DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila.
Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3-Par-6-aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs-Stardust (Sdt)-Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3-Par-6-aPKC and Crumbs-Sdt-Patj complexes based in the posttranslational modification of Crb by DaPKC.
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas and Univerisidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Sol
S
Sotillos
María Teresa
MT
Díaz-Meco
Eva
E
Caminero
Jorge
J
Moscat
Sonsoles
S
Campuzano
D000595:Amino Acid Sequence; D000818:Animals; D019521:Body Patterning; D016764:Cell Polarity; D018076:DNA, Complementary / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019108:Tight Junctions; D014921:Wings, Animal / Q000378:metabolism
D018076:DNA, Complementary; D029721:Drosophila Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C064360:crb protein, Drosophila; C118488:dlt protein, Drosophila; D005982:Glutathione Transferase; D011493:Protein Kinase C