title: QIKS--Quantitative identification of kinase substrates.
abstract: Signaling networks regulate cellular responses to external stimuli through post-translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large-scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel-screening platform developed for the proteome-wide substrate-analysis of specific kinases. Here, we aimed to identify substrates of mitogen-activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen-activated protein kinase cascade. An ATP analog-sensitive mutant of Mek1 (Mek1-as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC-MS/MS of IMAC-enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.
authors.affiliation: Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Sandra
authors.initials: S
authors.lastname: Morandell
authors.forename: Karin
authors.initials: K
authors.lastname: Grosstessner-Hain
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Roitinger
authors.forename: Otto
authors.initials: O
authors.lastname: Hudecz
authors.forename: Thomas
authors.initials: T
authors.lastname: Lindhorst
authors.forename: David
authors.initials: D
authors.lastname: Teis
authors.forename: Oliver A
authors.initials: OA
authors.lastname: Wrulich
authors.forename: Michael
authors.initials: M
authors.lastname: Mazanek
authors.forename: Thomas
authors.initials: T
authors.lastname: Taus
authors.forename: Florian
authors.initials: F
authors.lastname: Ueberall
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Lukas A
authors.initials: LA
authors.lastname: Huber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D011494:Protein Kinases