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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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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 were infected with adenovirus vector encoding aPKCλkn. 20 h after viral infection, the cells were subjected to calcium switch (+CS) or left untreated (−CS) (see Materials and Methods). After another 20 h, the cells were doubly labeled with anti–aPKCλ and anti–ASIP as indicated at the top (left and middle). Each photograph represents the projected view of optical sections (0.4 μm) obtained from the apical to basal membrane using a laser scanning confocal microscope (unless otherwise noted, the following data were obtained in the same way). Note that fluorescent signals for endogenous aPKCλ are not observed in the photographs because the anti–aPKCλ antibody used (λ2) is not sensitive enough to clearly detect the endogenous protein in MDCK II cells and because the exposure times were adjusted to cover the highly heterogenous level of aPKCλkn expression. (Right) Independent specimens stained by the anti–ZO-1 antibody. Bar, 25 μm. (b) Correlation between the expression level of aPKCλkn and the severity of ZO-1 mislocalization. Based on the fluorescent intensity of aPKCλ, cells were categorized as showing undetectable (−/+), low (+), medium (++), or high (+++) expression, and the ZO-1 staining pattern of the corresponding cell was estimated as complete (white), partially disappeared (hatched), or completely disappeared (black). Original statistical data are presented in Table . (c) LacZ- or aPKCλwt-expressing cells subjected to calcium switch were doubly stained as in a, with anti–aPKCλ and anti–ZO-1 antibodies. Bar, 25 μm.
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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 were infected with adenovirus vector encoding aPKCλkn. 20 h after viral infection, the cells were subjected to calcium switch (+CS) or left untreated (−CS) (see Materials and Methods). After another 20 h, the cells were doubly labeled with anti–aPKCλ and anti–ASIP as indicated at the top (left and middle). Each photograph represents the projected view of optical sections (0.4 μm) obtained from the apical to basal membrane using a laser scanning confocal microscope (unless otherwise noted, the following data were obtained in the same way). Note that fluorescent signals for endogenous aPKCλ are not observed in the photographs because the anti–aPKCλ antibody used (λ2) is not sensitive enough to clearly detect the endogenous protein in MDCK II cells and because the exposure times were adjusted to cover the highly heterogenous level of aPKCλkn expression. (Right) Independent specimens stained by the anti–ZO-1 antibody. Bar, 25 μm. (b) Correlation between the expression level of aPKCλkn and the severity of ZO-1 mislocalization. Based on the fluorescent intensity of aPKCλ, cells were categorized as showing undetectable (−/+), low (+), medium (++), or high (+++) expression, and the ZO-1 staining pattern of the corresponding cell was estimated as complete (white), partially disappeared (hatched), or completely disappeared (black). Original statistical data are presented in Table . (c) LacZ- or aPKCλwt-expressing cells subjected to calcium switch were doubly stained as in a, with anti–aPKCλ and anti–ZO-1 antibodies. Bar, 25 μm.

Mentions: 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 Fig. 1, 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% (Fig. 1, a and c, and Table ). 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 Fig. 1 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).


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)

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 were infected with adenovirus vector encoding aPKCλkn. 20 h after viral infection, the cells were subjected to calcium switch (+CS) or left untreated (−CS) (see Materials and Methods). After another 20 h, the cells were doubly labeled with anti–aPKCλ and anti–ASIP as indicated at the top (left and middle). Each photograph represents the projected view of optical sections (0.4 μm) obtained from the apical to basal membrane using a laser scanning confocal microscope (unless otherwise noted, the following data were obtained in the same way). Note that fluorescent signals for endogenous aPKCλ are not observed in the photographs because the anti–aPKCλ antibody used (λ2) is not sensitive enough to clearly detect the endogenous protein in MDCK II cells and because the exposure times were adjusted to cover the highly heterogenous level of aPKCλkn expression. (Right) Independent specimens stained by the anti–ZO-1 antibody. Bar, 25 μm. (b) Correlation between the expression level of aPKCλkn and the severity of ZO-1 mislocalization. Based on the fluorescent intensity of aPKCλ, cells were categorized as showing undetectable (−/+), low (+), medium (++), or high (+++) expression, and the ZO-1 staining pattern of the corresponding cell was estimated as complete (white), partially disappeared (hatched), or completely disappeared (black). Original statistical data are presented in Table . (c) LacZ- or aPKCλwt-expressing cells subjected to calcium switch were doubly stained as in a, with anti–aPKCλ and anti–ZO-1 antibodies. Bar, 25 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199212&req=5

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 were infected with adenovirus vector encoding aPKCλkn. 20 h after viral infection, the cells were subjected to calcium switch (+CS) or left untreated (−CS) (see Materials and Methods). After another 20 h, the cells were doubly labeled with anti–aPKCλ and anti–ASIP as indicated at the top (left and middle). Each photograph represents the projected view of optical sections (0.4 μm) obtained from the apical to basal membrane using a laser scanning confocal microscope (unless otherwise noted, the following data were obtained in the same way). Note that fluorescent signals for endogenous aPKCλ are not observed in the photographs because the anti–aPKCλ antibody used (λ2) is not sensitive enough to clearly detect the endogenous protein in MDCK II cells and because the exposure times were adjusted to cover the highly heterogenous level of aPKCλkn expression. (Right) Independent specimens stained by the anti–ZO-1 antibody. Bar, 25 μm. (b) Correlation between the expression level of aPKCλkn and the severity of ZO-1 mislocalization. Based on the fluorescent intensity of aPKCλ, cells were categorized as showing undetectable (−/+), low (+), medium (++), or high (+++) expression, and the ZO-1 staining pattern of the corresponding cell was estimated as complete (white), partially disappeared (hatched), or completely disappeared (black). Original statistical data are presented in Table . (c) LacZ- or aPKCλwt-expressing cells subjected to calcium switch were doubly stained as in a, with anti–aPKCλ and anti–ZO-1 antibodies. Bar, 25 μm.
Mentions: 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 Fig. 1, 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% (Fig. 1, a and c, and Table ). 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 Fig. 1 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).

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