Homeodomain-interacting protein kinase phosphorylates the Drosophila Paired box protein 6 (Pax6) homologues Twin of eyeless and Eyeless

Homeodomain-interacting protein kinase (Hipk), the Drosophila homologue of mammalian HIPK2, plays several important roles in regulating differentiation, proliferation, apoptosis, and stress responses and acts as a mediator for signals of diverse pathways, such as Notch or Wingless signalling. The Paired box protein 6 (Pax6) has two Drosophila homologues, Twin of eyeless (Toy) and Eyeless (Ey). Both stand atop the retinal determination gene network (RDGN), which is essential for proper eye development in Drosophila. Here, we set Hipk and the master regulators Toy and Ey in an enzyme-substrate relationship. Furthermore, we prove a physical interaction between Toy and Hipk in vivo using bimolecular fluorescence complementation. Using in vitro kinase assays with different truncated Toy constructs and mutational analyses, we mapped four Hipk phosphorylation sites of Toy, one in the paired domain (Ser(121)) and three in the C-terminal transactivation domain of Toy (Thr(395), Ser(410) and Thr(452)). The interaction and phosphorylation of the master regulator Toy by Hipk may be important for precise tuning of signalling within the RDGN and therefore for Drosophila eye development.