Leveraging Small Molecule Activators of Protein Phosphatase 2a (Pp2a) to Elucidate PP2As Role in Regulating DNA Replication and Apoptosis

• Main Author: Perl, Abbey Leigh
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2020
• Subjects: Biomedical engineering; Cellular biology; Pharmacology
• ISBN: 9798780648215

Aberrant signal transduction resulting from dysregulated phosphorylation is a hallmark of human cancer. Altered phosphorylation has broad implications on cancer biology. Much work has been done characterizing the effects of individual kinases with their cancer phenotypes. However, the structural complexity of their counterparts, phosphatases, has limited our knowledge of these signaling events. Protein Phosphatase 2A (PP2A), one such negative regulator of multiple oncogenic kinases, has been well characterized as a tumor suppressor protein that when inhibited can lead to cellular transformation. PP2A is a heterotrimeric complex whose substrate specificity is dependent on one of 23 different regulatory subunits that can bind to form over 60 distinct holoenzyme complexes. Although PP2A’s function as a general tumor suppressor is well studied, the role of PP2A on specific tumor suppressive signaling pathways and the specific holoenzymes mediating this signaling are not completely understood. Through chemical and genetic approaches, this work characterizes a new role for PP2A in the regulation of DNA replication, and links PP2A effects on replication with its ability to induce apoptosis.Utilizing both a gain of function chemical biology approach and loss of function genetic approaches to modulate PP2A activity, we demonstrate that increasing PP2A activity can interrupt ongoing DNA replication resulting in a collapse of replication forks, the induction of double-stranded DNA (dsDNA) breaks, and a replication stress response that is PP2A dependent. Additionally, we show that increasing PP2A activity during replication causes a dissociation of the replisome, a common mechanism of inhibiting ongoing replication. Furthermore, patients harboring mutations in PP2A are shown to have a higher fraction of their genome altered, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. Moreover, knockdown of the PR130 PP2A regulatory subunit renders cells resistant to PP2A-mediated apoptotic signaling both in vitro and in vivo, which coincides with the abrogation of the induction of replication stress, suggesting the resistance to PP2A mediated apoptotic signaling upon PR130 loss comes from an inability to induce PP2A-mediated replication stress.