Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca(2+) sensitization force of smooth muscle.
Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca(2+) sensitization force of smooth muscle.
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DOI：
10.1540/jsmr.58.22
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Kitazawa, Toshio
中科院分区：
文献类型：
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作者：
Eto, Masumi;Katsuki, Shuichi;Ohashi, Minami;Miyagawa, Yui;Tanaka, Yoshinori;Takeya, Kosuke;Kitazawa, Toshio
CPI-17调节肌球蛋白磷酸酶并介导激动剂诱导的平滑肌收缩。PKC和ROCK在Thr 38处磷酸化CPI-17，导致中央抑制结构域（PHIN结构域）的构象变化。CPI-17的N-和C-末端尾部被预测为非结构化环，并且它们的序列在哺乳动物中是保守的。在这里，我们的特点是CPI-17 N-和C-末端非结构化的尾巴，使用重组蛋白，缺乏药水。将生理水平（10 µM）的重组CPI-17蛋白掺入β-七叶皂苷渗透的平滑肌条中，用于Ca 2+致敏力测量。异位全长CPI-17增强PDBu-induced Ca 2+敏化力pCa6.3，表明肌球蛋白磷酸酶抑制。CPI-17的N-和C-末端尾部的缺失减弱了PDBu-induced Ca 2 +-敏化力的程度。N端缺失抑制了蛋白激酶C（PKC）在Thr 38的磷酸化，C端截短降低了对肌球蛋白磷酸酶的亲和力。在生理条件下，PKC和肌球蛋白磷酸酶可识别CPI-17的N-/C-末端非结构化尾部，诱导平滑肌细胞的Ca 2+增敏力。
CPI-17 regulates the myosin phosphatase and mediates the agonist-induced contraction of smooth muscle. PKC and ROCK phosphorylate CPI-17 at Thr38 leading to a conformational change of the central inhibitory domain (PHIN domain). The N- and C-terminal tails of CPI-17 are predicted as unstructured loops and their sequences are conserved among mammals. Here we characterized CPI-17 N- and C-terminal unstructured tails using recombinant proteins that lack the potions. Recombinant CPI-17 proteins at a physiologic level (10 µM) were doped into beta-escin-permeabilized smooth muscle strips for Ca2+ sensitization force measurement. The ectopic full-length CPI-17 augmented the PDBu-induced Ca2+ sensitization force at pCa6.3, indicating myosin phosphatase inhibition. Deletion of N- and C-terminal tails of CPI-17 attenuated the extent of PDBu-induced Ca2+-sensitization force. The N-terminal deletion dampened phosphorylation at Thr38 by protein kinase C (PKC), and the C-terminal truncation lowered the affinity to the myosin phosphatase. Under the physiologic conditions, PKC and myosin phosphatase may recognize CPI-17 N-/C-terminal unstructured tails inducing Ca2+ sensitization force in smooth muscle cells.