A strategy to disentangle direct and indirect effects on (de)phosphorylation by chemical modulators of the phosphatase PP1 in complex cellular contexts

ABSTRACT: Chemical activators and inhibitors are useful probes to identify substrates and downstream effects ofenzymes; however, due to the complex signaling environment within cells, it is challenging to distinguishbetween direct and indirect effects. This is particularly the case for phosphorylation, where a single(de)phosphorylation event can trigger rapid changes in many other phosphorylation sites. An additionalcomplication arises when a single catalytic entity, which acts in form of many different holoenzymes withdifferent substrates, is activated or inhibited, as it is unclear which holoenzymes are affected, and in turnwhich of their substrates are (de)phosphorylated. Direct target engaging MS-based technologies to studytargets of drugs do not address these challenges. Here, we tackle this by studying the modulation of proteinphosphatase-1 (PP1) activity by PP1-disrupting peptides (PDPs), as well as their selectivity toward PP1, byusing a combination of mass spectrometry-based experiments. By combining cellular treatment with the PDPwith in vitro dephosphorylation by the enzyme, we identify high confidence substrate candidates and beginto separate direct and indirect effects. Together with experiments analyzing which holoenzymes areparticularly susceptible to this treatment, we obtain insights into the effect of the modulator on the complexnetwork of protein (de)phosphorylation. This strategy holds promise for enhancing our understanding of PP1in particular and, due to the broad applicability of the workflow and the MS-based read-out, of chemicalmodulators with complex mode of action in general.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christina Ludwig  

LAB HEAD: Christina Ludwig

PROVIDER: PXD044415 | Pride | 2024-01-12

REPOSITORIES: Pride