Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation
Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation
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DOI：
10.1074/jbc.m503173200
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发表时间：
2005-09-23
影响因子：
4.8
通讯作者：
Wysolmerski, RB
中科院分区：
文献类型：
--
作者：
Goeckeler, ZM;Wysolmerski, RB
本研究确定了细胞内 cAMP 和 cAMP 依赖性蛋白激酶激活增加对内皮细胞基础和凝血酶诱导的等长张力发展的影响。 10 μM 毛喉素、40 μM 3-异丁基-1-甲基黄嘌呤诱导的 cAMP 升高和最大 cAMP 依赖性蛋白激酶激活导致肌球蛋白 II 调节轻链 (RLC) 磷酸化减少 50%，等长张力下降 35%，但它不会抑制凝血酶刺激的 RLC 磷酸化和等长张力增加 紧张。 cAMP 的升高不会改变肌球蛋白轻链激酶的催化活性。然而，用 KT5926 直接抑制肌球蛋白轻链激酶导致 RLC 磷酸化降低 90%，等长张力仅略有降低，但它阻止了凝血酶诱导的 RLC 磷酸化和等长张力发展的增加。我们发现，cAMP 升高会使 RhoA 的磷酸化增加 10 倍，同时 RhoA 活性降低 60%，RLC 磷酸酶活性增加 78%。有证据表明，正是 RhoA 的失活通过涉及丝切蛋白的途径调节等长张力的降低。经毛喉素/3-异丁基-1-甲基黄嘌呤处理的单层细胞提取物中，活化的丝动蛋白丝切蛋白与 F-肌动蛋白切断活性增加相关。用互变霉素（一种 1 型蛋白磷酸酶抑制剂）对培养物进行预处理，可阻断 cAMP 对 1) cofilin 去磷酸化、2) RLC 磷酸化减少和 3) 等长张力减少的影响。总之，这些数据提供了体内证据，表明细胞内 cAMP 升高通过抑制 RhoA 信号及其调节肌球蛋白 II 活性和肌动蛋白重组的下游途径来调节内皮细胞等长张力和 RLC 磷酸化。
This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 mu M forskolin, 40 mu M 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.