Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Peter S. Klein

Abstract

Glycogen Synthase Kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed kinase that acts as a critical regulator of many signaling pathways. These pathways, when dysregulated, have been implicated in many human diseases, including bipolar disorder (BD), cancer, and diabetes. Over 100 putative GSK-3 substrates have been reported, based on direct kinase assays or genetic and pharmacological manipulation of GSK-3, in diverse cell types. Many more have been predicted based upon on the prevalence of the GSK-3 consensus sequence. As a result, there remains an unclear picture of the complete GSK-3 phosphoproteome. We have therefore used a large-scale mass spectrometry approach to analyze global changes in phosphorylation and describe the repertoire of GSK-3 substrates in a single cell type. For our studies, we used stable isotope labeling of amino acids in culture (SILAC) to compare the phosphoproteome of wild-type mouse embryonic stem cells (ESCs) to ESCs completely lacking Gsk3a and Gsk3b expression (Gsk3 DKO). We used titanium oxide chromatography to enrich for phosphorylated peptides. From our analysis, we selected 65 phosphoproteins that exhibited significantly reduced phosphorylation in Gsk3 DKO ESCs as high-confidence candidate substrates of GSK-3. Our findings indicate that these candidate GSK-3 substrates can influence all levels of gene expression including chromatin modulators, transcription factors, RNA binding proteins, splicing factors, translational initiators and cell cycle regulators. Analysis of protein-protein interaction networks revealed enrichment of a cluster of proteins involved in alternative splicing. Our study is the first to discover a function for GSK-3 in alternative splicing. To further validate our top hits, we conducted in vitro kinase assays with recombinant proteins and identified the splicing factor RBM8A and an RNA processing protein NPM1 to be direct targets of GSK-3. Preliminary RNA sequencing results point to an overall increase in alternative splicing events when Gsk3 is deleted. Taken together, the research in this dissertation represents the first unbiased analysis of GSK-3 phosphorylation substrates in a single cell type and provides the first evidence of GSK-3 as a general regulator of alternative splicing.

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