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.
Sotillos
Sol
S
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas and Univerisidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Díaz-Meco
María Teresa
MT
Caminero
Eva
E
Moscat
Jorge
J
Campuzano
Sonsoles
S
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