Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways
Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways
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DOI：
10.1073/pnas.110149597
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发表时间：
2000-06-06
影响因子：
11.1
通讯作者：
Dixon, JE
中科院分区：
文献类型：
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作者：
Clemens, JC;Worby, CA;Dixon, JE
我们证明了双链RNA介导的干扰（RNAi）的基因表达在几个果蝇细胞系中产生特定蛋白质的“敲除”表型的功效。我们证明了这种技术的适用性，研究信号级联通过解剖特征良好的胰岛素信号转导通路。具体来说，我们证明抑制DSOR 1（促分裂原活化蛋白激酶，MAPKK）的表达可以阻止下游ERK-A（MAPK）的激活。相反，阻断ERK-A表达导致DSOR 1活化增加。我们还表明，果蝇AKT（DAKT）激活依赖于胰岛素受体底物。CHICO（IRS 1 -4）。最后，我们证明了阻断果蝇PTEN的表达会导致DAKT的激活。在所有情况下，RNAi对生化级联的干扰与途径中的已知步骤一致。我们将这种强大的技术扩展到研究两种蛋白质，DSH 3 PX 1和果蝇ACK（DACK）。DSH 3 PX 1是一种含有SH 3，phox同源结构域的蛋白，DACK与哺乳动物活化的Cdc 42酪氨酸激酶ACK同源。使用RNAi。我们证明DACK是DSH 3 PX 1磷酸化的上游。使DSH 3 PX 1成为ACK样酪氨酸激酶的鉴定的下游靶标/底物。这些实验强调了RNA干扰在剖析复杂的生化信号级联中的有用性，并为确定果蝇基因组测序项目中识别的基因的功能提供了一种高效的方法。
We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate. CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi. we demonstrate that DACK is upstream of DSH3PX1 phosphorylation. making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.