Figure 2.
Yrt is a substrate of aPKC. (A) Control embryos (daughterless [da]-GAL4) or embryos overexpressing Par-6 and aPKCCAAX (da-GAL4/UAS–Par-6 and UAS-aPKCCAAX) were homogenized at different developmental stages. Samples were treated or not treated with the λ phosphatase (λ PPase) and processed for SDS-PAGE. Western blotting using anti-Yrt antibodies showed the migration profile of Yrt, whereas Actin was used as loading control. (B) Western blot showing the migration profile of Yrt extracted from control (wild type) or aPKC maternal and zygotic mutant embryos (we used the allele aPKCpsu265 that encodes a kinase inactive protein; Kim et al., 2009). Actin was used as a loading control. (C and D) Radioactive kinase assays in which purified aPKC was incubated with GST coupled to full-length Yrt (FL; C) or an extended version of the FA domain of Yrt (FA; aa 330–415) and a mutant version of it in which S348, S358, T379, S387, and S392 were mutagenized to A residues (FA5A; D). Proteins were separated on a polyacrylamide gel, which was exposed to monitor protein phosphorylation and then colored with Coomassie blue to control the amount of substrate used in each sample. (E) Anti–Par-6 antibodies were used to immunoprecipitate Par-6 from wild-type embryo extracts (stages 10–13; immunoprecipitate [IP] Par-6), whereas normal guinea pig IgG (IgG) was used as a negative control. GST or GST-FA was added to precipitate along with radiolabeled ATP in the absence or presence of PKCtide, which is a high affinity substrate of aPKC. Proteins were separated by SDS-PAGE, and the gel was exposed to monitor protein phosphorylation. Then, proteins were transferred on a membrane to validate immunoprecipitation of Par-6 and coimmunoprecipitation of aPKC and to monitor the amount of substrate used in each reaction. (F) GST pull-down experiments were performed on wild-type embryo lysates using GST-FA (FA), the nonphosphorylatable GST-FA5A (FA5A), or the phosphomimetic GST-FA5D (FA5D). GST alone was used as a negative control. Western blotting detected pulled down aPKC and monitored the amount of GST or GST fusion proteins used in each experiment. (G) Alignment of the FA domain of mouse Lulu2 and Drosophila Yrt. Numbers indicate amino acid positions in the Yrt sequence. Arrows point to amino acids previously shown to be phosphorylated by aPKC in Lulu2 (Nakajima and Tanoue, 2011). Three of these residues are conserved in Yrt (black rectangles). Black circles indicate phosphorylated residues identified by MS in the FA domain of Yrt. Three of these residues are conserved in Lulu2 (orange rectangles). As per ClustalW nomenclature (Larkin et al., 2007), asterisks indicate positions that have fully conserved residues. Colons designate conservation between groups of strongly similar properties, and periods indicate conservation between groups of weakly similar properties.