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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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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) or anti–T7 (b) monoclonal antibodies, and the coimmunoprecipitated proteins were analyzed using with anti–aPKCλ (ι), anti–T7, anti–Flag, or anti–ASIP (C2-3AP) antibodies. (a) The Flag-PAR-6 immunoprecipitates contained T7-ASIPwt, but not T7-ASIP Δ30 lacking the sequence critical for the interaction with aPKC. (b) ASIPwt was not contained in PAR-6 ΔaPKCBD immunoprecipitates but in PAR-6wt or ΔCRIB/PDZ immunoprecipitates. (c) Coimmunoprecipitation of T7-ASIP with Flag-PAR-6 was enhanced by the coexpression aPKCλwt, but not aPKCλ ΔN47, which lacks the sequences in the PAR-6–binding region. (d) Schematic diagram of the PAR-6–aPKC-ASIP/PAR-3 ternary complex. PAR-6 interacts with ASIP through aPKCλ as a linker.
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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) or anti–T7 (b) monoclonal antibodies, and the coimmunoprecipitated proteins were analyzed using with anti–aPKCλ (ι), anti–T7, anti–Flag, or anti–ASIP (C2-3AP) antibodies. (a) The Flag-PAR-6 immunoprecipitates contained T7-ASIPwt, but not T7-ASIP Δ30 lacking the sequence critical for the interaction with aPKC. (b) ASIPwt was not contained in PAR-6 ΔaPKCBD immunoprecipitates but in PAR-6wt or ΔCRIB/PDZ immunoprecipitates. (c) Coimmunoprecipitation of T7-ASIP with Flag-PAR-6 was enhanced by the coexpression aPKCλwt, but not aPKCλ ΔN47, which lacks the sequences in the PAR-6–binding region. (d) Schematic diagram of the PAR-6–aPKC-ASIP/PAR-3 ternary complex. PAR-6 interacts with ASIP through aPKCλ as a linker.

Mentions: 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 (Fig. 8). As shown in Fig. 8 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 (Fig. 8 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, Fig. 8 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 (Fig. 8 d).


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)

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) or anti–T7 (b) monoclonal antibodies, and the coimmunoprecipitated proteins were analyzed using with anti–aPKCλ (ι), anti–T7, anti–Flag, or anti–ASIP (C2-3AP) antibodies. (a) The Flag-PAR-6 immunoprecipitates contained T7-ASIPwt, but not T7-ASIP Δ30 lacking the sequence critical for the interaction with aPKC. (b) ASIPwt was not contained in PAR-6 ΔaPKCBD immunoprecipitates but in PAR-6wt or ΔCRIB/PDZ immunoprecipitates. (c) Coimmunoprecipitation of T7-ASIP with Flag-PAR-6 was enhanced by the coexpression aPKCλwt, but not aPKCλ ΔN47, which lacks the sequences in the PAR-6–binding region. (d) Schematic diagram of the PAR-6–aPKC-ASIP/PAR-3 ternary complex. PAR-6 interacts with ASIP through aPKCλ as a linker.
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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) or anti–T7 (b) monoclonal antibodies, and the coimmunoprecipitated proteins were analyzed using with anti–aPKCλ (ι), anti–T7, anti–Flag, or anti–ASIP (C2-3AP) antibodies. (a) The Flag-PAR-6 immunoprecipitates contained T7-ASIPwt, but not T7-ASIP Δ30 lacking the sequence critical for the interaction with aPKC. (b) ASIPwt was not contained in PAR-6 ΔaPKCBD immunoprecipitates but in PAR-6wt or ΔCRIB/PDZ immunoprecipitates. (c) Coimmunoprecipitation of T7-ASIP with Flag-PAR-6 was enhanced by the coexpression aPKCλwt, but not aPKCλ ΔN47, which lacks the sequences in the PAR-6–binding region. (d) Schematic diagram of the PAR-6–aPKC-ASIP/PAR-3 ternary complex. PAR-6 interacts with ASIP through aPKCλ as a linker.
Mentions: 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 (Fig. 8). As shown in Fig. 8 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 (Fig. 8 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, Fig. 8 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 (Fig. 8 d).

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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