Regulation and Function of PP2A-B56delta

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Project Summary Protein phosphorylation is involved in the regulation of many biological processes, including the response to DNA damage. While kinase signaling has been extensively investigated, the role of phosphatases remains understudied. Phosphoprotein phosphatase 2A (PP2A) is responsible for the majority of phosphoserine and phosphothreonine dephosphorylation within a cell and confers its substrate specificity through its regulatory B subunit. The B56 family is the largest of the four families of PP2A regulatory subunits. All B56 isoforms recognize an LxxIxE short linear motif (SLiM) on their substrates and interactors. We have developed a SLiM-mediated competitive inhibitor approach to interrogate the PP2A-B56-dependent phosphoproteome, thus expanding the understanding of B56-specific substrates. However, our knowledge of the mechanisms that govern the activity of PP2A-B56 remains incomplete. Upon B56 inhibition, we found phosphoserines 88-90 of B56δ to be differentially regulated. Our preliminary data suggest that these sites are inactivating in nature. B56δ is encoded by the gene PPP2R5D, which is mutated in PPP2R5D-related developmental disorder characterized by cognitive impairment and epilepsy. Furthermore, we identified Oxidative Resistance 1 (OXR1), an LxxIxE motif-containing protein involved in the cellular response to oxidative stress and the maintenance of genome stability, to be a B56 substrate. Yet little is known about the regulation of OXR1. Here, we propose to determine how phosphorylation of S88-90 regulates B56δ and to expand upon our understanding of B56-dependent regulation of OXR1. Our central hypothesis is that S88-90 of B56δ are critical regulatory sites on B56δ, which serves an essential role in modulating DNA damage responses through OXR1 signaling. Specific Aim 1 investigates the regulation of B56δ S88-90 phosphosites, the kinase(s) that phosphorylate them, and their biological function using innovative approaches in mass spectrometry and in vitro and in cell studies. Specific Aim 2 seeks to determine the role of PP2A-B56 in the dephosphorylation of OXR1, the contribution of the OXR1 LxxIxE motif to this interaction, and the biological significance of the B56-OXR1 axis in DNA damage signaling. The successful completion of these aims will elucidate the phosphoregulation of B56δ and the role of PP2A-B56 in the dephosphorylation of OXR1. By understanding the regulation of these proteins, which are involved in a variety of human diseases, we will gain a better understanding of their disease-relevant physiology, potential therapeutic targets, and points for therapeutic intervention. The proposed projects and training plan in this application will enhance my skills in biochemical and cell biological techniques, experimental design, data analysis, scientific communication, mentoring, and translational research. The collaborative environment at Dartmouth College and the Norris Cotton Cancer Center and the strong network of mentors and collaborators provide an ideal environment for me to complete the research proposed in this application and support my development as a physician scientist.