn on TER development was more significant: similar to the immunostaining experiments shown in , the expression of ectopic proteins was induced in the cells cultured in LC medium, and calcium switch was applied 20 h after virus infection ( a, bottom). In this case, TER development of aPKCλkn-expressing cells was substantially suppressed until 48 h after calcium switch compared with LacZ- or aPKCλwt-expressing control cells, suggesting that the development of functional TJ is strongly suppressed in aPKCλkn-expressing cells.
Figure 5 Overexpression of aPKCλkn inhibits the development of TER of MDCK II cells after calcium switch. (a) Confluent MDCK II cells grown on a filter were infected with adenovirus vectors, and TER was measured. (Top) The ectopically expressed proteins (more ...)
The impairment of TJ barrier function in aPKCλkn-expressing cells was also demonstrated by measuring the paracellular diffusion of a nonionic solute ( b). In these experiments, the cells were prepared as in , 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 b, cells expressing aPKCλkn, but not LacZ or aPKCλwt, 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 aPKCλkn expression. These results indicate that aPKCλ activity is required for the development of TJ, which is essential for the barrier function of epithelial cells.
Disrupted Cell Surface Polarity in aPKCλkn-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 (van Meer and Simons 1986
). Therefore, the finding that functional depletion of aPKC blocks the development of the epithelia-specific junctional structures, including TJ, suggests the possibility that aPKCkn expression also results in the disruption of epithelial cell surface polarity. To confirm this possibility, we examined the two-dimensional diffusion of ectopically introduced fluorescent lipids in aPKCλkn-expressing cells using confocal microscopy. In 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 x–z sections of these cells demonstrated that only aPKCλkn-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.
Figure 6 Overexpression of aPKCλkn disrupts apico-basal cell surface polarity of MDCK II cells. (a) Two-dimensional diffusion of ectopically labeled fluorescent lipid from the apical to the basolateral domain. The apical surface of filter-grown MDCK II (more ...)
On the other hand, it has been demonstrated that several membrane proteins, such as a basolateral membrane marker, Na+
-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 (Ojakian and Schwimmer 1988
; McNeill et al. 1990
). Therefore, to assess the possibility that the overexpression of aPKCλkn 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 aPKCλkn-expressing cells. NKA is restricted to the lateral membrane in control cells expressing LacZ, whereas its polarized localization in cells expressing aPKCλwt is slightly disturbed to produce a leaky distribution in the apical membrane ( b). Significantly, cells that express aPKCλkn highly show an almost even distribution of NKA in both the apical and basolateral membrane domains ( 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 aPKCλkn overexpression ( c): cells expressing aPKCλkn 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 (Watts et al. 1996
; Tabuse et al. 1998
; Hung and Kemphues 1999
). Cloned human homologue of PAR-6 shows overall similarity with PAR-6 homologues in other species, sharing several stretches of conserved sequences (supplemental Fig. S1; c): (a) a sequence at the NH2
-terminal region containing two highly conserved regions, CR 1 and CR2; (b) a sequence similar to the CRIB motif (Burbelo et al. 1995
); and (c) the single PDZ domain in the COOH-terminal half that has been described previously (Hung and Kemphues 1999
). On the other hand, the COOH-terminal region after the PDZ domain is rarely conserved. Northern analysis of human tissues revealed that PAR-6 is expressed in a variety of human tissues (supplemental Fig. S2). To confirm the expression of PAR-6 in MDCK cells, we raised three kinds of anti–PAR-6 antibodies, GW2AP, GC2AP, and N12AP, which were raised against full length amino acids 126–346 and 1–125 of human PAR-6. As shown in a, immunoprecipitation analysis of MDCK cell lysate revealed the presence of a 43-kD protein reactive with GW2AP as well as N12AP, which is commonly immunoprecipitated with GW2AP and GC2AP. Taken together with the fact that this 43-kD protein comigrates with human PAR-6 (calculated to be 37.4 kD) overexpressed in COS1 cells ( a), we concluded that this 43-kD band represents endogenous PAR-6 protein in epithelial MDCK cells.
Figure 7 Identification of endogenous PAR-6 and its association with aPKCλ and ASIP in fully polarized epithelial cells. (a) Identification of endogenous PAR-6 protein in MDCK II cells. Semi-confluent MDCK cells were subjected to immunoprecipitation with (more ...)
Importantly, Western blot analysis further revealed that aPCKλ as well as ASIP/PAR-3 are specifically coimmunoprecipitated with PAR-6 ( b), raising a possibility that PAR-6 interacts with aPKC-ASIP/PAR-3 complex. Consistently, yeast two-hybrid analyses shown in c confirmed that the NH2-terminal 125 amino acid residues of PAR-6 interact with aPKCλ. 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 1–64, including CR1 but not CR2, nor 34–346, including CR2 but not CR1, interacts with aPKCλ ( c), suggesting that the NH2-terminal 125 amino acid sequence including CR1 and CR2 forms a structural domain (termed aPKCBD) required for protein–protein interaction. Another series of analyses also revealed that the NH2-terminal residues 22–113 of aPKCλ are sufficient for the interaction with PAR-6 ( d). Again, NH2- or COOH-terminal deletion of this region results in the disappearance of the interaction ( d), suggesting that this region, corresponding to the D1 region (diversed region 1) of aPKCλ, forms another structural domain for protein–protein interaction.
aPKC Mediates the Interaction between ASIP/PAR-3 and PAR-6 as a Linker
Previously, we demonstrated that aPKCλ directly binds to ASIP/PAR-3 through its kinase domain. Therefore, the above results raise the possibility that aPKCλ binds both ASIP/PAR-3 and PAR-6 simultaneously and mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex. To examine this possibility, we next performed a series of immunoprecipitation experiments in COS1 cells (). As shown in a, when Flag-tagged PAR-6 was overexpressed and immunoprecipitated with an anti–Flag antibody, coexpressed T7-tagged ASIP was coprecipitated together with endogenous aPKCλ. Significantly, when T7-tagged ASIP Δ30, which corresponds to an isoform lacking aPKC-binding region was coexpressed with Flag-tagged PAR-6 instead of wild-type ASIP, endogenous aPKCλ but not this ASIP isoform was coimmunoprecipitated with PAR-6, suggesting that ASIP indirectly associates with PAR-6 by way of aPKCλ. In fact, a T7-tagged PAR-6 mutant lacking NH2-terminal aPKCλ-binding region (ΔaPKCBD) does not show interactions not only with endogenous aPKCλ but also with ASIP ( b), although the other PAR-6 mutant (ΔCRIB/PDZ) lacking the CRIB and PDZ domains but retaining aPKCλ-binding region can interact with both proteins. Furthermore, c shows that overexpression of aPKCλ enhances the coprecipitation of T7-tagged ASIP with Flag-tagged PAR-6. On the other hand, overexpression of aPKCλ ΔN47 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 aPKCλ mutant on ASIP, inhibiting the indirect association of ASIP with PAR-6 by way of endogenous aPKCλ. 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 aPKCλ, ASIP/PAR-3, and PAR-6 ( d).
Figure 8 Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3. (a–c) COS cells were transfected with expression vectors as indicated (top). The cell lysates (Sup) were processed for immunoprecipitation (IP) with anti–Flag (a and c) (more ...)
Colocalization of PAR-6 as Well as aPKCλ and ASIP/PAR-3 to the Epithelial Junctional Complex with ZO-1
To evaluate the physiological significance of the physical interaction between aPKCλ, ASIP/PAR-3, and PAR-6 in epithelial cells, we next examined the intracellular localization of PAR-6 in MDCK cells. As shown in a, the anti–PAR-6 antibody, GW2AP, clearly stains the cell–cell boundary of confluent MDCK II cells. Since the similar result was obtained with the other independent antibody, GC2AP ( 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 cell–cell contact region with ZO-1 ( c). Since, as previously suggested (Izumi et al. 1998
), aPKCλ and ASIP/PAR-3 also localize to the corresponding region with ZO-1 ( c), these results strongly suggest that PAR-6 colocalizes with aPKCλ 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 aPKCλ and ASIP/PAR-3, PAR-6 also localizes to the most apical end of the junctional complex with ZO-1 ( d). In addition, like ASIP/PAR-3, the junctional localization of PAR-6 is severely disturbed in aPKCλkn-expressing cells, showing complete colocalization with ZO-1 ( e). Taken together with the physical interactions among these three proteins, the above results provide evidence supporting the idea that PAR-6, aPKCλ, and ASIP/PAR-3 asymmetrically localize in the apical junctional complex in polarized epithelial cells as a ternary protein complex.
Figure 9 Colocalization of PAR-6, aPKCλ, and ASIP/PAR-3 with ZO-1 at the apical end of cell–cell contact region of epithelial cells. (a and b) Immunofluorescence staining of MDCK cells with anti–PAR-6 polyclonal antibodies, GW2AP (a) and (more ...)