PKA: a portrait of protein kinase dynamics
PKA: a portrait of protein kinase dynamics
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DOI：
10.1016/j.bbapap.2003.11.029
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发表时间：
2004-03-11
影响因子：
3.2
通讯作者：
Anand, G
中科院分区：
文献类型：
--
作者：
Taylor, SS;Yang, J;Anand, G
蛋白激酶在真核细胞中信号网络的整合中起关键作用。cAMP依赖性蛋白激酶（PKA）是这个庞大且高度多样化的酶家族的原型。PKA的催化亚基提供了蛋白激酶如何识别其底物以及抑制剂的最佳例子，并且还显示了酶如何通过催化步骤移动。许多相关的构象状态与催化循环已被抓获的晶格在这里进行了总结。从这些结构，我们可以开始欣赏催化的分子事件以及催化亚基中关键残基的复杂编排。整个分子都参与其中。然而，要完全理解PKA的信号传导，需要了解大量相关蛋白质，而不仅仅是催化亚基。这包括作为cAMP受体的调节亚基和作为PKA支架的A激酶锚定蛋白（AKAP）。AKAP通过对接到调节亚基的N-末端将PKA定位到细胞中的特定位点，从而为PKA信号传导创造微环境。为了充分理解这些分子的多样性和整合性，不仅需要高分辨率的结构，还需要了解这些分子在溶液中的行为。因此，除了通过X射线晶体学和NMR获得高分辨率结构外，我们还使用荧光工具以及氢/氘交换与质谱联用来探测这些蛋白质的动态特性以及它们如何相互作用。描述了这些分子的分子特征。最后，我们描述了一种新的重组表达PKA报告，使我们能够监测活细胞中的PKA活性。(C)2004 Elsevier B.V保留所有权利。
Protein kinases play a critical role in the integration of signaling networks in eukaryotic cells. cAMP-dependent protein kinase (PKA) serves as a prototype for this large and highly diverse enzyme family. The catalytic subunit of PKA provides the best example of how a protein kinase recognizes its substrates, as well as inhibitors, and also show how the enzyme moves through the steps of catalysis. Many of the relevant conformational states associated with the catalytic cycle which have been captured in a crystal lattice are summarized here. From these structures, we can begin to appreciate the molecular events of catalysis as well as the intricate orchestration of critical residues in the catalytic subunit that contribute to catalysis. The entire molecule participates. To fully understand signaling by PKA, however, requires an understanding of a large set of related proteins, not just the catalytic subunit. This includes the regulatory subunits that serve as receptors for cAMP and the A kinase anchoring proteins (AKAPs) that serve as scaffolds for PKA. The AKAPs localize PKA to specific sites in the cell by docking to the N-terminus of the regulatory subunits, thus creating microenvironments for PKA signaling. To fully appreciate the diversity and integration of these molecules, one needs not only high-resolution structures but also an appreciation of how these molecules behave in solution. Thus, in addition to obtaining high-resolution structures by X-ray crystallography and NMR, we have used fluorescent tools and also hydrogen/deuterium exchange coupled with mass spectrometry to probe the dynamic properties of these proteins and how they interact with one another. The molecular features of these molecules are described. Finally, we describe a new recombinantly expressed PKA reporter that allows us to monitor PKA activity in living cells. (C) 2004 Elsevier B.V All rights reserved.