Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis.
Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis.
复制标题
DOI：
10.1111/j.1600-0854.2008.00788.x
复制
发表时间：
2008-09
期刊：
影响因子：
--
通讯作者：
Sorkin A
中科院分区：
文献类型：
--
作者：
Galperin E;Sorkin A
为了研究活细胞中丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK 1/2）信号级联的时空调节，产生了HeLa细胞系，其中MAPK激酶或ERK激酶（MEK）2（MAPK激酶）通过RNA干扰被敲低，并被绿色荧光蛋白（GFP）标记的MEK 2取代。在这些细胞中，MEK 2-GFP以与亲本细胞中内源性MEK 2相似的水平稳定表达。在EGF受体（EGFR）激活后，发现MEK 2-GFP库最初易位到质膜，然后在早期和晚期内体的子集中积累。然而，活化的MEK仅在质膜处检测到，而不在内体中检测到。令人惊讶的是，MEK 2-GFP内体不含活性EGFR，表明内体MEK 2-GFP与上游信号传导复合物分离。通过小干扰RNA（siRNA）敲低网格蛋白消除了MEK 2向内体的募集，但导致ERK的活化增加而不影响MEK 2-GFP的活性。MEK 2-GFP在内体中的积累也被siRNA消耗RAF激酶和MEK 1/2抑制剂UO 126阻断。我们认为MEK 2向核内体的募集可能是EGFR-MAPK信号通路通过内吞作用负反馈调节的一部分。
To study spatiotemporal regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling cascade in living cells, a HeLa cell line in which MAPK kinase of ERK kinase (MEK) 2 (MAPK kinase) was knocked down by RNA interference and replaced with the green fluorescent protein (GFP)-tagged MEK2 was generated. In these cells, MEK2–GFP was stably expressed at a level similar to that of the endogenous MEK2 in the parental cells. Upon activation of the EGF receptor (EGFR), a pool of MEK2–GFP was found initially translocated to the plasma membrane and then accumulated in a subset of early and late endosomes. However, activated MEK was detected only at the plasma membrane and not in endosomes. Surprisingly, MEK2–GFP endosomes did not contain active EGFR, suggesting that endosomal MEK2–GFP was separated from the upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in increased activation of ERK without affecting the activity of MEK2–GFP. The accumulation of MEK2–GFP in endosomes was also blocked by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the negative feedback regulation of the EGFR–MAPK signaling pathway by endocytosis.
登录
查看更多内容
影响因子：
5.3
作者：
Janssen, RAJ;Kim, PN;Morrison, DK
通讯作者：
Morrison, DK
影响因子：
3.5
作者：
BRUNET, A;PAGES, G;POUYSSEGUR, J
通讯作者：
POUYSSEGUR, J
影响因子：
5.7
作者：
Hao, Honglin;Muniz-Medina, Vanessa M.;Shields, Janiel M.
通讯作者：
Shields, Janiel M.
影响因子：
11.4
作者：
DIGUGLIELMO, GM;BAASS, PC;BERGERON, JJM
通讯作者：
BERGERON, JJM
影响因子：
48
作者：
Galperin, E;Verkhusha, V;Sorkin, A
通讯作者：
Sorkin, A