DOI
https://doi.org/10.25772/H9ZN-DQ97
Defense Date
2016
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
Dr. Kristoffer Valerie
Abstract
Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic stability and cell growth. We generated PP2A-Aα conditional knockout mouse embryonic fibroblasts expressing PP2A-Aα-WT, S401A (cannot be phosphorylated), or S401D phosphomimetic) transgenes by floxing out the endogenous PP2A-Aα alleles with Cre. The S401D mutant cells displayed increased ERK and AKT signaling, resulting in an enhanced growth rate. Phosphorylation of PP2A-Aα at S401 caused the dissociation of ATM with the holoenzyme, an effect that could be recapitulated with S401D. Additionally, the S401A and S401D mutants exhibited significantly more chromosomal aberrations and underwent increased mitotic catastrophe after radiation. Both the S401A and the S401D cells showed impaired DSB repair (Non-homologous end joining and Homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Time-lapse video and cellular localization experiments showed that the PP2A-Aα subunit was exported to the cytoplasm after radiation possibly by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects seen. In conclusion, our study demonstrates using a genetically defined system that ATM phosphorylation of a single, critical amino acid S401 is essential for regulating DDR. To study how the interplay between ATM and PP2A affects DDR in the brain, we are in the process of generating a brain specific PP2A-Aα conditional knockout mouse. Loss of many DDR related proteins like ATM and PP2A can lead to severe neuropathological effects. This model will be helpful in dissecting the PP2A-Aα/ATM regulatory circuit in the brain in response to DDR.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-14-2016