Abstract LB-052: Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification

Abstract

Protein kinases are attractive targets for pharmacological intervention through their frequent disease associated aberration of cellular signalling networks. In this context an understanding of the myriad of substrates on which a protein kinase operates can help to reveal potential pharmacodynamic and patient stratification biomarkers. Kinase Identification of Proximal Substrates (KIPS) utilises an innovative chemical genetics approach to identify novel proximal biomarkers of kinase target response. This approach relies on the generation of a desensitized mutant protein that retains its kinase function, but loses sensitivity to small-molecule mediated inhibition. Comparative phospho-proteomics is performed on immuno-complexes of tagged wild type and mutant protein kinases in the presence and absence of inhibitor to isolate specific phospho-proteomic changes in proximal substrates. To exemplify the KIPS approach we have focused on the identification of novel substrates of the atypical protein kinase C isoform, PKCι. Through structure based design we identified two acidic amino acid residues in the PKCι nucleotide binding site that are crucial for efficacy of a previously characterized PKCι inhibitor, CRT0066854. Upon mutation to non-acidic alanine residues the PKCι protein is rendered insensitive to compound-mediated inhibition, allowing a comparison of wild type and mutant responses to PKCι inhibition to be made. Using HCT116 cells, we identified Myosin X as a putative substrate for PKCι, and demonstrated its specificity to the PKCι signalling axis through alanine-scanning peptide arrays and generation of phospho-specific antibodies. We show that the key acidic amino acids utilised in KIPS are highly conserved across the AGC and CAMK kinase super families. Furthermore, extensive compound SAR, diverse kinome profiling and structural biology have identified a tool box of inhibitor compounds with a broad range of activities across these kinase families leading to broader applicability of KIPS to enable the identification of individual kinase specific substrates and response across approximately 20% of the human kinome.

Citation Format: jon roffey, Andrew Turnbull, Christian Dillon, Susan Boyd, Philippe Riou, Mark Linch, Peter Parker, Sven Kjaer, Neil McDonald. Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-052. doi:10.1158/1538-7445.AM2015-LB-052