Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: Impact on membrane blebbing
Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: Impact on membrane blebbing
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DOI：
10.1091/mbc.e02-04-0235
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发表时间：
2003-04-01
影响因子：
3.3
通讯作者：
Huot, J
中科院分区：
文献类型：
--
作者：
Houle, FO;Rousseau, S;Huot, J
氧化应激在内皮细胞中诱导应激激活蛋白激酶 2/p38 和细胞外信号调节激酶 (ERK) 丝裂原激活蛋白激酶快速且短暂的共激活。我们发现，抑制 ERK 通路会导致氧化应激 5 毫米内的粘着斑错误组装，其特征是关键蛋白（如桩蛋白）错误定位。在 H2O2 存在的情况下，用丝裂原激活蛋白激酶激酶 (MEK) 抑制剂 PDO98059（2'-氨基-3'-甲氧基黄酮）或 ERK 激酶阴性突变体抑制 ERK 后发生的粘着斑错误组装，导致快速而强烈的膜起泡，这与内皮的严重损伤有关。我们通过二维凝胶电泳分离了 38 kDa 的 PD098059 敏感磷蛋白，通过质谱分析将其鉴定为原肌球蛋白-1。事实上，H2O2 诱导原肌球蛋白发生时间依赖性磷酸化，该磷酸化对 PDO98059 和 UO126（1,4-二氨基-2,3-二氰基-1,4-双[2-氨基苯硫基]丁二烷）的抑制敏感。原肌球蛋白磷酸化也由 MEK1 (MEKCA) 的组成型激活形式的表达诱导，这证实其磷酸化是由 ERK 激活引起的。在未受刺激的情况下。在细胞中，原肌球蛋白-1 被发现弥漫在细胞中，而在 H2O2 刺激 ERK 或通过 MEKCA 表达后，它很快与肌动蛋白和应力纤维共定位。我们认为 ERK 下游原肌球蛋白-1 的磷酸化通过促进肌动蛋白丝的形成来增加细胞收缩性并促进粘着斑的形成。顺便说一句，ML-7（1-[5碘萘-1-磺酰基]高哌嗪，HCl）是一种细胞收缩力抑制剂，可抑制原肌球蛋白的磷酸化，并阻止应力纤维和粘着斑的形成，这也会在氧化应激存在时导致膜起泡。我们发现原肌球蛋白-1 在 ERK 下游被磷酸化，这是调节其与肌动蛋白相互作用的事件，可能有助于进一步了解该蛋白在调节与细胞骨架重塑相关的细胞功能中的作用。
Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 mm of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PDO98059 (2'-amino-3'-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H2O2 resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H2O2 induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PDO98059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEKCA), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated. cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H2O2 or by expression of MEKCA. We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7(1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.