Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach

Abstract

Characterizing protein kinase substrate specificity motifs represents a powerful step in elucidating kinase-signaling cascades. The protocol described here uses a bacterial system to evaluate kinase specificity motifs in vivo, without the need for radioactive ATP. The human kinase of interest is cloned into a heterologous bacterial expression vector and allowed to phosphorylate E. coli proteins in vivo, consistent with its endogenous substrate preferences. The cells are lysed, and the bacterial proteins are digested into peptides and phosphoenriched using bulk TiO . The pooled phosphopeptides are identified by tandem mass spectrometry, and bioinformatically analyzed using the pLogo visualization tool. The ProPeL approach allows for detailed characterization of wildtype kinase specificity motifs, identification of specificity drift due to kinase mutations, and evaluation of kinase residue structure-function relationships. © 2018 by John Wiley & Sons, Inc.

The Okur-Chung Neurodevelopmental Syndrome (OCNDS) mutation CK2K198Rleads to a rewiring of kinase specificity

“…ProPeL experiments were carried out as previously described 8 with the following conditions for in vivo proteome phosphorylation: all CK2 constructs were expressed in Escherichia coli OverExpress C43(DE3) cells (Lucigen) by IPTG induction. Optimal expression conditions were determined to be mid-log induction followed by expression for 24h at 37°C in TB media (data not shown).…”

“…7 To determine the impact of the CK2 K198R mutation we implemented the ProPeL method to generate a high-resolution motif of CK2 WT and CK2 K198R . 8 Additionally, we utilized a probabilistic strategy to determine the differential likelihood of modification for serine, threonine, and tyrosine residues on axonally localized ion channels under wild type and mutant kinase conditions.…”

Repurposing approved drugs on the pathway to novel therapies

“…Given the rising number of strains found to be resistant to drugs that affect pathways specific to infectious agents, many repurposing studies are now looking for more broad‐spectrum agents that block or activate host pathways, such as those targeted in cancer therapy . Drugs developed to inhibit intracellular kinases, phosphatases, or reduce inflammation may indeed have multivalent activity against several different infectious organisms. For example, drugs approved for different indications, chosen from a 400 compound library, inhibited the ability of several pathogenic bacteria to grow in macrophages.…”

Sequence and Structure-Based Analysis of Specificity Determinants in Eukaryotic Protein Kinases

“…When directed toward kinases belonging to a functional class, for example, the mitotic kinases, this approach can reveal systems-level mechanisms to ensure signaling fidelity—in this case, by the spatial separation of kinases with overlapping motifs and vice versa ( Alexander et al., 2011 ). The emergence of mass spectrometry (MS) technologies for the systematic profiling of kinase specificity promises to accelerate this field of research even further ( Barber et al., 2018 ; Imamura et al., 2014 ; Lubner et al., 2018 ; Sugiyama et al., 2019 ).…”