Role of N-end rule ubiquitin ligases UBR1 and UBR2 in regulating the leucine-mTOR signaling pathway
Role of N-end rule ubiquitin ligases UBR1 and UBR2 in regulating the leucine-mTOR signaling pathway
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DOI：
10.1111/j.1365-2443.2010.01385.x
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发表时间：
2010-04-01
期刊：
影响因子：
2.1
通讯作者：
Handa, Hiroshi
中科院分区：
文献类型：
--
作者：
Kume, Kanako;Iizumi, Yosuke;Handa, Hiroshi
在20种天然氨基酸中，亮氨酸对促进细胞蛋白质合成特别重要。亮氨酸的作用涉及哺乳动物雷帕霉素靶蛋白（mTOR），一种控制细胞生长的关键蛋白激酶。亮氨酸增强mTOR介导的S6 K1和4 E-BP磷酸化，从而促进蛋白质合成。然而，亮氨酸的存在是如何被感知并传递到mTOR的，人们对此知之甚少。在这里，我们证明了UBR 1和UBR 2可能是亮氨酸的细胞靶点。UBR 1和UBR 2是E3泛素连接酶，其识别N-末端残基的身份并根据N-末端规则促进靶蛋白的选择性去稳定化。使用亮氨酸固定的亲和珠，我们确定UBR 1和UBR 2作为亮氨酸结合蛋白的亮氨酸响应大鼠肝癌H4 IIE细胞。在氨基酸饥饿的人293 T细胞中，UBR 1或UBR 2的过表达导致mTOR依赖性S6 K1磷酸化的减少，而UBR 1或UBR 2的敲低增加了S6 K1磷酸化。我们还发现，亮氨酸结合到UBR 2的底物识别结构域，并抑制N-末端规则底物在体外的降解。这些发现表明，UBR 1和UBR 2是亮氨酸-mTOR信号通路的负调节剂。亮氨酸可能部分通过抑制它们的泛素连接酶活性来激活该途径。
Of 20 natural amino acids, leucine is particularly important for promoting cellular protein synthesis. The effect of leucine involves mammalian target of rapamycin (mTOR), a key protein kinase controlling cell growth. Leucine enhances mTOR-mediated phosphorylation of S6K1 and 4E-BP, thereby promoting protein synthesis. However, how the presence of leucine is sensed and transmitted to mTOR is poorly understood. Here, we show evidence that UBR1 and UBR2 might be cellular targets of leucine. UBR1 and UBR2 are E3 ubiquitin ligases that recognize the identity of N-terminal residues and contribute to selective destabilization of target proteins according to the N-end rule. Using leucine-immobilized affinity beads, we identified UBR1 and UBR2 as leucine-binding proteins from leucine-responsive rat hepatoma H4IIE cells. Over-expression of UBR1 or UBR2 resulted in a reduction in mTOR-dependent S6K1 phosphorylation, whereas knockdown of UBR1 or UBR2 increased S6K1 phosphorylation in amino acid-starved human 293T cells. We also found that leucine binds to the substrate-recognition domain of UBR2 and inhibits degradation of N-end rule substrates in vitro. These findings suggest that UBR1 and UBR2 are negative regulators of the leucine-mTOR signaling pathway. Leucine might activate this pathway in part through inhibition of their ubiquitin ligase activity.