Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.
Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.
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DOI：
10.1042/bj20141156
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发表时间：
2015-03-15
期刊：
影响因子：
--
通讯作者：
Kuang J
中科院分区：
文献类型：
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作者：
Sun S;Zhou X;Zhang W;Gallick GE;Kuang J
ESCRT-III介导的膜内陷和断裂是MVB分选泛素化膜受体的关键步骤，并且通常认为是溶酶体中这些受体降解所需的。衔接蛋白阿利克斯在哺乳动物细胞中参与了多种ESCRT-III介导的膜重塑过程。然而，阿利克斯敲低并不抑制哺乳动物细胞系中活化EGFR的降解，这导致人们普遍认为阿利克斯并不关键地参与哺乳动物细胞中泛素化膜受体的MVB分选。在这项研究中，我们证明，尽管阿利克斯在活化EGFR的降解中起着非必需的作用，但它在活化EGFR的MVB分选和沉默中起着关键作用。哺乳动物细胞系的EGF刺激通过阿利克斯V结构域诱导阿利克斯与泛素化EGFR的相互作用，并通过阿利克斯Bro 1结构域增加阿利克斯与膜结合CHMP 4的结合。在连续和脉冲追踪EGF刺激条件下，抑制阿利克斯与膜结合CHMP 4的相互作用、通过V结构域抑制阿利克斯二聚化或阿利克斯敲低显著抑制活化EGFR的MVB分选并促进ERK 1/2的持续活化。在连续EGF刺激条件下，这些细胞处理也延缓了活化EGFR的降解。这些发现表明阿利克斯在哺乳动物细胞中泛素化膜受体的MVB分选中起关键作用。
ESCRT-III mediated membrane invagination and scission is a critical step in MVB sorting of ubiquitinated membrane receptors and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein ALIX is critically involved in multiple ESCRT-III-mediated membrane remodeling processes in mammalian cells. However, ALIX knockdown does not inhibit degradation of activated EGFR in mammalian cell lines, leading to a widely held notion that ALIX is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In this study, we demonstrate that despite its non-essential roles in degradation of activated EGFR, ALIX plays a critical role in MVB sorting and silencing of activated EGFR. EGF stimulation of mammalian cell lines induces ALIX interaction with ubiquitinated EGFR through the ALIX V domain and increases ALIX association with membrane-bound CHMP4 through the ALIX Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of ALIX interaction with membrane-bound CHMP4, inhibition of ALIX dimerization through the V domain or ALIX knockdown dramatically inhibits MVB sorting of activated EGFR and promotes sustained activation of ERK1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of activated EGFR. These findings indicate that ALIX is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.