QIKS--Quantitative identification of kinase substrates.
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.
Morandell
Sandra
S
Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
Grosstessner-Hain
Karin
K
Roitinger
Elisabeth
E
Hudecz
Otto
O
Lindhorst
Thomas
T
Teis
David
D
Wrulich
Oliver A
OA
Mazanek
Michael
M
Taus
Thomas
T
Ueberall
Florian
F
Mechtler
Karl
K
Huber
Lukas A
LA
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
D011494:Protein Kinases