Sarkar Pabak, Davis Kaitlin A, Puhl Henry L, Veetil Jithesh V, Nguyen Tuan A, Vogel Steven S
Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland.
Biophys J. 2017 Mar 28;112(6):1270-1281. doi: 10.1016/j.bpj.2017.02.005.
While kinases are typically composed of one or two subunits, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is composed of 8-14 subunits arranged as pairs around a central core. It is not clear if the CaMKII holoenzyme functions as an assembly of independent subunits, as catalytic pairs, or as a single unit. One strategy to address this question is to genetically engineer monomeric and dimeric CaMKII and evaluate how their activity compares to the wild-type (WT) holoenzyme. Here a technique that combines fluorescence correlation spectroscopy and homo-FRET analysis was used to characterize assembly mutants of Venus-tagged CaMKIIα to identify a dimeric CaMKII. Spectroscopy was then used to compare how holoenzyme structure and function changes in response to activation with CaM in the dimeric mutant, WT-holoenzyme, and a monomeric CaMKII oligomerization-domain deletion mutant control. CaM triggered an increase in hydrodynamic volume in both WT and dimeric CaMKII without altering subunit stoichiometry or the net homo-FRET between Venus-tagged catalytic domains. Biochemical analysis revealed that the dimeric mutant also functioned like WT holoenzyme in terms of its kinase activity with an exogenous substrate, and for endogenous T286 autophosphorylation. We conclude that the fundamental functional units of CaMKII holoenzyme are paired catalytic-domains.
虽然激酶通常由一个或两个亚基组成，但钙调蛋白（CaM）依赖性蛋白激酶II（CaMKII）由8至14个亚基组成，围绕着一个中心核心成对排列。目前尚不清楚CaMKII全酶是作为独立亚基的集合体、催化对还是单个单元发挥作用。解决这个问题的一种策略是对单体和二聚体CaMKII进行基因工程改造，并评估它们的活性与野生型（WT）全酶相比如何。在这里，一种结合荧光相关光谱和同源FRET分析的技术被用于表征带有Venus标签的CaMKIIα的组装突变体，以鉴定二聚体CaMKII。然后使用光谱学来比较在二聚体突变体、WT全酶和单体CaMKII寡聚化结构域缺失突变体对照中，全酶结构和功能如何响应CaM激活而发生变化。CaM在WT和二聚体CaMKII中均引发了流体动力学体积的增加，而没有改变亚基化学计量或带有Venus标签的催化结构域之间的净同源FRET。生化分析表明，二聚体突变体在对外源底物的激酶活性以及内源性T286自身磷酸化方面也表现得像WT全酶。我们得出结论，CaMKII全酶的基本功能单元是成对的催化结构域。
