Investigating PLK-1 and PP2A: B56 recruitment mechanisms in C. elegans oocyte meiosis

The role of phosphorylation in regulating protein localisation and activity temporally and spatially is an essential aspect of successful cell division. Therefore, the localisation and activity of kinases and phosphatases must be tightly regulated to ensure specific substrates are phosphorylated at the appropriate time to carry out their functions. Data from our lab showed that the kinase PLK-1 and phosphatase PP2A:B56 play important roles in C. elegans oocyte meiosis I. Furthermore, we found that PLK-1 and PP2A:B56 both localise to meiotic chromosomes in prometaphase/metaphase I and the central spindle in anaphase. Specifically, in prometaphase/metaphase I PLK-1 localises broadly to the chromosome arms and midbivalent between the homologous chromosomes and PP2A:B56 localises specifically to the midbivalent and kinetochore. In this project, I have characterised the primary recruitment mechanisms of PLK-1 to meiotic chromosomes and PP2A:B56 to meiotic chromosomes and the anaphase central spindle in C. elegans oocytes. Analysis of nematode protein sequence alignments indicated the presence of conserved PLK-1 and PP2A:B56 interaction sites in BUB-1 and a conserved PLK-1 interaction site in CENP-CHCP-4. Using a combination of in vivo immunoprecipitation and in vitro recombinant protein purification, kinase assays, and various biochemical techniques to assess protein interactions, I found that C. elegans BUB-1 and CENPCHCP-4 directly interact with PLK-1 through phospho-dependent STP motifs. Additionally, I found that BUB-1 directly interacts with PP2A:B56 through a phospho-regulated B56 SLiM. In collaboration with other members of the Pelisch Lab, we generated point mutations of these interaction sites in vivo and found that BUB-1 specifically recruits PLK-1 to the midbivalent and kinetochore in prometaphase/metaphase I while CENP-CHCP-4 recruits PLK-1 to chromosome arms. Additionally, we found that the BUB-1 SLiM directly recruits PP2A:B56 to metaphase chromosomes and the anaphase central spindle in a phospho-dependent manner. Thus, my project has identified recruitment mechanisms of PLK-1 and PP2A:B56 in C. elegans that are important for oocyte meiosis.