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

Author : Liang Xue et al 

Date : July 14, 2014 

Post : Molecular & Cellular Proteomics, 13, 3199-3210. 

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 vitrophosphorylated proteins with heavy phosphates are further overlapped with in vivokinase-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.


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.

  • ↵* This project has been funded in part by an NSF CAREER award CHE-0645020 (W.A.T.), and by a National Institutes of Health grant GM088317 (WAT).

  • Graphic This article contains supplemental Figs. S1 to S7 and Tables S1 to S10.

  • ADDITIONAL INFORMATION: Supplementary data set containing 7 figures and 10 tables and annotated sequence spectra supporting identification of phosphorylated peptides can be downloaded are available on the internet through the MCP site. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (61) with the dataset identifier PXD000357.

  • Received February 12, 2014.
  • Revision received July 7, 2014.
  • © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.







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