title: FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms.
abstract: We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this assay we combined our full-length expressed stable isotope-labeled protein for quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multikinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n × 80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3β activity and permitting an advanced model for GSK3β-mediated signaling.
authors.affiliation: Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Sasha A
authors.initials: SA
authors.lastname: Singh
authors.forename: Dominic
authors.initials: D
authors.lastname: Winter
authors.forename: Parizad M
authors.initials: PM
authors.lastname: Bilimoria
authors.forename: Azad
authors.initials: A
authors.lastname: Bonni
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
authors.forename: Judith A
authors.initials: JA
authors.lastname: Steen
mesh_terms: D000089622:Doublecortin Domain Proteins; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000089622:Doublecortin Domain Proteins; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D048031:JNK Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3