Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1-Disrupting Peptides.

PP1-disrupting peptides (PDPs) are selective chemical modulators of protein phosphatase-1 (PP1) that liberate the active PP1 catalytic subunit from regulatory proteins, thus allowing it to dephosphorylate nearby substrates. Here, we optimized the original cell-active PDP3 for enhanced stability, and obtained insights into the chemical requirements for stabilizing this 23mer peptide for cellular applications. The optimized PDP-Nal was used to dissect the involvement of PP1 in the MAPK signaling cascade. Specifically, we demonstrate in human osteosarcoma (U2OS) cells that pMEK1/2 is a direct substrate of PP1, whereas dephosphorylation of pERK1/2 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.