Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches
Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches
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通过亚基定量和交联方法揭示抑制 cAMP 依赖性蛋白激酶的机制
DOI：
10.1073/pnas.1701782114
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
M. Gold
中科院分区：
文献类型：
--
作者：
R. Walker;Florian Stengel;M. Gold
camp依赖性蛋白激酶（PKA）磷酸化引发细胞变化，包括心脏细胞的战斗或逃跑反应和神经元的突触增强。PKA催化亚基活性失控是病理性的；然而，调控PKA在细胞中的作用机制尚不清楚。使用一种监测细胞PKA亚基相互作用的方法，我们表明-与最近的建议相反-催化亚基由cAMP从调控亚基释放。相反，我们确定了快速重新捕获释放的催化亚基的机制。调控亚基的表达比催化亚基高得多，以支持快速的催化亚基再结合。此外，对全局PKA结构的分析表明，II型调节亚基锚定与细胞膜内催化亚基释放和再捕获相容。环amp依赖性蛋白激酶（PKA）的蛋白磷酸化是关键细胞过程的基础，包括心脏细胞的交感刺激和神经元突触强度的增强。不受限制的PKA活性是病理性的，一个持久的挑战是了解PKA催化亚基的活性是如何在细胞中定向的。我们开发了一种光激活交联方法来监测活细胞中PKA亚基的时间精度相互作用。这使我们能够反驳最近提出的理论，即PKA催化亚基在cAMP升高期间仍然与调控亚基相连。相反，我们已经确定了PKA信号的其他特征，以减少催化亚基扩散和增加再捕获率。对人胚胎肾细胞和大鼠器官的蛋白质提取物进行的全面定量免疫印迹分析显示，调节亚基总是存在大量的催化亚基过剩（平均为17倍）。在大多数被检测的器官中，II型调节（RII）亚基被发现是主要的PKA亚基。我们还使用交联耦合质谱法研究了含有RII亚基的PKA配合物的结构。定量比较RIIβ和Cβ复合物内的交联，发现RIIβ的二聚化和对接结构域位于其第二个cAMP结合结构域之间。这种结构与锚定的RII亚基兼容，它将催化亚基的多柱化N端指向膜，以便在膜平面内释放和重新捕获。
Significance Protein phosphorylation by cAMP-dependent protein kinase (PKA) triggers cellular changes, including fight-or-flight responses in heart cells, and synaptic potentiation in neurons. Uncontrolled activity of PKA catalytic subunits is pathological; however, the mechanism for directing PKA in cells is unclear. Using an approach for monitoring cellular PKA subunit interactions, we show that—contrary to recent proposals—catalytic subunits are released from regulatory subunits by cAMP. Instead, we identify mechanisms for rapid recapture of liberated catalytic subunits. Regulatory subunits are expressed much more highly than catalytic subunits to support rapid catalytic subunit reassociation. Furthermore, analysis of global PKA architecture reveals that type II regulatory subunit anchoring is compatible with catalytic subunit release and recapture within the cell membrane. Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIβ and Cβ, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIβ is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.
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影响因子：
16
作者：
Gold, Matthew G.;Lygren, Birgitte;Barford, David
通讯作者：
Barford, David
影响因子：
8.8
作者：
Tillo SE;Xiong WH;Takahashi M;Miao S;Andrade AL;Fortin DA;Yang G;Qin M;Smoody BF;Stork PJS;Zhong H
通讯作者：
Zhong H
影响因子：
16
作者：
Kinderman, Francis S.;Kim, Choel;Taylor, Susan S.
通讯作者：
Taylor, Susan S.
异构体特异性十四烷基化开关将 II 型 PKA 全酶靶向膜。
DOI：
10.1016/j.str.2015.07.007
发表时间：
2015
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Zhang,Ping;Ye,Feng;Bastidas,AdamC;Kornev,AlexandrP;Wu,Jian;Ginsberg,MarkH;Taylor,SusanS
通讯作者：
Taylor,SusanS
DOI：
10.1073/pnas.0408608102
发表时间：
2005-01-11
影响因子：
11.1
作者：
Sastri, M;Barraclough, DM;Taylor, SS
通讯作者：
Taylor, SS