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

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase that regulates numerous cellular processes. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B) and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we generated HEK293 cells with homozygous knock-in of dTAG-PPP2CA, allowing for efficient and selective degradation of dTAG-PPP2CA using proteolysis-targeting chimeras (PROTACs) targeting dTAG. Unbiased global phospho-proteomics identified 6,280 phospho-peptides corresponding to 2,204 proteins with significantly increased abundance upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. Some among these are established PP2A substrates, but a vast majority are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in processes including spliceosome function, cell cycle, RNA transport and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.