Date of Award

5-23-2022

Publication Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

Keywords

DNA damage;GADD45A;kinase;phosphorylation;PLK4;substrate

Supervisor

John Hudson

Abstract

Polo-like kinase 4 (PLK4) is a serine/threonine kinase that is implicated in a variety of cellular processes including centrosome dynamics, cytokinesis, cell polarity, and epigenetics. PLK4 overexpression is sufficient to induce centrosome amplification, a hallmark of cancer. Plk4-null mice are non-viable at embryonic day 7.5, while Plk4+/– mice develop tumours of the liver and lung and have myeloid-biased hematopoiesis. Here, I have characterized the interaction between PLK4, and the protein known as growth arrest and DNA damage inducible protein 45 alpha (GADD45A). GADD45A is a protein whose expression is increased in response to a variety of DNA damaging agents including UV and IR. GADD45A is implicated in many facets of the DNA damage response including DNA repair, cell cycle arrest, and apoptosis. I have shown that PLK4 phosphorylates GADD45A at four serines and one tyrosine. I created phosphomimic and phospho-null versions of GADD45A on these sites and stably expressed them in cells. These cells were studied using a variety of different assays to assess how the expression of these mutants may change the cells. I found most markedly that phosphorylation of serine 93 of GADD45A seemed to modulate the migration ability of the cells. These data suggest that when serine 93 of GADD45A is phosphorylated, the migration ability of the cells is highly elevated, while when serine 93 is unphosphorylatable, the migration ability of the cells is low. Some phosphorylation sites seem to also alter the size and morphology characteristics of the cells and their nuclei. Additionally, I found some of the phosphorylation sites on GADD45A may be related to growth and apoptosis rates of the cells. Notably, I provide evidence that PLK4 may be a dual-specificity kinase, since I have identified a tyrosine phosphorylation site within GADD45A that is targeted by PLK4. Additionally, our data showed that when PLK4 is inhibited, tyrosine phosphorylation of GADD45A is reduced by around 90%. Therefore, tyrosine phosphorylation of GADD45A is dependent on PLK4 kinase activity. I also explore three other PLK4 interacting partners: PRMT5, DNMT3A, and NPM1. I have found no conclusive evidence that any of these are substrates of PLK4, but I present data towards understanding their potential to be PLK4 substrates or work in a complex with PLK4. Lastly, data relating to the fertility phenotypes observed in the Plk4+/– mouse model are shared as part of a collaborative project with other members of the lab. Together, we have found that Plk4+/– mice have smaller litters than wild-type mice. We identified round spermatid arrest in the seminiferous tubules of the male Plk4+/– mice and abnormal ovarian follicle development in the female Plk4+/–.

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