Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.
Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.
复制标题
DOI：
10.1083/jcb.146.1.149
复制
发表时间：
1999-07-12
期刊：
影响因子：
--
通讯作者：
Gonias SL
中科院分区：
文献类型：
--
作者：
Nguyen DH;Catling AD;Webb DJ;Sankovic M;Walker LA;Somlyo AV;Weber MJ;Gonias SL
尿激酶型纤溶酶原激活剂(UPA)在不同的细胞类型中激活丝裂原激活蛋白(MAP)激酶，即细胞外信号调节蛋白(ERK)1和2。在这项研究中，我们证明了uPA通过uPA受体(UPA)连接和ERK激活的机制刺激MCF-7乳腺癌细胞、HT1080纤维肉瘤细胞和uPAR高表达的MCF-7细胞的迁移。RAS和MAP激酶(MEK)是uPA诱导的ERK激活和刺激细胞迁移的必要条件和充分条件，这一点在这些信号蛋白的显性-负性和结构性活性突变体的实验中得到了证明。肌球蛋白轻链激酶(MLCK)也是uPA刺激的细胞迁移所必需的，这是在三种不同的MLCK抑制剂的实验中确定的。当用uPA处理MCF-7细胞时，MLCK通过MEK依赖的途径被磷酸化，并被明显激活，因为肌球蛋白II调节轻链(RLC)的丝氨酸磷酸化也增加。UPA诱导的MCF-7细胞迁移增加是由β1整合素(可能是αVβ1)和αVβ5介导的。当转染表达αVβ3的MCF-7细胞经uPA处理后，ERK仍被磷酸化，但细胞迁移没有增加。用封闭抗体中和αVβ3的功能，恢复了uPA促进细胞迁移的能力。因此，我们已经证明，uPA通过启动依赖于uPAR的信号级联来促进细胞迁移，其中RAS、MEK、ERK和MLCK作为重要的下游效应因子。
Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a β1-integrin (probably αVβ1) and αVβ5. When MCF-7 cells were transfected to express αVβ3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of αVβ3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.