ENGINEERING PROTEIN KINASES FOR SELECTIVE ACTIVATION OF CELLULAR PATHWAYS

Blake Tye , Benjamin Jester, and Indraneel Ghosh

ENGINEERING PROTEIN KINASES FOR SELECTIVE ACTIVATION OF CELLULAR PATHWAYS

Protein kinases are a large family of enzymes that catalyze the phosphorylation of a wide variety of target protein substrates. Phosphorylation of the protein substrate acts as a means to alter its activity through conformation or localization changes, or by altering interactions with other effectors. Subsequently, protein kinases play an essential role in cellular pathway transduction. However, dissection of pathways has previously relied on total inhibition of enzymes at various steps and observation of the resulting phenotypic change. In particular, small molecule inhibition at the protein level and RNA interference to silence the gene are frequently used. While both have proven useful in these efforts, they have the drawback of often affecting more than one pathway of interest, thereby making it complicated to definitively interpret the results.

Here, a potentially new approach is taken in dissection of cellular pathways: selective activation of a protein kinase towards one or several targets of interest. Structures deposited in the Protein Data Bank (PDB) of protein kinases with bound ligands and substrates were thoroughly explored to determine the regions that potentially correlate to binding specificity of each. It was observed that the two exhibit non-overlapping regions of residues within their binding proximities, suggesting possible residues of interest. Comparisons of residues between different kinases resulted in a reduction to 47 residues of interest surrounding the ligand-binding pocket. Sequence alignments were subsequently used to abstract these data to all kinases encoded in the human genome. These residues form the basis by which we are engineering kinases to generate chimeric kinases that maintain substrate specificity but have reversed phosphorylation activity (Tyr to Ser/Thr or vice versa). Subsequently, target substrates of interest could be mutated to be selectively phosphorylated by the chimeric kinase and the resulting outputs chemically and visually observed.

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