Agonist-induced changes in the phosphorylation of the myosin-binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle
Agonist-induced changes in the phosphorylation of the myosin-binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle
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DOI：
10.1042/bj20021040
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发表时间：
2003-01-01
影响因子：
4.1
通讯作者：
Ikebe, M
中科院分区：
文献类型：
--
作者：
Niiro, N;Koga, Y;Ikebe, M
抑制肌球蛋白轻链磷酸酶 (MLCP) 可增强恒定 [Ca2+] 下的平滑肌收缩。有两个成分，MLCP 的肌球蛋白结合亚基 (MBS) 和 CPI171，被认为负责外部刺激对 MLCP 的抑制。 MBS 在 Thr-641 处的磷酸化和 CPI17 在 Thr-38 处的磷酸化可抑制体外 MLCP 活性。在这里，我们使用磷酸化位点特异性抗体作为探针，测定了完整和透化平滑肌条中激动剂刺激后 MBS 和 CPI17 磷酸化的变化。在α-毒素外皮纤维和完整纤维中，激动剂刺激后，CPI17 磷酸化短暂增加。刺激后 CPI17 磷酸化增加的时间过程与肌球蛋白调节轻链 (MLC) 磷酸化的增加相关。 Rho 激酶抑制剂 Y27632 和蛋白激酶 C 抑制剂 GF109203alpha 显着减弱了 CPI17 磷酸化的增加，表明蛋白激酶 C 和 Rho 激酶途径均影响 CPI17 磷酸化的变化。另一方面，对于去皮纤维和完整纤维，在静息状态下观察到抑制位点 Thr-641 处的 MBS 磷酸化水平显着，并且激动剂刺激没有显着改变 Thr-641 处的 MBS 磷酸化水平。虽然去除激动剂显着降低了 MLC 磷酸化并诱导松弛，但 MBS 的磷酸化没有改变，而 CPI17 磷酸化则显着减弱。这些结果强烈表明，在激动剂诱导的肌球蛋白磷酸化和平滑肌收缩的增加中，CPI17 的磷酸化比在 Ca2+ 不依赖的平滑肌收缩激活机制中的 MBS 磷酸化发挥更重要的作用。
The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI171 thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203alpha, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.