Key pointsr Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca 2+ /calmodulindependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities.r MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro.r Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo.r Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.Abstract Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.
Background: MYPT1 is a regulatory subunit of myosin phosphatase. Results: Deleting MYPT1 in vascular smooth muscle enhances myosin phosphorylation, contractility, and blood pressure. Conclusion: Genetic evidence shows MYPT1 plays a role in modulating vascular smooth muscle contractility. Significance: Although MYPT1 is not essential for vascular smooth muscle contractility, it contributes to blood pressure maintenance in vivo through signaling to myosin phosphorylation.
Different interacting signaling modules involving Ca2؉ / calmodulin-dependent myosin light chain kinase, Ca 2؉ -independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K ؉ -depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.
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