Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling.
Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling.
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DOI：
10.1038/s41598-017-13021-y
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发表时间：
2017-10-26
影响因子：
4.6
通讯作者：
Zaccolo M
中科院分区：
文献类型：
--
作者：
Koschinski A;Zaccolo M
CAMP是一种普遍存在的第二信使，通过激活其主要效应物蛋白激酶A(PKA)，负责多种激素和神经递质的细胞效应。多项研究表明，在几种细胞类型中，cAMP的基础浓度约为1μM，这一值远高于报道的cAMP浓度，这是达到一半最大激活PKA所需的浓度，后者的范围在100-300 NM之间。人们提出了几种假设来解释这种明显的差异，包括不准确地测量细胞内游离cAMP，不准确地测量PKA的表观激活常数，或者通过将酶定位于基础cAMP浓度较低的微区来屏蔽PKA对大量胞浆cAMP的影响。然而，支持这些模型中任何一个的直接实验证据都是有限的，而且还缺乏明确的结论。在这项研究中，我们使用多个基于FRET的报告来检测完整细胞中cAMP和PKA的活性，我们发现，在细胞内测量时，PKA对cAMP的敏感性几乎比在体外测量时低20倍。我们的发现对于理解区分的阵营信号有重要的意义。
cAMP is a ubiquitous second messenger responsible for the cellular effects of multiple hormones and neurotransmitters via activation of its main effector, protein kinase A (PKA). Multiple studies have shown that the basal concentration of cAMP in several cell types is about 1 μM. This value is well above the reported concentration of cAMP required to half-maximally activate PKA, which measures in the 100–300 nM range. Several hypotheses have been suggested to explain this apparent discrepancy including inaccurate measurements of intracellular free cAMP, inaccurate measurement of the apparent activation constant of PKA or shielding of PKA from bulk cytosolic cAMP via localization of the enzyme to microdomains with lower basal cAMP concentration. However, direct experimental evidence in support of any of these models is limited and a firm conclusion is missing. In this study we use multiple FRET-based reporters for the detection of cAMP and PKA activity in intact cells and we establish that the sensitivity of PKA to cAMP is almost twenty times lower when measured in cell than when measured in vitro. Our findings have important implications for the understanding of compartmentalized cAMP signalling.
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影响因子：
3.7
作者：
Klarenbeek J;Goedhart J;van Batenburg A;Groenewald D;Jalink K
通讯作者：
Jalink K
DOI：
10.1038/nrd4228
发表时间：
2014-04
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.8
作者：
Gamanuma, M;Yuasa, K;Omori, K
通讯作者：
Omori, K
影响因子：
4.8
作者：
Saucerman, JJ;Brunton, LL;McCulloch, AD
通讯作者：
McCulloch, AD
影响因子：
0.5
作者：
Markova, O.;Real, E.;Bregestovski, P.
通讯作者：
Bregestovski, P.