Author ORCID Identifier

Date of Award

Winter 2-26-2024

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

Arminja Kettenbach


B56δ is a regulatory subunit of protein phosphatase PP2A, which is responsible for the majority of phosphoserine and phosphothreonine dephosphorylation within a cell. PPP2R5D-Related Developmental Disorder is characterized by intellectual disability, autism-spectrum disorder, hypotonia, and epilepsy and is caused by de novo single nucleotide changes in PPP2R5D, the gene that encodes B56δ, resulting in missense mutations (i.e. E198K, E420K, etc.). Yet, the molecular mechanisms impacted by these variants are unknown. To investigate these mechanisms as described in Chapters 2 and 3, we generated HEK293 cells harboring a single nucleotide change in the endogenous locus of PPP2R5D to recapitulate two pathogenic variants: E198K and E420K. Employing quantitative proteomic and phosphoproteomic approaches, we investigated global protein and phosphorylation changes in wild type-, heterozygous E198K- and heterozygous E420K-cell lines. These analyses revealed both unique and common changes in the E198K and E420K variants versus wild type cells in various signaling cascades. Based on mass spectrometry and western blot data, neither the E198K nor E420K mutation impacts the expression of PPP2R5D. However, we find that the variants bind less PP2A catalytic proteins, suggesting partial loss of PP2A holoenzyme assembly or reduced stability of the complex. The E420K and E198K variants also showed reduced activity compared to wild type in an in vitro dephosphorylation assay. Over- representation analysis of the E420K phosphoproteomic data revealed enrichment of the Protein Kinase B (AKT/PKB) consensus sequence. Hyperphosphorylation of AKT-mTOR pathway components such as RPS6 was also evident in western and LC-MS/MS analyses of the E420K variant. While RPS6 is also hyperphosphorylated in the E198K variant, activation of the AKT-mTOR pathway was not evident, suggesting that this variant results in a similar phenotype through convergent signaling pathways. Intriguingly, our data indicate ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, suggesting ERK/MAPK-mediated activation of mTORC1 as key signaling alterations in PPP2R5D-variant cells. These studies reveal molecular pathways that could be therapeutically targeted in PPP2R5D-Related Developmental Disorder. Finally in Chapter 4, we investigate the interaction between B56δ and Oxidative Resistance 1 (OXR1), a poorly characterized protein shown to be implicated in a various neurological diseases.