Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1
Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1
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DOI：
10.1021/pr400074f
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发表时间：
2013-05-01
影响因子：
4.4
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
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作者：
Hennrich, Marco L.;Marino, Fabio;Heck, Albert J. R.
为了理解细胞信号传导，对激酶-底物关系的清楚理解是必不可少的。这些关系中的一些由底物中存在的一致识别基序定义，使其可被指定的激酶磷酸化。在这里，我们探索了一种方法，该方法是基于强阳离子交换色谱结合差分稳定同位素标记的两个连续步骤，以高精度定义激酶的一致性基序。我们证明了我们的方法的价值，通过评估两个非常不同的激酶：cAMP调节蛋白激酶A（PKA）和人单极纺锤体1（Mps 1）激酶，也称为TTK的图案。PKA是一种被充分研究的嗜碱性激酶，具有相对明确的基序和许多已知的体外和体内底物。MPS 1是一种参与染色体分离的激酶，其特征尚不清楚。它的底物特异性尚不清楚，在这里，我们表明，Mps 1是一个嗜酸性激酶与一个惊人的倾向磷酸化的苏氨酸。我们工作的最终成果是PICA和Mps 1的高清晰度激酶共识基序。我们的通用方法，它利用蛋白水解细胞裂解物作为肽底物库的来源，可以实现对激酶组中存在的任何激酶。
In order to understand cellular signaling, a clear understanding of kinase-substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PICA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome.