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[Tymora] Identification of ERK1 Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

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2021-09-06
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Title : Identification of ERK1 Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics 

Author : Liang Xue et al 

Date : July 14, 2014 

Post : Current Issue 

Link : 

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 18O-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.

  • Kinases*

 Mass Spectrometry Phosphorylation QuantificationSerine/Threonine Kinases* Signal Transduction*

Footnotes

  • Author contributions: L.X., J.Z., and W.A.T. designed research; L.X. and P.W. performed research; L.X., P.C., and W.A.T. analyzed data; L.X. and W.A.T. wrote the paper.

  • Received February 12, 2014.
  • Accepted July 14, 2014.
  • Copyright © 2014, The American Society for Biochemistry and Molecular Biology

 





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