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Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.

Suzuki A, Yamanaka T, Hirose T, Manabe N, Mizuno K, Shimizu M, Akimoto K, Izumi Y, Ohnishi T, Ohno S - J. Cell Biol. (2001)

Bottom Line: 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.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-3-PAR-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
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.). 125:3607--3614]. 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. 143:95--106), 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-3-PAR-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.

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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 as described in Fig. 1 and, 20 h later, stained with anti–ZO-1 antibody. Total multiplicity of infection was normalized to 120 by mixing with a LacZ-encoding virus vector. The mixed ratios of the each vector are as follows: aPKCλkn:aPKCλwt:LacZ = 2:0:2 (top), 2:1:1 (middle), and 2:2:0 (bottom). Note that increasing amounts of coexpressed aPKCλwt increasingly rescue the phenotype caused by aPKCλkn. (b) MDCK II cells overexpressing aPKCζwt, ζkn, or δkn were similarly prepared as described in Fig. 1 and, 20 h later, doubly stained with anti–PKC antibodies corresponding to each PKC subtype (left) and anti–ZO-1 (right). Bar, 25 μm.
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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 as described in Fig. 1 and, 20 h later, stained with anti–ZO-1 antibody. Total multiplicity of infection was normalized to 120 by mixing with a LacZ-encoding virus vector. The mixed ratios of the each vector are as follows: aPKCλkn:aPKCλwt:LacZ = 2:0:2 (top), 2:1:1 (middle), and 2:2:0 (bottom). Note that increasing amounts of coexpressed aPKCλwt increasingly rescue the phenotype caused by aPKCλkn. (b) MDCK II cells overexpressing aPKCζwt, ζkn, or δkn were similarly prepared as described in Fig. 1 and, 20 h later, doubly stained with anti–PKC antibodies corresponding to each PKC subtype (left) and anti–ZO-1 (right). Bar, 25 μm.

Mentions: Fig. 2 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, Fig. 2 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.


Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.

Suzuki A, Yamanaka T, Hirose T, Manabe N, Mizuno K, Shimizu M, Akimoto K, Izumi Y, Ohnishi T, Ohno S - J. Cell Biol. (2001)

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 as described in Fig. 1 and, 20 h later, stained with anti–ZO-1 antibody. Total multiplicity of infection was normalized to 120 by mixing with a LacZ-encoding virus vector. The mixed ratios of the each vector are as follows: aPKCλkn:aPKCλwt:LacZ = 2:0:2 (top), 2:1:1 (middle), and 2:2:0 (bottom). Note that increasing amounts of coexpressed aPKCλwt increasingly rescue the phenotype caused by aPKCλkn. (b) MDCK II cells overexpressing aPKCζwt, ζkn, or δkn were similarly prepared as described in Fig. 1 and, 20 h later, doubly stained with anti–PKC antibodies corresponding to each PKC subtype (left) and anti–ZO-1 (right). Bar, 25 μm.
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Related In: Results  -  Collection

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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 as described in Fig. 1 and, 20 h later, stained with anti–ZO-1 antibody. Total multiplicity of infection was normalized to 120 by mixing with a LacZ-encoding virus vector. The mixed ratios of the each vector are as follows: aPKCλkn:aPKCλwt:LacZ = 2:0:2 (top), 2:1:1 (middle), and 2:2:0 (bottom). Note that increasing amounts of coexpressed aPKCλwt increasingly rescue the phenotype caused by aPKCλkn. (b) MDCK II cells overexpressing aPKCζwt, ζkn, or δkn were similarly prepared as described in Fig. 1 and, 20 h later, doubly stained with anti–PKC antibodies corresponding to each PKC subtype (left) and anti–ZO-1 (right). Bar, 25 μm.
Mentions: Fig. 2 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, Fig. 2 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.

Bottom Line: 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.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-3-PAR-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.

ABSTRACT
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.). 125:3607--3614]. 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. 143:95--106), 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-3-PAR-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.

Show MeSH
Related in: MedlinePlus