A chemical-genetics approach to study the role of atypical Protein Kinase C in Drosophila

Studying the function of proteins using genetics in cycling cells is complicated by the fact that there is often a delay between gene inactivation and the time point of phenotypic analysis. This is particularly true when studying kinases that have pleiotropic functions and multiple substrates. Drosophila neuroblasts (NBs) are rapidly dividing stem cells and an important model system for the study of cell polarity. Mutations in multiple kinases cause NB polarity defects, but their precise functions at particular time points in the cell cycle are unknown. Here, we use chemical genetics and report the generation of an analogue-sensitive allele of Drosophila atypical Protein Kinase C (aPKC). We demonstrate that the resulting mutant aPKC kinase can be specifically inhibited in vitro and in vivo. Acute inhibition of aPKC during NB polarity establishment abolishes asymmetric localization of Miranda, whereas its inhibition during NB polarity maintenance does not in the time frame of normal mitosis. However, aPKC helps to sharpen the pattern of Miranda, by keeping it off the apical and lateral cortex after nuclear envelope breakdown.