Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Present address: Graduate Institute of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd, San Ming District, Kaohsiung, Taiwan.
J Physiol. 2017 Oct 1;595(19):6231-6247. doi: 10.1113/JP274715. Epub 2017 Aug 24.
Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.
Ca /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1 , MYPT1 and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.
平滑肌肌球蛋白调节轻链（RLC）由 Ca/钙调蛋白依赖性肌球蛋白轻链激酶磷酸化，并由肌球蛋白轻链磷酸酶（MLCP）去磷酸化。气管平滑肌除了含有 MLCP 调节亚基 MYPT1 外，还含有大量的肌球蛋白结合亚基 85（MBS85），另一种肌球蛋白磷酸酶靶向亚基（MYPT）家族成员。牛气管平滑肌对乙酰胆碱的浓度/时间反应，对 RLC 的磷酸化、MYPT1、MBS85 和桩蛋白的磷酸化显示出相似的敏感性。电刺激通过从神经释放 ACh 来增加 RLC 的磷酸化，但不增加 MYPT1 或 MBS85 的磷酸化。因此，神经介导的信号转导模块对 RLC 磷酸化的儿茶酚胺反应与加入浴液的激动剂的药理学反应不同。MYPT1 的条件敲除或在小鼠中的 T853A 基因突变对分离的气管组织中的毒蕈碱力反应没有影响。MLCP 活性可能来自 MYPT1 和 MBS85 之间功能共享的作用，导致 MYPT1 敲除对收缩的影响最小。
肌球蛋白轻链激酶（MLCK）的 Ca/钙调蛋白激活引发肌球蛋白调节轻链（RLC）磷酸化，从而引发平滑肌收缩，随后由肌球蛋白轻链磷酸酶（MLCP）介导的 RLC 去磷酸化引起松弛，MLCP 包含调节（MYPT1）和催化（PP1cδ）亚基。RLC 磷酸化依赖性力发展受到涉及蛋白质磷酸化的不同信号转导模块的调节。我们研究了牛气管平滑肌对胆碱能激动剂处理与电刺激（EFS）神经刺激的反应。乙酰胆碱的浓度/时间反应表明，力发展与 RLC 磷酸化之间存在紧密的偶联，但 MLCK、MYPT1 T853、MYPT1 T696、肌球蛋白结合亚基 85（MBS85）、桩蛋白和 CPI-17（PKC 增强的蛋白磷酸酶 1 抑制剂蛋白 17 kDa）磷酸化的敏感性存在差异。EFS 增加了 RLC、CPI-17 和 MLCK 的力和磷酸化。在胆碱酯酶抑制剂新斯的明存在的情况下，EFS 导致 MYPT1 T853、MYPT1 T696、MBS85 和桩蛋白的磷酸化进一步增加。因此，在作用于 RLC 磷酸化和力反应的信号转导模块中，存在着明显的药理学与生理学反应，这可能与退化的 G 蛋白信号网络有关。使用基因修饰小鼠进行了研究。另一种 MYPT1 家族成员 MBS85 在小鼠和牛气管平滑肌中的表达丰富。MYPT1、MYPT1 和含有非磷酸化 MYPT1 T853A 的突变体小鼠的气管中，乙酰胆碱的浓度/时间力反应相似，RLC 磷酸化没有差异。因此，MYPT1 T853 磷酸化对于调节气道平滑肌的 RLC 磷酸化不是必需的。此外，MLCP 活性可能来自 MYPT1 和 MBS85 之间功能共享的作用，导致 MYPT1 敲除对收缩的影响最小。
