aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity.
BACKGROUND
aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC.


RESULTS
We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development.


CONCLUSIONS
These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.
Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan. abell@med.yokohama-cu.ac.jp
Atsushi
A
Suzuki
Maki
M
Hirata
Katsusi
K
Kamimura
Rika
R
Maniwa
Tomoyuki
T
Yamanaka
Keiko
K
Mizuno
Masaru
M
Kishikawa
Hiroko
H
Hirose
Yoshiko
Y
Amano
Natsuko
N
Izumi
Yoshihiro
Y
Miwa
Shigeo
S
Ohno
D000818:Animals; D002458:Cell Fractionation; D016764:Cell Polarity / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004847:Epithelial Cells / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D004854:Herpesvirus 4, Human; D006801:Humans; D009154:Mutation / Q000235:genetics; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D019108:Tight Junctions / Q000235:genetics / Q000502:physiology; D014162:Transfection
D018076:DNA, Complementary; C094670:MARK2 protein, human; D017346:Protein Serine-Threonine Kinases; C115015:PKC-3 protein; D011493:Protein Kinase C