Reconstituted Human Myosin Light Chain Phosphatase Reveals Distinct Roles of Two Inhibitory Phosphorylation Sites of the Regulatory Subunit, MYPT1

Abstract

The myosin light chain phosphatase (MLCP) is a cytoskeleton-associated protein phosphatase-1 (PP1) holoenzyme and a RhoA/ROCK effector, regulating cytoskeletal reorganization. ROCK-induced phosphorylation of the MLCP regulatory subunit (MYPT1) at two sites, Thr696 and Thr853, suppresses the activity, although little is known about the difference in the role. Here, we developed a new method for the preparation of the recombinant human MLCP complex and determined the molecular and cellular basis of inhibitory phosphorylation. The recombinant MLCP partially purified from mammalian cell lysates retained characteristics of the native enzyme, such that it was fully active without Mn2+ and sensitive to PP1 inhibitor compounds. Selective thio-phosphorylation of MYPT1 at Thr696 with ROCK inhibited the MLCP activity 30%, whereas the Thr853 thio-phosphorylation did not alter the phosphatase activity. Interference with the docking of phospho-Thr696 at the active site weakened the inhibition, suggesting selective autoinhibition induced by phospho-Thr696. Both Thr696 and Thr853 sites underwent autodephosphorylation. Compared with that of Thr853, phosphorylation of Thr696 was more stable, and it facilitated Thr853 phosphorylation. Endogenous MYPT1 at Thr696 was spontaneously phosphorylated in quiescent human leiomyosarcoma cells. Serum stimulation of the cells resulted in dissociation of MYPT1 from myosin and PP1C in parallel with an increase in the level of Thr853 phosphorylation. The C-terminal domain of human MYPT1(495–1030) was responsible for the binding to the N-terminal portion of myosin light meromyosin. The spontaneous phosphorylation at Thr696 may adjust the basal activity of cellular MLCP and affect the temporal phosphorylation at Thr853 that is synchronized with myosin targeting.

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

“…21 Currently, phosphorylation of T696 is thought to directly inhibit MLCP activity towards MLC, via pseudosubstrate inhibition, whereas T853 phosphorylation is believed to cause dissociation of MLCP from myosin and/or inhibit MLCP activity directly. 68 The latter hypothesis is supported by the recent study using a genetic approach to knock-in nonphosphorylatable Ala for T852 or T694. 69 The MYPT1 T694A mutation was found to significantly inhibit sustained force as well as MLC phosphorylation, while the T852A mutation had no significant effect on maximal force development and little effect on force maintenance in neonatal bladder smooth muscle.…”

Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells

“…In terms of bladder smooth muscle, Mizuno et al [24] reported that carbachol stimulation increased MYPT1 Thr696 phosphorylation in mice, being consistent with our current observations. More recently, a report from Khasnis et al [8] demonstrated that selective phosphorylation of MYPT1 at Thr696 with ROCK inhibited the MLCP activity 30%, whereas the Thr853 phosphorylation did not alter the phosphatase activity. Their results suggested that phosphorylation of Thr696 was more stable compared with that of Thr853, and it may facilitate Thr853 phosphorylation.…”

“…MLCP is a heterotrimer that consists of three subunits: a 110-kDa regulatory myosin phosphatase-targeting subunit (MYPT1), a 38-kDa catalytic subunit of type 1 phosphatase (PP1c), and a 20-KDa subunit (M20) with unknown function [7]. Lines of evidence suggest that the regulatory MYPT1 subunit play the central role in the inhibition of cellular MLCP, which is essential for smooth muscle contraction [6,8]. Moreover, the inhibition of MLCP activity by Rho-associated protein kinase (ROCK) mediated MYPT1 phosphorylation is thought to act a key role in Ca2+-sensitized contractions of different smooth muscles [9].…”

Inhibition of protein phosphatase-1 and -2A decreases the chemosensitivity of leukemic cells to chemotherapeutic drugs

“…Anticancer drugs are known to induce apoptosis by activating 47 proapoptotic regulators and at the same time suppressing survival 48 factors [1]. Several signaling pathways may influence the efficacy of the activation of some MAP-kinase types might contribute to the 60 destructive cellular effects.…”