Identifying a kinase network regulating FGF14:Nav1.6 complex assembly using split-luciferase complementation.
Identifying a kinase network regulating FGF14:Nav1.6 complex assembly using split-luciferase complementation.
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DOI：
10.1371/journal.pone.0117246
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Laezza F
中科院分区：
文献类型：
--
作者：
Hsu WC;Nenov MN;Shavkunov A;Panova N;Zhan M;Laezza F
激酶在大脑中起着重要作用。通过复杂的信号通路，激酶调节蛋白质的强度：蛋白质相互作用（PPI）影响细胞周期、信号转导和神经元的电活动。由激酶引起的神经元兴奋性、突触可塑性和大脑连通性的变化与复杂的大脑疾病有关，但这些细胞事件背后的分子机制在很大程度上仍是难以捉摸的。为了进一步了解大脑疾病，需要在细胞背景下快速测量激酶途径的新方法。基于生物发光的荧光素酶互补测定（LCA）是一个强大的，多功能的工具，用于探索PPI。LCA依赖于两个萤火虫荧光素酶蛋白片段的互补，这两个片段通过两个相互作用的结合伙伴在功能上重组为完整的荧光素酶。本研究中，我们在活细胞中应用LCA检测了12种激酶通路作为PPI复合物的调节因子，PPI复合物由电压门控钠通道Nav1.6及其多价辅助蛋白成纤维细胞生长因子14 （FGF14）及其跨膜离子通道形成。Nav1.6是一种诱导神经元动作电位并介导突触传递的跨膜离子通道。通过对结构多样的激酶抑制剂的剂量依赖性验证和分层聚类，我们确定了PI3K/Akt通路、细胞周期调节剂Wee1激酶和蛋白激酶C （PKC）是通过调节FGF14:Nav1.6复合物调控神经元兴奋性的前瞻性调控节点。独创性通路分析显示这些通路在糖原合成酶激酶3 （GSK3）上趋同，功能分析表明抑制GSK3会损害海马神经元的兴奋性。这种结合的方法为快速测量PPI信号提供了一个通用的工具包，允许发现以GSK3为中心的新的模块化途径，这可能是正常和患病大脑之间功能改变的基础。
Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.
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影响因子：
46.9
作者：
通讯作者：
--
影响因子：
2
作者：
Gildish, Iness;Manor, David;Rosenblum, Kobi
通讯作者：
Rosenblum, Kobi
DOI：
10.1017/s1461145704004535
发表时间：
2004-12-01
影响因子：
4.8
作者：
Gould, TD;Einat, H;Manji, HK
通讯作者：
Manji, HK
DOI：
10.1073/pnas.0708800104
发表时间：
2007-12-18
影响因子：
11.1
作者：
Fedorov, Oleg;Marsden, Brian;Knapp, Stefan
通讯作者：
Knapp, Stefan
影响因子：
4.1
作者：
Budni, Josiane;Lobato, Kelly R.;Rodrigues, Ana Lucia S.
通讯作者：
Rodrigues, Ana Lucia S.