《The Journal of Physiology》 14:3031-3051 （2014）
Constitutive phosphorylation of myosin phosphatase targeting subunit-1 in smooth muscle: MYPT1 regulation and function in smooth muscle
Tsai, Ming-Ho; Chang, Audrey N.; Huang, Jian; He, Weiqi; Sweeney, H. Lee; Zhu, Minsheng; Kamm, Kristine E.; Stull, James T.
Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca2+–calmodulin-dependent myosin light chain kinase (MLCK) andmyosin light chain phosphatase (MLCP).We have investigated the physiological role of theMLCP regulatory subunitMYPT1 in bladder smooth muscle containing a smooth muscle-specific deletion of MYPT1 in adult mice. Deep-sequencing analyses of mRNA and immunoblotting revealed that MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in totalhomogenates fromwild-type orMYPT1-deficient tissues. IsolatedMYPT1-deficient tissues from MYPT1SM−/− mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5-fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation ofMLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuatedMLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist-induced MYPT1 phosphorylation to inhibitMLCP activity. In summary, MYPT1 deficiency may not cause significant derangement of smooth muscle contractility because the effective MLCP activity is not changed.