Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification.

Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A‐B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A‐B55. We use specific inhibition of PP1 and PP2A‐B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A‐B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation. Synopsis: MRBLE:Dephos a new method for high‐throughput investigation of peptide dephosphorylation using spectral encoded beads. Using this method, the active site preferences of PP1 and PP2A‐B55 are investigated and compared to mitotic exit substrates identified by phosphoproteomics. MRBLE:Dephos is a novel high‐throughput dephosphorylation method.MRBLE:Dephos provides insights into PP1 and PP2A‐B55 sequence preferences.Mitotic exit substrate screens for PP1 and PP2A‐B55 identifies thousands of regulated sites.Binding of PP1 to RVxF motifs can modulate active site preferences. [ABSTRACT FROM AUTHOR]