A unique platform for H-Ras signaling involving clathrin-independent endocytosis
A unique platform for H-Ras signaling involving clathrin-independent endocytosis
复制标题
DOI：
10.1091/mbc.e07-08-0841
复制
发表时间：
2008-03-01
影响因子：
3.3
通讯作者：
Donaldson, Julie G.
中科院分区：
文献类型：
--
作者：
Porat-Shliom, Natalie;Kloog, Yoel;Donaldson, Julie G.
检查H-Ras的运输以确定其是否可以通过网格蛋白非依赖性内吞作用（CIE）进入细胞。H-Ras与CIE货物蛋白（I类主要组织相容性复合体）共定位，并且它被隔离在Arf 6的活性突变体Q67 L表达后形成的空泡中。激活Ras，无论是通过表皮生长因子刺激或表达的活性突变体Ras，G12 V，诱导质膜皱褶和巨胞饮，刺激形式的CIE。表达H-RasG 12 V和荧光蛋白嵌合体的细胞的实时成像显示，进入的巨胞饮体含有磷脂酰肌醇4，5-二磷酸（PIP 2）和磷脂酰肌醇3，4，5-三磷酸（PIP 3）。PIP 2从大胞饮体的损失之后是Rab 5（Ras的下游靶标）的募集，然后是PIP 3损失。我们的研究支持一种模型，即Ras可以通过三个不同的阶段：PIP 2/PIP 3，PIP 3/Rab 5和Rab 5在大胞饮体上发出信号。在表达Arf 6 Q67 L的细胞中形成的寡核苷酸在第一阶段捕获Ras信号传导，募集Ras效应物细胞外信号调节激酶和蛋白激酶B（Akt）的活性形式，但不募集Rab 5。Arf 6刺激巨胞饮作用还涉及通过不同的脂质相，但未观察到Akt的募集。
Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatiylinositol 3,4,5-trisphosphate (PIP3). PIP2 loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP3 loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP2/PIP3, PIP3/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.