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

Chemical activators and inhibitors are useful probes to identify substrates and downstream effects of enzymes; however, due to the complex signaling environment within cells, it is challenging to distinguish between 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 additional complication arises when a single catalytic entity, which acts in the form of many different holoenzymes with different substrates, is activated or inhibited, as it is unclear which holoenzymes are affected, and in turn which of their substrates are (de)phosphorylated.