Cloning and characterization of an androgen receptor N-terminal-interacting protein with ubiquitin-protein ligase activity
Cloning and characterization of an androgen receptor N-terminal-interacting protein with ubiquitin-protein ligase activity
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DOI：
10.1677/jme.0.0290041
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发表时间：
2002-08-01
影响因子：
3.5
通讯作者：
Trifiro, MA
中科院分区：
文献类型：
--
作者：
Beitel, LK;Elhaji, YA;Trifiro, MA
雄激素受体（AR） n端结构域在雄激素应答基因调控中起关键作用。利用酵母双杂交系统分离到一种新的AR n端相互作用蛋白（ARNIP），并通过谷胱甘肽s转移酶下拉试验证实其与人AR正常区或相应区11-172氨基酸相互作用。从NSC-34（小鼠神经母细胞瘤/脊髓）或PC-3（人前列腺腺癌）mRNA克隆的ARNIP cdna编码高度同源的30 kDa（261个氨基酸）富含半胱氨酸的蛋白，具有RING-H-2 （C3H2C3锌指）结构域；该基序在其他几种物种预测的arip同源蛋白中高度保守。Northern blotting在多种组织中检测到类似于1.7 kb的ARNIP mRNA的表达，但在小鼠睾丸、肾脏和几种神经细胞系中表达最多。此外，人类ARNIP蛋白被发现在4q21染色体上由9个长32 kb的外显子编码。在COS-1细胞中，ARNIP和AR的共表达不影响AR配体结合动力学，ARNIP在反活化试验中也不作为共激活因子或共抑制因子。然而，ARNIP的存在降低了AR n端：c端相互作用。有趣的是，ARNIP，特别是它的RING-H2结构域，在体外泛素结合酶Ubc4-1存在的情况下作为泛素蛋白连接酶起作用。ARNIP环- h2结构域（Cys145Ala）中单个半胱氨酸残基的突变使这种E3泛素连接酶活性丧失。荧光蛋白标记研究显示，在COS-1细胞中AR-ARNIP的相互作用是不依赖于激素的，这表明在雄激素加入后AR和ARNIP在细胞核中的共定位可能使ARNIP在核过程中发挥作用。因此，鉴定一种具有泛素连接酶活性的新型ar相互作用蛋白将刺激进一步研究泛素化和泛素-蛋白酶体系统在ar介导的细胞功能中的作用。
The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H-2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the similar to1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.