A novel approach towards identification of PP2A substrates

Protein phosphatase 2A (PP2A) is a prime example for the multisubunit architecture of protein-serinehreonine phosphatases (PSTPs). PP2A comprises in vivo more than 70 different heterotrimeric holoenzymes, each of which consists of a catalytic C subunit, a constant regulatory A subunit and one of several additional regulatory B-type subunits. The regulatory B-type subunits are responsible for substrate specificity and intracellular localization of PP2A holoenzymes. From the large number of differently assembled PP2A holoenzymes we can predict the existence of at least as many different PP2A substrates in a cell. Indeed, genetic studies in yeast and studies in mammalian cells using PP2A inhibitors show that PP2A holoenzymes regulate many different cellular processes indicating the existence of many different intracellular PP2A substrates. The identification of these substrates proved to be difficult because with the current methods it is impossible to detect the transient interactions between enzyme and substrates during catalysis. Therefore, the major goal of the proposed project is to adapt and optimize M-TRACKing for the identification of the transient PP2A interactom in yeast. M-TRACKing is a novel two-hybrid system based on a concept by G. Ammerer (University of Vienna), K. Nasmyth and T. Jenuwein (formerly IMP, Vienna) that has been pioneered by the Ammerer lab for the detection of protein interactions in the HOG stress pathway. M-TRACKing possesses the advantage over existing systems that an interaction partner is marked by an enzyme-catalyzed protein modification that is transferred with the specificity, efficiency and speed of an enzymatic reaction. Because an interaction is detected by an enzyme-catalyzed reaction, I reasoned that it should be possible to trap with M-TRACKing also PP2A substrates, which usually only transiently interact with PP2A and which cannot not be identified by currently existing methods. Our preliminary data show that we can indeed detect a transient PP2A-substrate interaction with M-TRACKing. Thus, emanating from our preliminary results we plan to optimize experimentally the M- TRACKing system for the detection of such short-lived interactions. As a complementary strategy to speeding up M-TRACKing (the detection tool) we will slow down PP2A catalysis in vivo by replacing wild-type PP2A with catalytically impaired mutants. We will apply the optimized M-TRACKing system in a proteome-wide screen for the identification of PP2A substrates. The results should provide new insights into PP2A-regulated pathways and at the same time provide proof of principle for M-TRACKing as the method of choice for the detection of transient interactions in vivo.

Proteins are the major constituents of a cell and exert their functions by physically interacting with each other and other cellular components. The identification of the interaction network, in particular the protein-protein interaction network, is a key prerequisite for the elucidation of the molecular mechanisms underlying all processes taking place in cells. Many of these interactions are very short-lived, for example, the interactions between enzymes and their substrates during catalysis. These interactions, however, are extremely difficult and often impossible to detect with the methods that existed when we started the project, and thus the knowledge on the identity of substrates was very limited. This was also true for protein phosphatase 2A (PP2A), an important enzyme family that catalyses the dephosphorylation of protein substrates and whose biogenesis and regulation we study in the lab. In order to fill the gaps on the PP2A interaction map, in particular those regarding transient interactions, I proposed to use a detection method that by itself is built on a short-lived enzyme-substrate reaction. Thus, we adapted a two-hybrid system termed M-Track (Methyl-Tracking), in which a PP2A subunit was fused to a histone lysine methyltransferase (the bait) and a potential PP2A substrate was fused to the histone H3 peptide (the prey), which is the substrate of the methyltransferase. As hypothesized, we were able to detect in vivo and for the first time short-lived PP2A-substrate interactions. Not only could we discriminate direct from indirect PP2A targets but we also provided proof on the molecular level for the substrate specificity of different PP2A holoenzymes. We published our findings in the top journal Nature Methods and the first authors of this paper were awarded the Sanofi-Aventis prize 2012 at the Medical University of Vienna. Subsequently, we validated additional PP2A substrates with M-Track and developed a method termed M-Tracker, with which a protein that has undergone an M-Track interaction can be visualized in vivo. Moreover, we could show that the methylation mark can be used to follow a protein's path in a protein network. In addition, there was also a translational research aspect of this project. We developed highly specific monoclonal antibodies that were an essential prerequisite for the successful application of M-Track. As these antibodies with their specificity for the histone H3 methylation states can also be used for epigenetic analyses, we were able to conclude licensing agreements with 11 international biotech companies. In summary, we established a method for the in vivo detection of short-lived protein interactions. With this tool at hands it will now be possible to determine the hierarchical structure of interaction networks, leading to a better understanding of signaling cascades.