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
详细信息 查看全文
- 作者：Marco L. Hennrich ; Fabio Marino ; Vincent Groenewold ; Geert J. P. L. Kops ; Shabaz Mohammed ; Albert J. R. Heck
- 刊名：Journal of Proteome Research
- 出版年：2013
- 出版时间：May 3, 2013
- 年：2013
- 卷：12
- 期：5
- 页码：2214-2224
- 全文大小：467K
- 年卷期：v.12,no.5(May 3, 2013)
- ISSN：1535-3907
文摘
In order to understand cellular signaling, a clear understanding of kinase鈥搒ubstrate 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.