Limits...
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.

Show MeSH
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 1 μg of affinity-purified anti–PAR-6 rabbit polyclonal antibodies (GW2AP, GC2AP) or control normal rabbit IgG. The resultant immunoprecipitates were subjected to Western blot analysis using the antibodies indicated in parentheses (right). GW2AP and GC2AP specifically immunoprecipitate a 43-kD protein comigrating with PAR-6 expressed in COS cells, which was recognized by N12AP as well as GW2AP. (b) Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3 in vivo. Anti–PAR-6 (GW2AP) immunoprecipitate (IP), prepared as in a, was analyzed using anti–aPKCλ monoclonal or anti–ASIP polyclonal antibody. PAR-6 immunoprecipitates specifically contain endogenous aPKCλ, full-length ASIP, and its splicing variant (bottom band). (c and d) Summary of the yeast two-hybrid assays to analyze the PAR-6–aPKCλ interaction. The interaction was examined by growth on culture plates lacking histidine. The NH2-terminal region including CR1 and 2 of PAR-6 is sufficient for the interaction with aPKCλ (c), while NH2-terminal residues 22–113 of aPKCλ are sufficient for the interaction with PAR-6 (d).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199212&req=5

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 1 μg of affinity-purified anti–PAR-6 rabbit polyclonal antibodies (GW2AP, GC2AP) or control normal rabbit IgG. The resultant immunoprecipitates were subjected to Western blot analysis using the antibodies indicated in parentheses (right). GW2AP and GC2AP specifically immunoprecipitate a 43-kD protein comigrating with PAR-6 expressed in COS cells, which was recognized by N12AP as well as GW2AP. (b) Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3 in vivo. Anti–PAR-6 (GW2AP) immunoprecipitate (IP), prepared as in a, was analyzed using anti–aPKCλ monoclonal or anti–ASIP polyclonal antibody. PAR-6 immunoprecipitates specifically contain endogenous aPKCλ, full-length ASIP, and its splicing variant (bottom band). (c and d) Summary of the yeast two-hybrid assays to analyze the PAR-6–aPKCλ interaction. The interaction was examined by growth on culture plates lacking histidine. The NH2-terminal region including CR1 and 2 of PAR-6 is sufficient for the interaction with aPKCλ (c), while NH2-terminal residues 22–113 of aPKCλ are sufficient for the interaction with PAR-6 (d).

Mentions: 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; Fig. 7 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 Fig. 7 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 (Fig. 7 a), we concluded that this 43-kD band represents endogenous PAR-6 protein in epithelial MDCK cells.


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)

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 1 μg of affinity-purified anti–PAR-6 rabbit polyclonal antibodies (GW2AP, GC2AP) or control normal rabbit IgG. The resultant immunoprecipitates were subjected to Western blot analysis using the antibodies indicated in parentheses (right). GW2AP and GC2AP specifically immunoprecipitate a 43-kD protein comigrating with PAR-6 expressed in COS cells, which was recognized by N12AP as well as GW2AP. (b) Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3 in vivo. Anti–PAR-6 (GW2AP) immunoprecipitate (IP), prepared as in a, was analyzed using anti–aPKCλ monoclonal or anti–ASIP polyclonal antibody. PAR-6 immunoprecipitates specifically contain endogenous aPKCλ, full-length ASIP, and its splicing variant (bottom band). (c and d) Summary of the yeast two-hybrid assays to analyze the PAR-6–aPKCλ interaction. The interaction was examined by growth on culture plates lacking histidine. The NH2-terminal region including CR1 and 2 of PAR-6 is sufficient for the interaction with aPKCλ (c), while NH2-terminal residues 22–113 of aPKCλ are sufficient for the interaction with PAR-6 (d).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199212&req=5

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 1 μg of affinity-purified anti–PAR-6 rabbit polyclonal antibodies (GW2AP, GC2AP) or control normal rabbit IgG. The resultant immunoprecipitates were subjected to Western blot analysis using the antibodies indicated in parentheses (right). GW2AP and GC2AP specifically immunoprecipitate a 43-kD protein comigrating with PAR-6 expressed in COS cells, which was recognized by N12AP as well as GW2AP. (b) Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3 in vivo. Anti–PAR-6 (GW2AP) immunoprecipitate (IP), prepared as in a, was analyzed using anti–aPKCλ monoclonal or anti–ASIP polyclonal antibody. PAR-6 immunoprecipitates specifically contain endogenous aPKCλ, full-length ASIP, and its splicing variant (bottom band). (c and d) Summary of the yeast two-hybrid assays to analyze the PAR-6–aPKCλ interaction. The interaction was examined by growth on culture plates lacking histidine. The NH2-terminal region including CR1 and 2 of PAR-6 is sufficient for the interaction with aPKCλ (c), while NH2-terminal residues 22–113 of aPKCλ are sufficient for the interaction with PAR-6 (d).
Mentions: 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; Fig. 7 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 Fig. 7 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 (Fig. 7 a), we concluded that this 43-kD band represents endogenous PAR-6 protein in epithelial MDCK cells.

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