The tumour suppressor PTEN mediates a negative regulation of the E3 ubiquitin-protein ligase Nedd4
The tumour suppressor PTEN mediates a negative regulation of the E3 ubiquitin-protein ligase Nedd4
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DOI：
10.1042/bj20071403
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发表时间：
2008-06-01
影响因子：
4.1
通讯作者：
Lee, Cheolju
中科院分区：
文献类型：
--
作者：
Ahn, Younghee;Hwang, Chae Young;Lee, Cheolju
肿瘤抑制基因PTEN(第10号染色体上缺失的磷酸酶和张力蛋白同源物，是一种磷脂酰肌醇3-磷酸酶)是一种多功能蛋白，在许多类型的癌症中都是去调控的。这表明，许多与PTEN功能或物理上相关的蛋白质尚未被发现。为了寻找可能在调控PTEN中起关键作用的PTEN相互作用蛋白，我们采用了一种基于蛋白质组学的方法。将表达PTEN的NIH3T3细胞裂解产物用于亲和层析，然后用LC-ESI-MS/MS(LC-ESI-MS-Tandem MS)进行分析。共鉴定出93种蛋白质。在已鉴定的蛋白质中，我们集中在E3泛素蛋白连接酶Nedd4(神经前体细胞表达，发育下调基因4)上，并使用HeLa细胞进行了后续的验证实验。Nedd4抑制PTEN诱导的细胞死亡，反之，PTEN下调Nedd4水平。PTEN催化位点的突变(C124S)减弱了下调效应。PI3K(磷酸肌醇3-激酶)抑制剂LY294002也降低了Nedd4的表达，表明这种调节依赖于PTEN-PI3K/Akt通路的磷酸酶活性。实时荧光定量聚合酶链式反应分析表明，Nedd4基因受PTEN转录调控。因此，我们的结果对于PTEN在E3辅素连接酶Nedd4中作为负反馈调节因子和底物的作用具有重要的意义。
The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) is a multifunctional protein deregulated in many types of cancer. It is suggested that a number of proteins that relate with PTEN functionally or physically have not yet been found. In order to search for PTEN-interacting proteins that might be crucial in the regulation of PTEN, we exploited a proteomics-based approach. PTEN-expressing NIH 3T3 cell lysates were used in affinity chromatography and then analysed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem MS). A total of 93 proteins were identified. Among the proteins identified, we concentrated on the E3 ubiquitin-protein ligase Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated gene 4), and performed subsequent validation experiments using HeLa cells. Nedd4 inhibited PTEN-induced apoptotic cell death and, conversely, the Nedd4 level was down-regulated by PTEN. The down-regulation effect was diminished by a mutation (C124S) in the catalytic site of PTEN. Nedd4 expression was also decreased by a PI3K (phosphoinositide 3-kinase) inhibitor, LY294002, suggesting that the regulation is dependent on the phosphatase-kinase activity of the PTEN-PI3K/Akt pathway. Semi-quantitative real-time PCR analysis revealed that Nedd4 was transcriptionally regulated by PTEN. Thus our results have important implications regarding the roles of PTEN upon the E3 ubquitin ligase Nedd4 as a negative feedback regulator as well as a substrate.