Examining the mechanism of Erk nuclear translocation using green fluorescent protein
Examining the mechanism of Erk nuclear translocation using green fluorescent protein
复制标题
DOI：
10.1016/s0014-4827(03)00037-5
复制
发表时间：
2003-05-01
影响因子：
3.7
通讯作者：
Stork, PJS
中科院分区：
文献类型：
--
作者：
Horgan, AM;Stork, PJS
在神经细胞中，丝裂原活化蛋白激酶(MAP)级联是神经营养素信号从细胞表面受体传递到细胞核的重要中介，从而导致基因表达的变化。ERK的核定位被认为是这些效应的必要条件。为了研究ERK核转位的机制和调控，我们创建了一个绿色荧光蛋白(GFP)标记的ERK2结构，它提供了一种敏感的手段来跟踪ERK在受体介导的MAP激酶激活后从细胞质到细胞核的运动。在PC12细胞中使用这个系统，我们研究了一些与调节ERK转位有关的机制。在PC12细胞中，NGF和EGF诱导需要RAS和MEK的GFP-Erk的快速易位。我们发现ERK的长时间磷酸化不是生长因子刺激后ERK快速和早期进入细胞核所必需的。此外，GFP-Erk在内流后迅速返回细胞质，而不受其磷酸化状态的影响。ERK从其胞浆激活剂MEK释放，然后Erk二聚化，足以刺激核摄取，而Erk激酶活性是必不可少的。据报道，在PC12细胞中，ERK的转位需要PKA的活性。然而，PKA活性也不是NGF或RAS将Erk早期移位到核中所必需的，但它能够诱导少量的Erk内流，这可以用GFP-ERK2来测量。(C)2003年埃尔塞维尔科学公司(美国)。版权所有。
In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2. (C) 2003 Elsevier Science (USA). All rights reserved.