Abstract
Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which (18)O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.
Highlights
Majority of protein kinases [3]; mapping comprehensive kinase-substrate relationships is essential to understanding biological mechanisms and uncovering new drug targets [4]
The method was applied to identify direct substrates of extracellular signal-regulated kinase 1 (ERK1), a serine/threonine kinase acting as an essential component of the Mitogen-activated protein kinase (MAPK) signal transduction pathway [21]
ERK1 can be activated by growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGF) [26]
Summary
Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*□S. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. Bioinformatics, and downstream biochemical assays are mandatory for the substrate verification [12, 13] Another strategy uses purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins. The results support the hypothesis that ERK1 plays complex roles in multiple pathways that are essential for the cell growth regulation
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