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PLoS One. 2012;7(12):e52747. doi: 10.1371/journal.pone.0052747. Epub 2012 Dec 26.

Using bacteria to determine protein kinase specificity and predict target substrates.

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1
Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

Abstract

The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

PMID:
23300758
PMCID:
PMC3530509
DOI:
10.1371/journal.pone.0052747
[Indexed for MEDLINE]
Free PMC Article
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