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 (Lead / Corresponding author)

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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.

Original languageEnglish
Article number109302
Number of pages24
JournaliScience
Volume27
Issue number3
Early online date19 Feb 2024
DOIs
Publication statusPublished - 15 Mar 2024

Keywords

  • PP2A
  • PPP2CA
  • phosphatase
  • phosphorylation
  • kinase
  • phospho-proteomics
  • substrate landscape
  • dTAG
  • PROTAC
  • targeted protein degradation
  • Properties of biomolecules
  • Protein
  • Proteomics
  • Enzymology

ASJC Scopus subject areas

  • General

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