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 p

SUBMITTER: Xue L 

PROVIDER: S-EPMC4223502 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature