New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation

Abstract

Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca2+], and hence, a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9 ± 1 % under resting conditions (PSS) to 36 ± 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8 s, which was associated with an initial decline in RLC phosphorylation to 18 % within 2 s and a rebound to 34 % after 30 s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone.

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle

“…9 A ), monophosphorylated smRLC (denoted as P3), a mixture of diphosphorylated smRLC and unphosphorylated nonmuscle RLC (nmRLC; P2), and monophosphorylated nmRLC (P1, the most acidic spot in Fig. 9 A ; Yuen et al, 2009 ; Puetz et al, 2012 ; Zhang and Gunst, 2017 ). A combination of 2-D Phos-Tag PAGE and Western blot analysis using phosphospecific antibodies against pS19 indicated that spot U represented unphosphorylated RLC.…”

Role of serine-threonine phosphoprotein phosphatases in smooth muscle contractility

“…MLC20 can be diphosphorylated at Ser 19 and Thr 18 , and Thr 18 phosphorylation may sustain contractile force by reducing the rate of MLC20 dephosphorylation (228). However, Thr 18 phosphorylation alone does not affect contractile force (191,228). MLC20 phosphorylation is modulated by smooth muscle serine/threonine myosin phosphatases (MPs), which dephosphorylate MLC20 at Ser 19 and Thr 18 , thereby causing relaxation (72,93).…”

Investigating the role of phosphorylated regulatory light chains during heart failure progression

“…Supporting this, a study has reported that MLCK levels were significantly decreased 3 weeks post-MI, in HF mouse model (88). Cardiac isoforms are phosphorylated on a minute timescale, while smooth and fast skeletal RLCs are phosphorylated on a timescale of seconds in response to stimuli (293,(295)(296)(297). However, the heart is purely made of cardiac specific fibres, with no other muscle isotypes such as smooth, fast-twitch or slow-twitch skeletal muscle (298).…”