Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA

Abstract

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase that regulates a plethora of cellular processes. PP2A operates as a holoenzyme complex, comprising one each of the scaffolding (A), regulatory (B) and catalytic (C) subunits. PPP2CA is the principal catalytic subunit of the PP2A holoenzyme complex. Although previous studies have reported many substrates of specific PP2A holoenzyme complexes, the full scope of PP2A substrates in cells remains to be defined. To address this, we generated HEK293 cells in which PPP2CA was homozygously knocked in with a dTAG, allowing for efficient and selective degradation of dTAG-PPP2CA with proteolysis-targeting chimeras (PROTACs) targeting the dTAG. By employing an unbiased global phospho-proteomic analysis, we identified 6,280 phospho-peptides corresponding to 2,204 proteins that showed a significant increase in abundance upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. Among these, some were established PP2A substrates, while most were novel. Bioinformatic analyses revealed the involvement of the identified potential PPP2CA substrates in many cellular processes, including spliceosome function, the cell cycle, RNA transport and ubiquitin-mediated proteolysis. We show that a pSP/pTP motif is a predominant target for PPP2CA. We confirmed some of our phospho-proteomic data with immunoblotting, by utilising commercially available phospho-specific antibodies. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.