Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Amanchy, Ramars; Zhong, Jun; Molina, Henrik; Chaerkady, Raghothama; Iwahori, Akiko; Kalume, Dário Eluan; Grønborg, Mads; Joore, Jos; Cope, Leslie; Pandey, Akhilesh

doi:10.1021/pr800198w

Cited by 61 publications

(67 citation statements)

References 46 publications

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“…Accompanied with advances of high-speed and high-resolution mass spectrometry, the technique of kinase substrate screening using proteomic strategy is quickly evolving (5)(6)(7). Mass spectrometry has been extensively used for kinasesubstrate interaction mapping (8) and global phosphorylation profiling (9).…”

mentioning

confidence: 99%

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

Xue¹,

Wang²,

Cao³

et al. 2014

Molecular & Cellular Proteomics

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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

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confidence: 99%

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

Xue¹,

Wang²,

Cao³

et al. 2014

Molecular & Cellular Proteomics

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“…Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,14). These attempts commonly used purified, active kinases to phosphorylate cell lysate (or fractions of cell lysate) in vitro, followed by mass spectrometric analysis to identify phosphoproteins.…”

mentioning

confidence: 99%

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Xue¹,

Wang²,

Iliuk³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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138

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Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.P rotein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions, subcellular localization, and ultimately cellular functions (1, 2). Deregulation of the signaling network often leads to disease states such as human malignancies, diabetes, and immune disorders. Although many kinases are excellent therapeutic targets, the precise connection between protein kinases and their direct substrates has not been fully elucidated for a majority of protein kinases. Besides classical genetic and biochemical methods, there have been a number of high throughput approaches for the identification of potential kinase substrates. Common methods include in vitro kinase assays using libraries of synthetic peptides (3), phase expression libraries (4), protein/peptide arrays (5-7), or cell extracts (8, 9), but these methods can often be misleading and provide many false positive results. The discovery of physiological substrates for specific protein kinases has remained challenging, even with recent advances in mass spectrometry.Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,1...

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“…c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. Amanchy et al employed peptide microarrays approach and identified tyrosine phosphorylation sites in c-Src substrates (Amanchy et al, 2008). They designed custom peptide microarrays containing all possible tyrosine-containing peptides and their mutant counterparts containing a Tyr → Phe substitution from the identified substrates.…”

Section: Arrays-based Applicationmentioning

confidence: 99%

Phosphoproteomics: Detection, Identification and Importance of Protein Phosphorylation

Jia¹,

Lin²,

Souchelnytskyi³

2012

Integrative Proteomics

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Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Cited by 61 publications

References 46 publications

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

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

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Phosphoproteomics: Detection, Identification and Importance of Protein Phosphorylation

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