Expanding the substantial interactome of NEMO using protein microarrays.
Expanding the substantial interactome of NEMO using protein microarrays.
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DOI：
10.1371/journal.pone.0008799
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发表时间：
2010-01-20
期刊：
影响因子：
3.7
通讯作者：
Prehn JH
中科院分区：
文献类型：
--
作者：
Fenner BJ;Scannell M;Prehn JH
NF-kappaB通路的信号转导是一系列细胞对细胞外和细胞内信息反应的关键调节因子。NEMO接头蛋白位于该通路的顶端，作为分子通道，将上游传感器传递的信号连接到下游NF-kappaB转录因子和随后的基因激活。NEMO在该通路中的位置使其成为一个有吸引力的靶标，从中寻找将NF-kappaB信号连接到其他通路和上游效应物的新蛋白。在这项工作中，我们使用蛋白质微阵列来鉴定新的NEMO相互作用物。共鉴定出112种蛋白质相互作用物，其中最具统计学意义的是典型的NEMO相互作用物IKKbeta， IKKalpha也被鉴定出来。在新的相互作用中，超过30%是激酶，而至少25%参与信号转导。通过GST下拉试验和共免疫沉淀，NEMO与CALB1、CDK2、SAG、SENP2和SYT1等相互作用物结合，验证了最初的筛选方法。在人胚胎肾细胞中，CALB1、CDK2和SAG的过表达刺激NF-kappaB的转录激活，而SYT1的过表达抑制tnfalpha依赖性NF-kappaB的转录激活。与这一发现相对应的是，CDK2、SAG和SENP2的RNA沉默降低了NF-kappaB的转录激活，支持了这些蛋白在NF-kappaB通路中的积极作用。许多新的NEMO相互作用物的鉴定为提高对这一重要细胞信号通路的理解开辟了新的研究机会。
Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.