Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen
Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen
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DOI：
10.1073/pnas.1401556111
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发表时间：
2014-03-04
影响因子：
11.1
通讯作者：
Sarikas, Antonio
中科院分区：
文献类型：
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作者：
Hartmann, Thomas;Xu, Xinsong;Sarikas, Antonio
猴病毒40(SV40)大肿瘤抗原(LT)通过抑制关键的肿瘤抑制蛋白(包括P53和视网膜母细胞瘤家族成员)而触发致癌转化。此外，SV40的转化还需要LT与cullin 7(CUL7)结合，cullin 7是cullin环E3泛素连接酶7(CRL7)的核心成分。然而，LT-CUL7相互作用的致病机制作用大多尚不清楚。在此，我们报道了SV40 LT抑制CRL7泛素连接酶功能的体外和体内实验证据。我们发现，SV40 LT，而不是CUL7结合缺陷突变体(LT Delta 69-83)，损害了26S蛋白酶体依赖的CRL7靶蛋白胰岛素受体底物1(IRS1)的蛋白分解，IRS1是胰岛素和胰岛素样生长因子1信号通路的组成部分。SV40 LT的表达导致IRS1的蓄积和半衰期延长。在体外，纯化的SV40 LT以浓度依赖的方式减少CRL7依赖的IRS1泛素化。SV40 LT的表达或CUL7的RNA干扰导致IRS1下游信号通路磷脂酰肌醇-3激酶/AKT和ERK丝裂原激活通路的激活，以及下游靶基因c-fos的上调。最后，SV40 LT阳性的癌胚抗原424/SV40 LT转基因小鼠表现出IRS1蛋白水平升高和下游信号的激活。综上所述，这些数据表明，SV40 LT保护IRS1免受CRL7介导的降解，从而维持高水平的促有丝分裂IRS1下游信号通路。
Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT Delta 69-83), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.