Analysis of the structure and function of protein phosphatase 2A

Abstract

One of the hallmarks of a living organism is the ability to respond to intra- or extracellular changes. These responses involve panoply of enzymes mediating signals through the cell and regulating distinct cellular functions. Protein kinases and protein phosphatases are important antagonists in this finely balanced process. Protein phosphatase 2A (PP2A) is one of the major serine/threonine-specific phosphatases and has the most diverse substrate specificity of all protein serine/threonine phosphatases in the cell. PP2A consists of a core dimer made up of the 36-kDa catalytic subunit C tightly complexed with the scaffold regulatory subunit PR65/A. This complex associates with any one of the second or variable regulatory subunits PR55/B, PR61/B’, PR72/B” or PR110/B’’’ to form an extensive array of trimeric holoenzymes. PP2A impacts on all major signaling pathways by reversing the functions of protein kinases and is, therefore, considered to be a central regulator of eucaryotic signal transduction. Dysfunction of this molecule may have severe consequences for the organism and it is, therefore, not surprising that PP2A has become an important target in the investigation of various diseases. We investigated the function of invariant active-site residues of PP2A that are crucial for catalytic function of the enzyme. A baculovirus system using High Five insect cells was developed that allowed high level expression of active PP2A which was used for structural and functional analysis. Site-directed mutagenesis of PP2Ac and purification of mutant proteins from insect cells combined with functional analysis in yeast provided a powerful system for structure–function analysis of PP2Ac. Mutation of the active-site residues Asp88 or His118 within the human PP2A catalytic α subunit impaired catalytic activity in vitro and in vivo indicating an important role for these residues in catalysis. As PP2A containing the PR55/B regulatory subunit is known to be involved in the pathogenesis of neurodegenerative disorders, we characterized the PR55/B family with particular emphasis on its distribution and developmental regulation in the brain. The study revealed new aspects of genomic organization and variability, as well as hitherto unknown expression patterns of the PR55/B family in the brain. We also found distinct subcellular localizations of PR55/B isoforms in areas of the brain known to be affected by Alzheimer’s disease. In addition, our results suggest a distinct role for PR55/Bα in astrocytosis, given that this isoform is highly expressed in activated astrocytes. Interestingly, astrocyte activation is an early step in the pathogenesis of Alzheimer’s disease and related disorders. In addition, we attempted to define the transcriptional effects of the PP2A-inhibitor okadaic acid (OA) on promoter complexes using Affymetrix GeneChips. Based on known target genes and further target genes that we identified, we suggest that OA mainly stimulates transcription activators and/or inhibits transcription repressors, probably by inhibition of PP2A. In order to investigate genes that are transcriptionally coregulated by OA, we developed a software tool we named “StampCollector” that predicts potential transcription factor pairs (TF pairs) involved in the regulation of genes based on their promoter sequences. Taken together, the results presented in this thesis underline the significance of PP2A in the regulation of cellular events. We combined various approaches in order to characterize the precise role of PP2A and its PR55/B regulatory subunits in gene regulation. Considering the putative role of PP2A in the pathogenesis of human disease, our results may lead eventually to the discovery of therapeutic agents for specifically counteracting PP2A dysfunction.