A chemical-genetics approach to study the role of atypical protein kinase C in Drosophila

Abstract

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.

Lgl cortical dynamics are independent of binding to the Scrib-Dlg complex but require Dlg-dependent restriction of aPKC

“…Long-term disruption of aPKC activity enables constitutive binding of unphosphorylated Lgl at the plasma membrane, which could mask a role for Dlg in Lgl cortical recruitment. We therefore inactivated aPKC acutely using a recent analogue-sensitive aPKC allele (Hannaford et al, 2019). Live imaging of endogenously expressed Lgl-GFP shows that aPKC inhibition induces a quick reallocation of Lgl from the cytoplasm to the cortex in dlg mutant cells ( Figure 3H).…”

“…UAS-Lgl 3A -GFP and UAS-Lgl-GFP (Betschinger et al, 2003) for overexpression conditions; (Betschinger et al, 2003); aPKC K06403 (Wodarz et al, 2000); apkc as4 , an analogue-sensitive allele used for acute inhibition of aPKC (Hannaford et al, 2019, kindly provided by Jens Januschke); scrib 2 . We used a recently generated dlg 1A mutant allele, which was produced with low concentrations of ethyl methylsulphonate to diminish secondary alterations Yamamoto et al, 2014).…”

Neural stem cell dynamics: the development of brain tumours

“…Recent studies have revealed that the phosphorylation state of Mira is important for cortical localisation [22]. During interphase, Mira is non-phosphorylated and is uniformly associated with the plasma membrane, but aPKC-mediated phosphorylation at metaphase prevents the localisation of Mira at the apical pole [22,23]. Furthermore, the asymmetric localisation of Mira is maintained through interactions with its cognate mRNA [24].…”

aPKC in neuronal differentiation, maturation and function

“…Thus, the exquisite regulation of spatial (apical) aPKC activity in the cortex is critical for the establishment of neural stem cell versus progenitor identity, in part through the asymmetric segregation of Numb [81]. Another study generated an analog-sensitive mutant of DaPKC [82]. Analog-sensitive mutants substitute a structurally conserved bulky amino acid such as methionine, leucine, phenylalanine or threonine in the active site of kinases termed as 'gatekeeper residue' to a residue with a much smaller side chain [83].…”

“…This enables binding of kinase inhibitors with large bulky groups, such as C3-tolyl ring, specifically to the engineered kinase while sparing wild-type kinases [83]. Employing the analog-sensitive DaPKC mutants, the authors were able to demonstrate that acute inhibition of DaPKC during neuroblast polarity establishment abolished the asymmetric localization of Miranda, as well as Numb, while PARD3 and DLG (discs large) were mostly unaffected [82].…”