title: 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.
abstract: 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.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584CH, Utrecht, The Netherlands.
authors.forename: Marco L
authors.initials: ML
authors.lastname: Hennrich
authors.forename: Fabio
authors.initials: F
authors.lastname: Marino
authors.forename: Vincent
authors.initials: V
authors.lastname: Groenewold
authors.forename: Geert J P L
authors.initials: GJ
authors.lastname: Kops
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008745:Methylation; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C075840:TTK protein, human