Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1.

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), RQSRRS(pT696)QGVTL (P-Thr696-MYPT1), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1 was dephosphorylated by PP1c slowly (t = 81.6-87.9 min), which was further impeded (t = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t = 1.69 min) significantly (t = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1) or phosphoserine (PP1c-P-Ser696-MYPT1) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.