Substrate mapping and activity regulation of PP1 holoenzymes

Protein phosphatase 1 (PP1) is an essential protein Ser/Thr phosphatase, which regulates various cellular processes in eukaryotic cells. The activity and substrate specificity of PP1 is strictly controlled by associated polypeptides, which are known as Regulatory Interactors of Protein Phosphatase One (RIPPOs). Currently, more than 200 RIPPOs have been described, all acting as subcellular targeting subunits, substrate recruiters, and/or activity modulators for associated PP1. Knowledge of the physiological substrates of each individual PP1 holoenzymes would massively enhance the functional understanding of these complexes. At the start of my research project, a straightforward strategy for substrate mapping of PP1 holoenzymes was not available. In the first part of my PhD project, I developed a tool to trap substrates of individual PP1:RIPPO holoenzymes. I found that a fusion of an hypoactive point mutant of PP1 and a RIPPO accumulates with its associated substrate, due to a lack of efficient dephosphorylation. The associated substrates were subsequently trapped and identified by Mass Spectrometry analysis. The identified substrates (novel and known proteins) were independently validated by in vitro dephosphorylation experiments. In this way, I performed substrate trapping, followed by substrate identification, for PP1:NIPP1, PP1:PNUTS, PP1:MYPT and PP1:RepoMan holoenzymes using the corresponding hypoactive PP1-RIPPO fusions. Furthermore, I found that RepoMan itself was a substrate of associated PP1. Together, my results suggest that hypoactive fusions represent an easy-to-use tool for substrate identification of multimeric protein Ser/Thr phosphatases individual holoenzymes.

Jaar van publicatie:2022

Toegankelijkheid:Closed