Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery
Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery
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DOI：
10.1161/01.atv.0000116028.42230.4c
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发表时间：
2004-03-01
影响因子：
8.7
通讯作者：
Kanaide, H
中科院分区：
文献类型：
--
作者：
Hirano, K;Derkach, DN;Kanaide, H
目的-平滑肌肌球蛋白磷酸酶的110 kDa的调节亚基（MYPT 1）的区域参与收缩的调节在生理conditions.Methods和结果-使用HIV达特蛋白介导的蛋白转导，MYPT 1的N-末端片段被引入到完整的猪冠状动脉条。与3 mumol/L TAT-MYPT 1（1-374）（一种含有达特肽和MYPT 1残基1至374的构建体）预孵育15分钟，可增强（2.4倍）在118 mmol/L K+去极化下添加1.25 mmol/L细胞外Ca 2+诱导的随后收缩，但[Ca 2 +]（i）升高没有增强。删除达特肽MYPT 1（1-374），消除了增强效应。TAT-MYPT 1（1-296）表现出较弱但显著的扩增（1.7倍）。然而，TAT-MYPT 1（1-171）、TAT-MYPT 1（39- 374）、TAT-MYPT 1（39 - 296）和TAT-MYPT 1（297 - 374）没有增强活性。肌球蛋白轻链磷酸化水平作为细胞外Ca 2+浓度的函数，在用TAT-MYPT 1（1-374）预处理的条带中与control.Conclusions相比向左移动-区域1至296是参与增强收缩的最小区域，区域297至374起补充作用。这些结果表明，主要是催化亚基与MYPT 1之间的相互作用在完整平滑肌内源性肌球蛋白磷酸酶的调节中起关键作用。
Objective - The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions.Methods and Results - Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 mumol/L TAT-MYPT1(1-374), a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca2+ under 118 mmol/L K+ depolarization, with no augmentation of the [Ca2+](i) elevation. The deletion of the Tat peptide, MYPT1(1-374), abolished the augmenting effect. TAT-MYPT1(1-296) demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1(1-171), TAT-MYPT1(39- 374), TAT-MYPT1(39 - 296), and TAT-MYPT1(297 - 374) had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1(1-374) compared with the control.Conclusions - Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.