Using bacteria to determine protein kinase specificity and predict target substrates

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

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Casein Kinase II / biosynthesis
  • Casein Kinase II / chemistry*
  • Casein Kinase II / genetics
  • Consensus Sequence
  • Cyclic AMP-Dependent Protein Kinases / biosynthesis
  • Cyclic AMP-Dependent Protein Kinases / chemistry*
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Molecular Sequence Data
  • Peptide Library
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • ROC Curve
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Sequence Analysis, Protein
  • Signal Transduction
  • Substrate Specificity

Substances

  • Escherichia coli Proteins
  • Peptide Library
  • Recombinant Proteins
  • Casein Kinase II
  • Cyclic AMP-Dependent Protein Kinases