DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila - PubMed (original) (raw)

DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila

Sol Sotillos et al. J Cell Biol. 2004.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

The kinase activity of DaPKC is required for epithelial cell polarity. In wild-type embryos (A, D, G, J, and K), DaPKC (A), Crb (D), and Patj (G) are localized at the apicolateral cell membrane, whereas Scrib (J) and Nrt (K) mark the basolateral membrane domain. (B, E, H, and L) In embryos with maternal VP16V67-Gal4 driven overexpression of UAS-DaPKC CAAXDN (B), Crb (E), and Patj (H) fail to localize to the cell membrane, concomitant with apical mislocalization of the basolateral protein Scrib (L, asterisks). (C, F, I, and M) Overexpression of membrane targeted wild-type DaPKC with maternal VP16V67-Gal4, shown in C, causes the redistribution of Crb (F) and Patj (I) to the whole cell contour, and interferes with the localization of Nrt (M). B, E, C, and F show the localization of DaPKC (purple) or Crb (green) in the same embryo. Bars, 10 μm. A–C, E, F, H, I, and M, stage 13 embryos; D, G, J–L, stage 11 embryos. Embryonic stages are according to Campos-Ortega and Hartenstein (1985). Apical is up in all confocal images.

Figure 2.

Figure 2.

Physical interaction between the apical complexes. (A) Drosophila embryonic extracts were incubated with GST or with the indicated GST-fusion proteins (GST-Crb wild-type intracellular domain, GST-Crbi, and GST-Patj) bound to a glutathione matrix. Immunoblotting of bound proteins, resolved by SDS-PAGE, with anti-aPKC antibody, indicates the interaction of a protein complex containing DaPKC with Crbi and Patj. First lane (Lysate) shows a 13% of the extract used in the assay. (B) Direct binding of DaPKC to Crbi and Patj. His-tagged DaPKC was bound to Ni-NTA-agarose and incubated with purified GST, GST-Crbi, and GST-Patj. Bound proteins were revealed by immunoblotting with anti-GST antibody. Bottom panel is a loading control. Half the amount of His-tagged DaPKC used in the experiment was run in a parallel gel and revealed with anti-Xpress epitope antibody. (C) Pull-down experiments of embryonic extracts were performed as in A with GST or with the indicated GST-fusion proteins: GST-Crbi, GST-CrbiT6AT9AS11AS13A, GST-CrbiT6DT9D, and GST-CrbiΔERLI. Bound proteins were resolved by SDS-PAGE and immunoblotted with anti-aPKC and anti-Patj antibodies. First lane (Lysate) shows a 10% of the extract used in the assay. Molecular weight markers are shown on the left.

Figure 3.

Figure 3.

Crb is a phosphorylation target of DaPKC. (A) In vitro kinase assay. The indicated GST-fusion proteins were subjected to aPKCζ phosphorylation in vitro and resolved by SDS-PAGE followed by autoradiography. Arrow points to phosphorylated Crbi. Asterisk marks autophosphorylated aPKC. Patj (position indicated by arrowhead) is not phosphorylated. Note that the presence of Patj largely prevents Crbi phosphorylation. Position of molecular weight markers is indicated on the left. Immunoblotting with anti-aPKC is shown in the bottom left panel. (Right) Staining with Coomassie blue of the total protein loaded. (B) Alignment of the amino acid sequences of the cytoplasmic domains of Drosophila (DmCrbi), mouse Crb3 (MmCrbi3), human Crb3 (HCrbi3), and C. elegans Crb1 (CeCrbi1). The conserved Thr and Ser residues (T6, T9, S11, and S13) are indicated with asterisks. All of these amino acids were mutated to A in the CrbiT6AT9AS11AS13A protein. T6 and T9 were mutated to D in DmCrbiT6DT9D and to A in DmCrbiT6AT9A. (C) Mutagenesis analysis of the phosphorylation sites of Crbi. aPKC-dependent in vitro phosphorylation was performed with the indicated fusion proteins. aPKC is unable to phosphorylate the mutated Crbi devoid of its four Ser/Thr residues (top left, CrbiT6AT9AS11AS13A). Phosphorylation is restored only after reversion in the mutated Crbi protein of Ala6 or Ala9 to Thr (top middle, named ABT6 and ABT9) Mutated CrbiT6AT9A is not phosphorylated by aPKC (top right). Total proteins, stained with Coomassie blue, are shown in the bottom panels as loading control.

Figure 4.

Figure 4.

Regulation of Crbi activity by DaPKC-dependent phosphorylation. Different forms of Crbi were overexpressed in embryos (B–G, Ubx-Gal4 line) and in the wing disc (J–N_, en-Gal4_ line). (A) Embryos expressing UAS-LacZ under the control of Ubx-Gal4 line were stained with anti–β-galactosidase antibody to reveal the domain of expression of the Ubx-Gal4 line. Similar regions to the one marked in A are shown in B–G. In these panels, the extent of the Ubx-Gal4 expressing domain is indicated by the white bar. (B–G) Embryos of the indicated genotype were stained with an antibody raised against the intracellular domain of Crb to reveal the distribution of both the endogenous and overexpressed Crb (purple) and costained (green) for DaPKC (B and F), Nrt (C and G), Patj (D), and Scrib (E). (B and C) Embryos expressing wild-type Crbi showed extensive relocalization of DaPKC to the whole cell contour in the cells overexpressing Crb (B). The localization of the basolateral marker Nrt was not affected (C). (D and E) On the contrary, overexpression of Crbi T6AT9AS11AS13A was associated with loss from the apical membrane of Crb and Patj (D) and with ectopic apical localization of Scrib (E, asterisks). (F and G) Overexpression of Crbi T6DT9D caused, similarly to Crbi, relocalization of DaPKC to the whole cell cortex (F) without affecting Nrt distribution (G). (H) en-gal4/UAS-LacZ wing stained with X-gal to show (blue) the posterior compartment of the wing. (I–N) Wild-type (I) and mutant wings expressing the indicated UAS transgenes driven by en-Gal4 (J–N). Overexpression of UAS-Crbi WT (J) or UAS-Crbi T6DT9D (K) causes strong cuticular defects. Overexpression of UAS-DaPKC CAAXDN does not affect wing cuticle deposition (L) but its coexpression suppresses the dominant phenotype of UAS-Crbi WT (M) but not that of UAS-Crbi T6DT9D (N). Bars: (A) 20 μm; (G, small white bar) 10 μm; (N) 10 μm.

References

    1. Ashburner, M. 1989. Drosophila. Transformation. A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1017–1063.
    1. Bachmann, A., M. Schneider, B. Theilenberg, F. Grawe, and E. Knust. 2001. Drosophila stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature. 414:638–643. - PubMed
    1. Benton, R., and D.S. Johnston. 2003. Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells. Cell. 115:691–704. - PubMed
    1. Berra, E., M.T. Diaz-Meco, I. Dominguez, M.M. Municio, L. Sanz, J. Lozano, R.S. Chapkin, and J. Moscat. 1993. Protein kinase C zeta isoform is critical for mitogenic signal transduction. Cell. 74:555–563. - PubMed
    1. Betschinger, J., K. Mechtler, and J.A. Knoblich. 2003. The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl. Nature. 422:326–330. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources