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

Abigail Brewer; Gajanan Sathe; Billie E Pflug; Rosemary G Clarke; Thomas J Macartney; Gopal P Sapkota

doi:10.1016/j.isci.2024.109302

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

iScience. 2024 Feb 19;27(3):109302. doi: 10.1016/j.isci.2024.109302. eCollection 2024 Mar 15.

Authors

Abigail Brewer¹, Gajanan Sathe¹, Billie E Pflug¹, Rosemary G Clarke¹, Thomas J Macartney¹, Gopal P Sapkota¹

Affiliation

¹ Medical Research Council (MRC) Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

Abstract

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. 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 employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in 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.

Keywords: Enzymology; Properties of biomolecules; Protein; Proteomics.