Mechanism of myosin phosphatase inhibition via phosphorylation of MYPT1 subunit by RhoA/ROCK

Smooth muscle tone is determined by reciprocal activities of myosin light chain kinase and phosphatase in response to agonist stimulation and nitric oxide production. Myosin phosphatase, a heterotrimeric form of protein phosphatase‐1, is inhibited upon activation of RhoA/ROCK, and the inhibition is cancelled in response to cyclic nucleotide signal. MYPT1, a regulatory subunit of myosin phosphatase, is phosphorylated at Thr696 and 853 during agonist‐induced contraction. Here, we investigated mechanisms underlying the inhibition of myosin phosphatase through phosphorylation of MYPT1, using GST‐fusion peptides of MYPT1 segments including phosphorylation sites. GST‐MYPT1(654‐880) peptide including both Thr696 and 853 and shorter peptides including either phosphorylation site, 654‐714 with Thr696 and 697‐880 with Thr853, induced the contraction of beta‐escin permeabilized rabbit ileum at constant Ca 2+ of pCa 6.3. The MYPT1 peptide‐induced force completely declined by addition of ROCK inhibitors, as well as addition of 8Br‐cGMP. Purified myosin phosphatase was inhibited by phosphorylated GST‐MYPT1(654‐714) and (696‐880) peptides, with IC50 of 1 nM and 20 nM, respectively. These peptides directly docked at the active site of myosin phosphatase catalytic subunit. Thus, phosphorylation of MYPT1 at Thr696 and 853 converts the region into autoinhibitory domain of myosin phosphatase that mediates agonist stimulation into smooth muscle contraction.