Abstract
The phosphoprotein phosphatase family is responsible for a vast amount of dephosphorylation events on phosphoserine and -threonine in cells. As such, they are involved in key cellular processes, and consequently, disruption of their function contributes to the etiology and progression of diseases. Many of these phosphatases work as holoenzymes, where the catalytic subunit is complexed with regulatory proteins. How these phosphatases are regulated, how they recognize their substrates, and how substrates can be identified are long-standing questions in the field. Here, we lay out recently emerged concepts addressing these questions using examples of the phosphatases PP1, PP2A, and PP5. These new concepts include substrate recruitment through distal complexed proteins, the use of tailored peptide probes and mass spectrometry for substrate identification, substrate recognition through short helical motifs, and insights into holoenzyme assembly, as well as mechanisms of substrate release and phosphatase activation. Furthermore, we discuss future directions enabled by these new insights.