Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells
Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells
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EGF 刺激细胞中内源性荧光标记 RAF1 的定位动态
DOI：
10.1091/mbc.e18-08-0512
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发表时间：
2019
影响因子：
3.3
通讯作者：
Heldin, Carl-Henrik
中科院分区：
文献类型：
--
作者：
Surve, Sachin V.;Myers, Paul J.;Clayton, Samantha A.;Watkins, Simon C.;Lazzara, Matthew J.;Sorkin, Alexander;Heldin, Carl-Henrik
表皮生长因子（EGF）受体（EGFR）在细胞表面的活化通过RAS-RAF-MAPK/ERK 1/2途径和受体内吞作用启动信号传导。这种信号传导是否从内体继续仍然不清楚，因为RAS主要位于质膜上，并且内源性RAF激酶（RAS的下游效应物）的定位未被定义。为了检查RAF定位，我们使用基因编辑用mVenus标记内源性RAF 1。从10%到15%的RAF 1-mVenus（<2000分子/细胞），最初完全是胞质的，在EGF刺激后瞬时易位到质膜。在易位的早期爆发之后，膜相关的RAF 1-mVenus通过显微镜或亚细胞分级分离检测不到，并且该池估计为每个细胞<200个分子。相反，RAF 1-mVenus持续EGF依赖性转运至质膜是由RAF抑制剂索拉非尼驱动的，索拉非尼增加了Ras-GTP：RAF 1相互作用的亲和力。RAF 1-mVenus在任何条件下均未在含EGFR的内体中发现。RAF 1动力学的计算模型显示，RAF 1膜丰度控制最显着的协会和RAS-GTP和RAS-GTP浓度的解离速率。该模型进一步表明，与RAF 1膜定位相比，RAF-MEK 1/2-ERK 1/2模块的相对延长的激活可能涉及多轮细胞溶质RAF 1与膜上的活性RAS再结合。
Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.
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影响因子：
3.5
作者：
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Muratcioglu, Serena;Chavan, Tanmay S.;Freed, Benjamin C.;Jang, Hyunbum;Khavrutskii, Lyuba;Freed, R. Natasha;Dyba, Marzena A.;Stefanisko, Karen;Tarasov, Sergey G.;Gursoy, Attila;Keskin, Ozlem;Tarasova, Nadya I.;Gaponenko, Vadim;Nussinov, Ruth
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Nussinov, Ruth
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3.3
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Huang, FT;Sorkin, A
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通讯作者：
Morrison, Deborah K.
影响因子：
3.3
作者：
Jiang, XJ;Sorkin, A
通讯作者：
Sorkin, A