Subunit specific inhibitors of protein phosphatase 2A

Many publications on protein phosphatase 2A (PP2A) start with a sentence like: "PP2A, a highly conserved serinehreonine phosphatase, is involved in the regulation of many cellular processes." The data leading to such conclusions were primarily based on the usage of low molecular weight inhibitors, which bind directly and with similar affinities to the catalytic subunits of PP2A and other related phosphatases thereby inhibiting catalysis. With the currently used compounds it is impossible to assign a certain function to PP2A because several other related phosphatases including protein phosphatase 4 (PP4) and protein phosphatase 6 (PP6) are also inhibited by these toxins. In addition the substrate specificity of PP2A is determined by the interaction of the catalytic subunit with further regulatory subunits. Over 70 different holoenzymes can be assembled from the known set of subunits. The in vivo functions of these distinct PP2A enzymes are mostly unknown because of the lack of specific inhibitors. Therefore I propose to generate inhibitors that bind specifically to the B or B` regulatory subunit of PP2A and inhibit the catalytic activity of B or B` containing PP2A complexes. In order to isolate such interactors/inhibitors we will screen a combinatorial peptide aptamer library using the yeast two-hybrid system. Peptide aptamers are antibody like recognition molecules that display conformationally constrained dodecapeptides (of random sequence) on scaffolds like E.coli thioredoxin (TrxA). In contrast to other selection methods (e.g. phage display) the aptamer library screen is carried out in vivo, e.g. in S.cerevisiae. Aptamers isolated in such a way automatically fulfill the basic requirements for intracellular use: they are non-toxic, stably expressed and most importantly they interact with the target protein in the reducing intracellular environment. The isolated peptide aptamers will be characterized in vitro and in vivo for their interaction with B-type subunits, their binding specificities and their inhibitory potential. Such inhibitors will allow defining and clarifying the regulatory role of a specific PP2A complex in certain cellular processes. The proposed project will be a step forward in our understanding of the multifunctional enzyme PP2A.

Protein phosphatase 2A (PP2A) is a prime example for the multi-subunit architecture of protein-serinehreonine phosphatases (PSTPs). Multisubunit enzymes like PP2A consist in vivo of a catalytic subunit and one to several regulatory subunits that are responsible for substrate specificity. From the known PP2A subunits more than 70 PP2A enzymes can be assembled combinatorially, each probably with unique substrate specificity. The in vivo functions, however, of these distinct PP2A enzymes are mostly unknown because of the lack of inhibitors that target one specific PP2A enzyme. Several screens of random peptide-aptamer libraries to isolate such specific PP2A inhibitors were unsuccessful. Thus we aimed to understand how the assembly of PP2A enzymes is regulated in vivo in order to being able in the future to generate - based on this knowledge - specific PP2A inhibitors. We analyzed the biogenesis of this highly conserved enzyme family in the model organism yeast. In this project we obtained evidence for the existence of the catalytic subunit in a low activity conformation that requires an activator for the switch into the active conformation. This requirement suggested that the existing model of PP2A biogenesis was incomplete because it could not explain how the unspecific phosphatase activity of the catalytic subunit is kept in check until complex assembly with substrate-targeting subunits can occur. In this study we were able to show that the generation of active catalytic subunit is coupled and regulated with holoenzyme assembly. We propose a novel model of protein phosphatase biogenesis, in which a tightly controlled activation cascade protects cells from the potential risk of unspecific dephosphorylation events by PP2A. Moreover, in the course of the project we generated several new mono- and polyclonal antibodies against PP2A subunits. For their commercialization I could conclude material license agreements with two international bio-tool companies. Thus, the project was not only scientifically but also commercially successful.