A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.
A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.
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DOI：
10.1042/bj20131174
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发表时间：
2014-01-15
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影响因子：
--
通讯作者：
Lucet IS
中科院分区：
文献类型：
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作者：
Murphy JM;Zhang Q;Young SN;Reese ML;Bailey FP;Eyers PA;Ungureanu D;Hammaren H;Silvennoinen O;Varghese LN;Chen K;Tripaydonis A;Jura N;Fukuda K;Qin J;Nimchuk Z;Mudgett MB;Elowe S;Gee CL;Liu L;Daly RJ;Manning G;Babon JJ;Lucet IS
蛋白激酶类结构域缺乏已知催化磷酸化转移的保守残基，称为假蛋白激酶，已成为所有生命王国中重要的信号域。虽然预测的主要功能是作为非催化的蛋白质相互作用模块，但几个假激酶结构域被认为具有意想不到的催化功能，经常存在争议。我们建立了一种热位移法作为一种基准技术来定义激酶样结构域的核苷酸结合特性。与体外酶分析不同的是，该方法对微量污染酶的存在不敏感，否则可能会导致对假激酶酶催化功能的错误归类。我们通过将31个不同的假激酶结构域分成四组：没有可检测到的核苷酸或阳离子结合；阳离子非依赖的核苷酸结合；阳离子结合；以及阳离子促进的核苷酸结合，证明了这种方法的实用性。虽然9个假激酶以二价阳离子依赖的方式与ATP结合，但超过一半的研究对象没有检测到与核苷酸结合，这表明假激酶结构域在信号网络中主要作为非催化蛋白质相互作用模块发挥作用，并且只有一小部分具有潜在的催化活性。我们建议从今以后采用热位移法作为标准技术来确定激酶样结构域的核苷酸结合和催化潜力。
Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.