Monoubiquitination of pro-amphiregulin regulates its endocytosis and ectodomain shedding
Monoubiquitination of pro-amphiregulin regulates its endocytosis and ectodomain shedding
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DOI：
10.1016/j.bbrc.2012.02.156
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发表时间：
2012-04-06
影响因子：
3.1
通讯作者：
Higashiyama, Shigeki
中科院分区：
文献类型：
--
作者：
Fukuda, Shinji;Nishida-Fukuda, Hisayo;Higashiyama, Shigeki
表皮生长因子（EGF）家族的所有成员均以跨膜前体的形式表达于细胞表面，然后通过解整合素和金属蛋白酶（亚当斯）蛋白水解转化为EGF受体（EGFR）的可溶性配体。由于酶-底物复合物的形成对于这种“胞外域脱落”是必不可少的，细胞表面保留的改变可能影响它们与亚当斯的物理相互作用，并最终有助于脱落效率。在这里，我们表明，前双调蛋白（pro-AREG，EGFR配体）的单泛素化加速了其在细胞表面的半衰期。在作为主要受体位点的pro-AREG的赖氨酸240处发生单泛素化。使用pro-AREG和单体泛素突变体（pro-AREGmUb）的嵌合蛋白，免疫细胞化学分析和细胞表面生物素化测定显示，pro-AREGmUb的显著部分在细胞表面上表达，立即被内吞，并且主要定位于早期内体。重要的是，与野生型pro-AREG相比，由十四酰佛波醇乙酸酯诱导的pro-AREG mUb的胞外域脱落显著减少。这些结果表明，pro-AREG monoubiquitination和随后的运输到细胞内细胞器是一种新的脱落调节机制，有助于分泌EGFR配体的生长因子信号传导。(C)2012 Elsevier Inc. All rights reserved.
All members of epidermal growth factor (EGF) family are expressed as transmembrane precursors on cell surfaces and then proteolytically converted to soluble ligands for EGF receptor (EGFR) by a disintegrin and metalloproteases (ADAMs). As enzyme-substrate complex formation is essential for this "ectodomain shedding", alteration of cell surface retention could affect their physical interaction with ADAMs and eventually contribute to shedding efficiency. Here, we showed that monoubiquitination of pro-amphiregulin (pro-AREG, an EGFR ligand) accelerated its half-life on cell surface. Monoubiquitination occurred at lysine 240 of pro-AREG as the primary acceptor site. Using a chimeric protein of pro-AREG and a monomeric ubiquitin mutant (pro-AREGmUb), immunocytochemical analysis and a cell surface biotinylation assay revealed that a significant portion of pro-AREGmUb was expressed on the cell surface, immediately endocytosed, and predominantly localized to early endosomes. Importantly, ectodomain shedding of pro-AREGmUb induced by tetradecanoyl phorbol acetate was significantly reduced in comparison to wild-type pro-AREG. These results suggested that pro-AREG monoubiquitination and the subsequent trafficking to intracellular organelles is a novel shedding regulatory mechanism that contributes to the secretion of EGFR ligands in growth factor signaling. (C) 2012 Elsevier Inc. All rights reserved.