Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584CH, Utrecht, The Netherlands.
J Proteome Res. 2013 May 3;12(5):2214-24. doi: 10.1021/pr400074f. Epub 2013 Apr 3.
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 PKA 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.
为了理解细胞信号转导，清晰地认识激酶-底物关系是必不可少的。其中一些关系是由底物中存在的共识识别基序定义的，这使得它们可以被指定的激酶进行磷酸化。在这里，我们探索了一种基于两步强阳离子交换层析结合差异稳定同位素标记的方法，以高精度定义激酶的共识基序。我们通过评估两种截然不同的激酶（cAMP 调节蛋白激酶 A（PKA）和人单极纺锤体 1（Mps1）激酶，也称为 TTK）的基序来证明我们方法的价值。PKA 是一种研究充分的嗜碱性激酶，其基序相对明确，并且在体外和体内有许多已知的底物。Mps1 是一种参与染色体分离的激酶，其底物特异性尚不清楚，我们在这里表明 Mps1 是一种酸性激酶，具有强烈的磷酸化苏氨酸的倾向。我们工作的最终结果是 PKA 和 Mps1 的高清晰度激酶共识基序。我们的通用方法利用蛋白水解细胞裂解物作为肽-底物文库的来源，可以应用于激酶组中存在的任何激酶。
