Medicine (St Vincent's) - Theses
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ItemRoles of cyclin-dependent kinase substrates: cell cycle and beyond( 2015)The cyclin-dependent kinases (CDKs) are serine/threonine specific kinases that are key regulators of the cell cycle. However, several reports indicated their roles in other pathways. Therefore, it is important to identify novel CDK substrates in order to gain a better understanding of the pathways they regulate and in turn, the diseases where they are deregulated. In this thesis, I describe two substrates; Brahma (Brm), an ATPase subunit of the SWI/SNF chromatin remodeling complex and Breast Cancer Metastasis Suppressor 1 (BRMS1), a metastasis suppressor in human cancers. Brm has long been modeled to be part of the pRb/E2F complex, regulating entry into S phase of the cell cycle. In addition, several studies have indicated that Brm interacts with Cyclin E and CDKs. Here, I demonstrate that Drosophila Brm is phosphorylated in vitro by both Cyclin A and Cyclin E/CDK2. Furthermore, using Drosophila as an animal model, a phospho-mimic of Brm was able to bypass the developmental G1 arrest in the wing discs’ zone of non-proliferating cells (ZNC), indicating its role in inhibiting S phase entry. In addition, based on the phenotypes obtained from the expression of Brm phospho-mutants and the current literature, I postulate that CDK-mediated phosphorylation of Brm also has roles in maintaining genomic stability, cell signaling and expression of cell adhesion systems. Our laboratory had previously identified the regulation of Drosophila EGFR/Ras signaling by Brm-DN and its antagonism by Gem. In this thesis, I have further characterised the roles of Brm-DN and Gem in Drosophila development and have identified Rhomboid (a positive regulator of EGFR/Ras signaling) to be the target gene that is transcriptionally regulated by Brm and Gem. Finally, BRMS1 is a metastasis suppressor, better known to exert its functions by being a part of the SAP30/mSin3/HDAC complex to modulate transcription. Furthermore, it was previously reported to be in complex with RBP1, a CDK substrate, which inhibits S phase entry. In this thesis, we have found that BRMS1 is phosphorylated by CDKs both in vivo and in vitro. Using Mass Spectrometry, we have further identified the phosphorylated site to be Serine 237. Interestingly, mutation of this phosphorylation site had no impact on cell cycle progression and BRMS1’s role as a transcriptional regulator, however, it modulated BRMS1’s role in inhibiting cell migration. Taken together, this thesis confirms the CDK-mediated phosphorylation of two proteins and has expanded our understanding of the various roles that CDK substrates have beyond the cell cycle. Overall, this work provides further insights into the role of CDK substrates in cellular behaviour, tissue growth and differentiation, and the development of cancer and metastasis.