Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity.
Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity.
复制标题
DOI：
10.1038/onc.2012.388
复制
发表时间：
2013-08-08
期刊：
影响因子：
8
通讯作者：
Hayes, J. D.
中科院分区：
文献类型：
--
作者：
Chowdhry, S.;Zhang, Y.;McMahon, M.;Sutherland, C.;Cuadrado, A.;Hayes, J. D.
转录因子NF-E2 p45相关因子2（Nrf 2）被抑制的可调节机制的鉴定将允许设计策略，以对抗与其在Kelch样ECH相关蛋白-1（Keap 1）中的体细胞突变相关的肿瘤中的上调相关的耐药性，Keap 1是编码Cul 3-Rbx 1/Roc 1泛素连接酶的联合衔接子和底物受体的基因。我们现在发现，小鼠Nrf 2含有两个β-transducin repeat-containing蛋白（β-TrCP）的结合位点，β-TrCP作为Skp 1-Cul 1-Rbx 1/Roc 1泛素连接酶复合物的底物受体。Nrf 2中任一结合位点的缺失均降低了β-TrCP介导的转录因子泛素化。两个β-TrCP结合位点中的一个作为降解决定子的能力可以通过操纵糖原合成酶激酶-3（GSK-3）活性来增加和降低。生物素化肽下拉测定将DSGIS 338和DSAPGS 378鉴定为Nrf 2中的两个β-TrCP结合基序。值得注意的是，我们的下拉试验表明，β-TrCP与磷酸化版本的DSGIS的结合比其非磷酸化对应物更紧密，而DSAPGS的情况并非如此。这些数据表明，DSGIS，而不是DSAPGS，包含一个功能性GSK-3磷酸化位点。通过抑制磷酸肌醇3-激酶（PI 3 K）-蛋白激酶B（PKB）/Akt途径，在Keap 1缺失的小鼠胚胎成纤维细胞（MEF）或含有突变体Keap 1的人肺A549细胞中激活GSK-3，显著降低内源性Nrf 2蛋白，并将原型Nrf 2调节酶的mRNA水平降低至正常水平的10-50%，包括谷氨酸-半胱氨酸连接酶催化和修饰亚基、谷胱甘肽S-转移酶α-1和Mu-1、血红素加氧酶-1和NAD（P）H：醌氧化还原酶-1。用LY 294002 PI 3 K抑制剂或MK-2206 PKB/Akt抑制剂预处理Keap 1 −/− MEFs或A549细胞，使其对丙烯醛、苯丁酸氮芥和顺铂的敏感性增加1.9倍至3.1倍，同时用GSK-3抑制剂CT 99021预处理，这一点基本上减弱。
Identification of regulatable mechanisms by which transcription factor NF-E2 p45-related factor 2 (Nrf2) is repressed will allow strategies to be designed that counter drug resistance associated with its up-regulation in tumours that harbour somatic mutations in Kelch-like ECH-associated protein-1 (Keap1), a gene that encodes a joint adaptor and substrate receptor for the Cul3-Rbx1/Roc1 ubiquitin ligase. We now show that mouse Nrf2 contains two binding sites for β-transducin repeat-containing protein (β-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased β-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two β-TrCP-binding sites to serve as a degron could be both increased and decreased by manipulation of glycogen synthase kinase-3 (GSK-3) activity. Biotinylated-peptide pull-down assays identified DSGIS338 and DSAPGS378 as the two β-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that β-TrCP binds a phosphorylated version of DSGIS more tightly than its non-phosphorylated counterpart, whereas this was not the case for DSAPGS. These data suggest that DSGIS, but not DSAPGS, contains a functional GSK-3 phosphorylation site. Activation of GSK-3 in Keap1-null mouse embryonic fibroblasts (MEFs), or in human lung A549 cells that contain mutant Keap1, by inhibition of the phosphoinositide 3-kinase (PI3K) – protein kinase B (PKB)/Akt pathway markedly reduced endogenous Nrf2 protein and decreased to 10-50% of normal the levels of mRNA for prototypic Nrf2-regulated enzymes, including the glutamate-cysteine ligase catalytic and modifier subunits, glutathione S-transferases Alpha-1 and Mu-1, heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Pre-treatment of Keap1−/− MEFs or A549 cells with the LY294002 PI3K inhibitor or the MK-2206 PKB/Akt inhibitor increased their sensitivity to acrolein, chlorambucil and cisplatin between 1.9-fold and 3.1-fold, and this was substantially attenuated by simultaneous pre-treatment with the GSK-3 inhibitor CT99021.
登录
查看更多内容
影响因子：
5.3
作者：
Lassot, I;Ségéral, E;Margottin-Goguet, F
通讯作者：
Margottin-Goguet, F
影响因子：
5.3
作者：
Kobayashi, Makoto;Li, Li;Yamamoto, Masayuki
通讯作者：
Yamamoto, Masayuki
影响因子：
11.4
作者：
Aberle, H;Bauer, A;Kemler, R
通讯作者：
Kemler, R
影响因子：
3.8
作者：
Hwang, Yong Pil;Kim, Hyung Gyun;Jeong, Hye Gwang
通讯作者：
Jeong, Hye Gwang
影响因子：
4.8
作者：
Huang, HC;Nguyen, T;Pickett, CB
通讯作者：
Pickett, CB