Luminescent kinase activity biosensors based on a versatile bimolecular switch.
Luminescent kinase activity biosensors based on a versatile bimolecular switch.
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DOI：
10.1021/ja1117396
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发表时间：
2011-04-20
影响因子：
15
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
--
作者：
Herbst, Katie J.;Allen, Michael D.;Zhang, Jin
实时跟踪生命系统中的激酶活性揭示了将信号信息编码为时空活动模式的新模式，并为筛选激酶调节剂开辟了新的途径。然而，通常与基于FRET的读数相耦合的激酶活性检测的灵敏度通常是一个限制因素。在这里，我们展示了一种激酶诱导的双分子开关，由目标激酶的底物和磷酸氨基酸结合域组成，可以被设计来检测不同的激酶活性，并连接到不同的读数，从而允许以更高的灵敏度和多功能性来检测动态的激酶活性。具体地说，我们证明了设计用于检测蛋白激酶A(PKA)或蛋白激酶C(PKC)活性的双分子开关可以开启FRET以及生物发光信号。值得注意的是，与单分子传感器相比，基于FRET的传感器获得了更大的动态范围；基于PKA和PKC的新型生物发光报告显示出高灵敏度和独特的检测基础激酶活性的能力，应该能够在活体成像和高通量化合物筛选中实现新的应用。因此，这种通用化的设计推动了激酶活性检测的分子工具箱的发展，并提供了一种在各种生物系统中检测激酶活性的通用和灵敏的手段。
Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into the spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a FRET-based readout, has often been a limiting factor. Here we show a kinase-inducible bimolecular switch, consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain, can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense Protein Kinase A (PKA) or Protein Kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges when compared to their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.
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影响因子：
64.8
作者：
Fuller, Brian G.;Lampson, Michael A.;Kapoor, Tarun M.
通讯作者：
Kapoor, Tarun M.
影响因子：
--
作者：
Depry, Charlene;Allen, Michael D.;Zhang, Jin
通讯作者：
Zhang, Jin
通过CA2+-CAMP-PKA振荡电路实现的PKA的信号多样性。
影响因子：
14.8
作者：
Ni, Qiang;Ganesan, Ambhighainath;Aye-Han, Nwe-Nwe;Gao, Xinxin;Allen, Michael D.;Levchenko, Andre;Zhang, Jin
通讯作者：
Zhang, Jin
DOI：
10.1083/jcb.200302125
发表时间：
2003-06-09
期刊：
The Journal of cell biology
影响因子：
--
作者：
Violin JD;Zhang J;Tsien RY;Newton AC
通讯作者：
Newton AC
影响因子：
4
作者：
Allen, Michael D.;DiPilato, Lisa M.;Zhang, Jin
通讯作者：
Zhang, Jin