Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity
Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity
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DOI：
10.1038/sj.onc.1208743
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发表时间：
2005-09-01
期刊：
影响因子：
8
通讯作者：
Enrich, C
中科院分区：
文献类型：
--
作者：
Grewal, T;Evans, R;Enrich, C
膜联蛋白A6是一种钙依赖性膜结合蛋白，与信号蛋白相互作用，包括GTP酶激活蛋白p120 GAP，Ras最重要的灭活剂之一。由于我们已经证明，膜联蛋白A6抑制EGF和TPA诱导的Ras信号转导，我们调查是否通过膜联蛋白A6调节Ras活性介导的p120 GAP的亚细胞定位改变。首先，我们利用我们的观察，高密度脂蛋白（HDL）可以激活Ras/MAP激酶途径。膜联蛋白A6的表达导致HDL诱导的Ras和Raf-1活化显著减少。膜联蛋白A6促进p120 GAP在体外的膜结合，和在活细胞中的质膜靶向p120 GAP，两者都以Ca 2+依赖的方式，这与膜联蛋白A6促进p120 GAP-Ras在质膜上的Ca 2+依赖性组装一致。然后，我们将这些研究扩展到其他细胞类型和刺激。A431细胞中膜联蛋白A6的表达减少，而HeLa细胞中RNAi介导的膜联蛋白A6的抑制增强EGF诱导的Ras和Erk活化。重要的是，在RNAi介导的p120 GAP水平降低后，表达膜联蛋白A6的A431细胞中Ras活化的增强比对照更显著，表明膜联蛋白A6对Ras的作用是通过p120 GAP介导的。最后，我们证明，膜联蛋白A6促进质膜靶向的p120 GAP在A431细胞响应于各种刺激，导致与H-Ras共定位。这些发现表明膜联蛋白A6在调节p120 GAP的质膜定位和Ras活性中的重要作用。
Annexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF- and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDL-induced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca2+-dependent manner, which is consistent with annexin A6 promoting the Ca2+-dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF- induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity.