Chemical proteomics and functional proteomics strategies for protein kinase inhibitor validation and protein kinase substrate identification: Applications to protein kinase CK2 ☆ Laszlo Gyenis a , Jacob P. Turowec a , Maria Bretner b, c , David W. Litchfield a, d, ⁎ a Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada N6A 5C1 b Chemistry Department, Warsaw University of Technology, Warsaw, Poland c Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland d Department of Oncology, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada N6A 5C1 abstract article info Article history: Received 14 January 2013 Accepted 4 February 2013 Available online xxxx Keywords: CK2 Protein kinase inhibitor TBB Chemoproteomics Functional proteomics Inhibitor profiling Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To over- come these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhib- itors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhib- itors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sen- sitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012). © 2013 Elsevier B.V. All rights reserved. 1. Introduction Protein kinases play universal roles in the regulation of many biolog- ical processes and have been implicated in a large number of human diseases including many forms of malignancy [1,2]. In fact, a recent analysis of available genomics data revealed nearly one thousand kinase mutations in ~70 single-gene disorders, the majority of which (50) are associated with cancer [3]. With the ongoing explosion of genome sequencing, it is anticipated that the number of kinase mutations asso- ciated with cancer and other diseases will continue to mount [4,5]. The remarkable success of Gleevec/imatinib, the first protein tyrosine kinase inhibitor drug to receive FDA approval, for the treatment of Chronic Myelogenous Leukemia (CML) was a seminal advance demonstrating the utility of kinase inhibitors as therapeutic agents [6,7]. This revelation that kinases are “druggable” together with the striking prevalence of kinase alterations in disease has spurred intense interest in the develop- ment of kinase inhibitor drugs [2,5,8]. Furthermore, the universal in- volvement of phosphorylation as a regulatory mechanism has resulted in widespread use of protein kinase inhibitors as agents for interrogating the involvement of protein kinases in a vast array of experimental systems. Despite their promise as therapeutic agents and routine use in exper- imental studies, a number of significant challenges arise both in the design and in the utilization of protein kinase inhibitors. First of all, pro- tein kinases represent a large family comprising more than 500 members within the human genome. Consequently, the conserved architecture and shared catalytic machinery of these enzymes present major hurdles in the development of specific inhibitors, especially for ATP-competitive kinase inhibitors that continue to represent the majority of kinase inhib- itors which are currently available. It is also evident that in many in- stances, the inhibitory characteristics of protein kinase inhibitors are primarily derived from studies performed with purified, often recombi- nant, components that may not accurately reflect physiological condi- tions within living cells. Overall, while protein kinase inhibitors offer Biochimica et Biophysica Acta xxx (2013) xxx–xxx ☆ This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012). ⁎ Corresponding author at: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada N6A 5C1. Tel.: +1 519 661 4186; fax: +1 519 661 3175. E-mail address: litchfi@uwo.ca (D.W. Litchfield). BBAPAP-39019; No. of pages: 7; 4C: 3, 4, 5 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.02.006 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap Please cite this article as: L. Gyenis, et al., Chemical proteomics and functional proteomics strategies for protein kinase inhibitor validation and protein kinase substrate identification: Applications ..., Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.02.006