Figure 1. aPKC binds and phosphorylates Miranda.

(A) Miranda domain architecture. (B) The Miranda cortical localization domain is specifically phosphorylated by aPKC. Phosphorimage and Coomassie stained SDS gel of GST and GST:Miranda fusion proteins in the presence of recombinant HIS:aPKC and ³²P. GST:Miranda 1–290 is phosphorylated whereas all other constructs are not. Commassie stain of gel as loading control. (C) Miranda interacts with aPKC in transfected S2 cells. Immunoblot of S2 cell lysates from cells transfected with aPKC:myc and HA:Miranda immunoprecipitated with anti-aPKC, -HA, or -Pins antibodies. Protein G and anti-Pins antibody used as controls. (D) Miranda interacts with aPKC in Drosophila embryonic extracts. Immunoblot of embryonic lysate immunoprecipitated with anti-aPKC, -Par-6, -Miranda, or -Pins antibodies. Controls as in panel C. (E) Identification of residues within Miranda 1–290 phosphorylated by aPKC in vitro. Purple-highlighted residues indicate coverage by LC/MS/MS, red lettering indicates residues identified as phosphorylated, and stars indicate residues tested by point mutation as in Figure 2. Phosphorylation prediction programs predicted residues 141 and 143 to be highly likely for phosphorylation by aPKC.