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.
Highlights
Protein phosphorylation provides one of the primary means of transducing cellular signals, and as such has been utilized by a majority of organisms that span all domains of life [1]
The methodology resulted in the detection of 806 phosphorylation sites in E. coli expressing Protein Kinase A (PKA), and 467 phosphorylation sites in E. coli expressing Casein Kinase II (CK II)
Following removal of known endogenous phosphorylation sites obtained from both negative controls in the present study and an additional study of E. coli phosphorylation [14], 794 PKA phosphorylation sites and 458 CK II phosphorylation sites remained, which served as the data sets for motif analyses
Summary
Protein phosphorylation provides one of the primary means of transducing cellular signals, and as such has been utilized by a majority of organisms that span all domains of life [1]. Extensive research has been carried out to uncover the existence and specific location of phosphorylation sites on proteins as a means of understanding protein function and regulation. Advances in enrichment and detection technologies have led to an exponential increase in known phosphorylation sites on substrate proteins over the past decade [2], an important limitation of these strategies is that they do not provide information on the kinases responsible for the phosphorylation events. Kinase specificity motifs have been of significant importance in elucidating kinase function and cellular signaling mechanisms
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