Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET)
Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET)
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DOI：
10.1016/j.cellsig.2006.01.013
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发表时间：
2006-10-01
影响因子：
4.8
通讯作者：
Herberg, Friedrich W.
中科院分区：
文献类型：
--
作者：
Prinz, Anke;Diskar, Mandy;Herberg, Friedrich W.
不需要分离步骤的均匀蛋白-蛋白相互作用测定是揭示活细胞中信号转导事件的重要工具。我们建立了基于生物发光共振能量转移（BRET）的异构体特异性蛋白激酶A （PKA）亚基相互作用测定方法。标记人类r - α (I)-，。分别以Renilla luciferase （Rluc）为生物发光供体或以绿色荧光蛋白（GFP(2)）为能量受体的PKA的r - α (I)-和C - α -亚基，可以直接探测PKA亚基在活细胞和细胞总提取物中的相互作用，从而并行研究PKA I型和II型全酶动力学。几个新型的基因编码cAMP传感器和PKA I型传感器首次产生。当C和r亚基在细胞内组装成各自的全酶复合物时，BRET发生的信号比背景高三倍。内源性cAMP水平的增加以及cAMP类似物8-Br-cAMP的处理通过表达野生型蛋白的细胞中剂量依赖性的BRET信号减少来反映。与II型相反，在cAMP水平最高的细胞中，PKA I型全酶复合物的解离从未完全完成。两个传感器在细胞裂解后用8-Br-cAMP处理后完全解离，与使用纯化的PKA亚基组装的全酶的体外激活实验一致。有趣的是，与PKA拮抗剂rp -8- br - camp孵育的细胞导致表达PKA I型或II型亚型的细胞中BRET信号显著增加，表明体内全酶复合物稳定。研究了降低（hRI α - r21ok）或消除（hRI α - g200e /G324E） cAMP结合能力的突变体RI亚基，以量化RI- bret传感器的最大信噪比。利用BRET，我们证明PKA II型全酶对异丙肾上腺素能受体的刺激不敏感，当锚定在COS-7细胞的质膜上被Ht31肽或膜胆固醇消耗破坏时。(c) 2006爱思唯尔公司版权所有。
Homogeneous protein-protein interaction assays without the need of a separation step are an essential tool to unravel signal transduction events in live cells. We have established an isoform specific protein kinase A (PKA) subunit interaction assay based on bioluminescence resonance energy transfer (BRET). Tagging human R-alpha(I)-,. R-alpha(I)-, as well as C alpha-subunits of PKA with Renilla luciferase (Rluc) as the bioluminescent donor or with green fluorescent protein (GFP(2)) as the energy acceptor, respectively, allows to directly probe PKA subunit interaction in living cells as well as in total cell extracts in order to study side by side PKA type I versus type II holoenzyme dynamics.Several novel, genetically encoded cAMP sensors and-for the first time PKA type I sensors-were generated. When C- and R-subunits are assembled to the respective holoenzyme complexes inside the cell, BRET occurs with a signal up to three times above the background. An increase of endogenous cAMP levels as well as treatment with the cAMP analog 8-Br-cAMP is reflected by a dose-dependent BRET signal reduction in cells expressing wild type proteins. In contrast to type II, the dissociation of the PKA type I holoenzyme complex was never complete in cells with maximally elevated cAMP levels. Both sensors dissociated completely upon treatment with 8-Br-cAMP after cell lysis, consistent with in vitro activation assays using holoenzymes assembled from purified PKA subunits. Interestingly, incubation of cells with the PKA antagonist Rp-8-Br-cAMPS leads to a significant BRET signal increase in cells expressing PKA type I or type II isoforms, indicating a stabilization of the holoenzyme complexes in vivo. Mutant RI subunits with reduced (hRI alpha-R21OK) or abolished (hRI alpha-G200E/G324E) cAMP binding capability were studied to quantify maximal signal to noise ratios for the RI-BRET sensor.Utilizing BRET we demonstrate that PKA type II holoenzyme was rendered insensitive to p-adrenergic receptor stimulation with isoproterenol when anchoring to the plasma membrane of COS-7 cells was disrupted by either using Ht31 peptide or by depletion of membrane cholesterol. (c) 2006 Elsevier Inc. All rights reserved.