Prinz Anke, Diskar Mandy, Erlbruch Andrea, Herberg Friedrich W
University of Kassel, Department of Biochemistry, Heinrich Plett Str. 40, D-34132 Kassel, Germany.
Cell Signal. 2006 Oct;18(10):1616-25. doi: 10.1016/j.cellsig.2006.01.013. Epub 2006 Mar 9.
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 Ralpha(I)-, Ralpha(II)-, as well as Calpha-subunits of PKA with Renilla luciferase (Rluc) as the bioluminescent donor or with green fluorescent protein (GFP2) 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 (hRIalpha-R210K) or abolished (hRIalpha-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 beta-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.
无需分离步骤的均相蛋白质-蛋白质相互作用分析方法是揭示活细胞中信号转导事件的重要工具。我们基于生物发光共振能量转移（BRET）建立了一种同工型特异性蛋白激酶A（PKA）亚基相互作用分析方法。分别用海肾荧光素酶（Rluc）作为生物发光供体或绿色荧光蛋白（GFP2）作为能量受体标记人PKA的Rα（I）-、Rα（II）-以及Cα亚基，可直接在活细胞以及全细胞提取物中探测PKA亚基相互作用，以便同时研究I型和II型PKA全酶的动力学。我们构建了几种新型的、基因编码的cAMP传感器，并且首次构建了I型PKA传感器。当C亚基和R亚基在细胞内组装成各自的全酶复合物时，会发生BRET，信号强度比背景高可达三倍。在表达野生型蛋白的细胞中，内源性cAMP水平的升高以及用cAMP类似物8-Br-cAMP处理会导致BRET信号呈剂量依赖性降低。与II型不同，在cAMP水平最高的细胞中，I型PKA全酶复合物的解离从未完全发生。细胞裂解后用8-Br-cAMP处理时，两种传感器均完全解离，这与使用由纯化的PKA亚基组装的全酶进行的体外激活分析结果一致。有趣的是，用PKA拮抗剂Rp-8-Br-cAMPS处理细胞会导致表达I型或II型PKA同工型的细胞中BRET信号显著增加，这表明体内全酶复合物得到了稳定。我们研究了cAMP结合能力降低（hRIα-R210K）或丧失（hRIα-G200E/G324E）的突变RI亚基，以量化RI-BRET传感器的最大信噪比。利用BRET我们证明，当通过使用Ht31肽或耗尽膜胆固醇破坏其与COS-7细胞质膜的锚定作用时，II型PKA全酶对异丙肾上腺素的β-肾上腺素能受体刺激变得不敏感。
