Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

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. Molecular & Cellular

Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

“…, hitchhiker phosphoproteins from the ABL1-PP and ABL1-Kin − immunopurification, were excluded from the data as the contribution from such proteins only occurred for ‘light’ sites. Consistent with Xue et al 10,. our assay is more robust and sensitive than with normal ATP.…”

“…FSBA kinase inactivation minimally affects the native state of the proteins; whereas phosphatase and heat treatment10 should be avoided as ABL1 recognition of substrates is partially coordinated via the phosphotyrosine-binding SH2-domain2324. One potential drawback of preserving the phosphorylation state, however, is a reduced sensitivity towards substrates that are already present in the cells in the major phosphorylated form.…”

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

“…The in vitro kinase reaction was performed based on previous reports [20, 21] with some modifications. The Lys-C digested peptides (200 μg) were treated with a thermosensitive alkaline phosphatase (TSAP) (Roche, Madison, WI) in a 1:100 (w/w) enzyme-to-peptide ratio at 37 °C overnight for dephosphorylation, and the dephosphorylated peptides were desalted using Sep-Pak C18 column.…”

Recent advances in phosphoproteomics and application to neurological diseases

“…The KALIP approach has been used to extensively examine the direct substrates of the spleen tyrosine kinase SYK 63, 156 as well as the S/T kinase ERK1. 157 In the case of ERK1, a stable isotope labeled form of ATP was introduced to further enhance the sensitivity of the in vitro reaction; since cell lysate is used as the candidate substrate pool, addition of a heavy phosphate group makes it significantly easier to differentiate between ERK1 activity and background endogenous phosphorylation.…”