Physiological signalling to myosin phosphatase targeting subunit-1 phosphorylation in ileal smooth muscle
Physiological signalling to myosin phosphatase targeting subunit-1 phosphorylation in ileal smooth muscle
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DOI：
10.1113/jp271703
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发表时间：
2016-06-15
影响因子：
5.5
通讯作者：
Stull, James T.
中科院分区：
文献类型：
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作者：
Gao, Ning;Chang, Audrey N.;Stull, James T.
肌球蛋白调节轻链 (RLC) 磷酸化引发的平滑肌收缩取决于 Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶 (MLCK) 和肌球蛋白轻链磷酸酶 (MLCP) 的相对活性。我们研究了 MLCP 调节亚基 MYPT1 在成年小鼠回肠平滑肌中的生理作用，其中（1）平滑肌特异性删除 MYPT1； (2)含有T853A敲入突变的不可磷酸化的MYPT1； (3)测量电场刺激引发的胆碱能神经刺激的力和蛋白质磷酸化反应。来自 MYPT1(SM-/-) 小鼠的分离的 MYPT1 缺陷组织迅速收缩和松弛，对 KCl 和卡巴胆碱处理和冲洗的持续反应分别有适度差异。同样，对收缩期间负责 RLC 磷酸化的调节蛋白的测量也揭示了适度的变化。正常小鼠组织与 MYPT1 T853A 敲入小鼠组织之间对卡巴胆碱的收缩或 RLC 磷酸化反应没有差异。从数量上讲，在静息条件下，野生型组织中存在大量 MYPT1 T696 和 T853 磷酸化，这预示着 MLCP 磷酸酶的高度抑制。 MYPT1 缺陷组织中 PP1c 活性的降低可能与野生型组织中 MYPT1 组成型磷酸化导致的 MLCP 活性减弱相似。电场刺激增加了野生型小鼠组织中的 RLC 磷酸化和强迫发育，但未增加 MYPT1 磷酸化。因此，回肠平滑肌中的生理性 RLC 磷酸化和力量发展似乎依赖于 MLCK 和 MLCP 活性，而不改变 MYPT1 磷酸化的组成型。
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) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1c activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation.