LAUSR

Protein phosphatase‐1 (PP1)‐disrupting peptides (PDPs) are selective chemical modulators of PP1 that liberate the active PP1 catalytic subunit from regulatory proteins; thus allowing the dephosphorylation of nearby substrates. We have optimized the original cell‐active PDP3 for enhanced stability, and obtained insights into the chemical requirements for stabilizing this 23‐mer peptide for cellular applications. The optimized PDP‐Nal was used to dissect the involvement of PP1 in the MAPK signaling cascade. Specifically, we have demonstrated that, in human osteosarcoma (U2OS) cells, phosphoMEK1/2 is a direct substrate of PP1, whereas dephosphorylation of phosphoERK1/2 is indirect and likely mediated through enhanced tyrosine phosphatase activity after PDP‐mediated PP1 activation. Thus, as liberators of PP1 activity, PDPs represent a valuable tool for identifying the substrates of PP1 and understanding its role in diverse signaling cascades.