University of Cambridge
Investigating how ubiquitin-mediated proteolysis of AURKA contributes to its activity in the cell cycle
Abstract
dc:description.abstractAurora kinase A (AURKA) is a major mitotic regulatory kinase required for mitotic entry, the formation of a bipolar mitotic spindle, and the completion of cytokinesis. In recent years AURKA has been identified as an upstream regulator for many interphase functions such as cilia disassembly and mitochondrial fragmentation. AURKA is overexpressed in many tumours and has a pivotal role in the acquisition of malignant cell phenotypes. Therefore, it is considered a highly attractive drug target for anti-cancer therapy. The activity of AURKA is regulated by phosphorylation at the active loop or the interaction with binding partners. TPX2 is a well-known binding partner of AURKA. It activates AURKA through stabilizing the T-loop and is required for targeting AURKA to the mitotic spindle. Phosphorylation and binding partners may act synergistically to induce hyperactivity of the kinase. Previous research from my lab has highlighted that AURKA is frequently co-expressed with TPX2 in human cancers and proposed AURKA/TPX2 complex as an oncogenic holoenzyme in a variety of cancers. AURKA protein is targeted for proteasome-mediated degradation by the FZR1 activated form of APC/C at the end of mitosis. This study focuses on characterisation of AURKA degrons, the contribution of APC/C-FZR1 in the timing of AURKA inactivation, identifying the physiological consequences of AURKA deregulation outside mitosis, and examining the role of Short Linear motifs (SLiMs) within AURKA N-terminal domain in regulating its stability and activity. I show that the previously known D-box-like motif (R371xxL374) within C-terminal is not a functional degron. I also reveal that the A-box motif may act as an atypical D-box that is sufficient to drive protein degradation. I use a new tool CRISPR/Cas9 FZR1 knockout cell line and a FRET-based biosensor for measuring AURKA activity to investigate directly whether AURKA inactivation is regulated simply by destruction. These, in combination with time-lapse imaging, show that inactivation of AURKA is identical in wild-type and FZR1KO cells, despit¬e the difference in protein levels between the two cell lines. I demonstrate that the timing of AURKA inactivation is regulated via the degradation of its activator TPX2 at mitotic exit. Moreover, the destruction of AURKA is required to regulate its interphase activity. I also identify that extra AURKA activity can have consequences on the morphology of the mitochondrial network outside of mitosis. My time-lapse imaging reveals that FZR1-restricted degradation of AURKA controls mitochondrial dynamics. This mechanism links the destruction machinery, through AURKA signaling to the mitochondrial dynamics of the cell. I further explore the role of the N-terminal domain in the regulation of AURKA activity through the detailed analysis of the potential SLiMs. I find that K23RVL has a role in mediating the autoinhibition of AURKA. I then investigate the hypothesis that calmodulin (CaM) protects AURKA from degradation through its binding to the A-box SLiM. I find that AURKA degradation is not affected by inhibition of Ca2+/CaM. In summary, this work sheds light not only on the molecular mechanisms of AURKA activity and stability but also on the physiological relevance outside mitosis, which is urgently needed in the field to understand the oncogenic activity of AURKA and to improve therapeutic applications of cancer patients.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Abdelbaki, Ahmed Mohamed
- Advisor dc:contributor.advisor
-
- Lindon, Catherine
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.69713
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/322253