Identification and validation of inhibitor-responsive kinase substrates using a new paradigm to measure kinase-specific protein phosphorylation index.

ABSTRACT: Regulation of all cellular processes requires dynamic regulation of protein phosphorylation. We have developed an unbiased system to globally quantify the phosphorylation index for substrates of a specific kinase by independently quantifying phosphorylated and total substrate molecules in a reverse in-gel kinase assay. Non-phosphorylated substrate molecules are first quantified in the presence and absence of a specific stimulus. Total substrate molecules are then measured after complete chemical dephosphorylation, and a ratio of phosphorylated to total substrate is derived. To demonstrate the utility of this approach, we profiled and quantified changes in phosphorylation index for Protein Kinase CK2 substrates that respond to a small-molecule inhibitor. A broad range of inhibitor-induced c

SUBMITTER: Li X 

PROVIDER: S-EPMC4133982 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature