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
| Autor: | Hennrich, M.L., Marino, F., Groenewold, V., Kops, G.J.P.L., Mohammed, S., Heck, A.J.R., Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Sub Biomol.Mass Spect. and Proteomics |
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| Přispěvatelé: | Biomolecular Mass Spectrometry and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Sub Biomol.Mass Spect. and Proteomics |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
Proteomics
Amino Acid Motifs Molecular Sequence Data Cell Cycle Proteins Protein Serine-Threonine Kinases Biology Methylation Biochemistry Substrate Specificity MAP2K7 03 medical and health sciences 0302 clinical medicine Tandem Mass Spectrometry Consensus Sequence Consensus sequence Humans Kinome Phosphorylation Protein kinase A Enzyme Assays 030304 developmental biology 0303 health sciences Binding Sites Chromatography Protein-Serine-Threonine Kinases Kinase General Chemistry Protein-Tyrosine Kinases Chromatography Ion Exchange Phosphoproteins Cyclic AMP-Dependent Protein Kinases HEK293 Cells Isotope Labeling Cyclin-dependent kinase complex Protein Processing Post-Translational 030217 neurology & neurosurgery HeLa Cells |
| Zdroj: | Journal of Proteome Research Journal of Proteome Research, 12, 2214. American Chemical Society |
| ISSN: | 1535-3893 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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