| Elevated CK2 activity has been associated with the malignant transformation of several tissues, and is associated with aggressive tumor behavior. While the precise roles of CK2 in tumorigenesis remain incompletely understood, mounting evidence suggests a role for CK2 in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity. Consequently, CK2 has emerged as a potential therapeutic target, and strategies to inhibit CK2 have been ongoing in pre-clinical trials.;The convergence of protein kinases and caspase signaling pathways has become increasingly evident, as phosphorylation of a number of caspase substrates within the caspase recognition motif has been shown to prevent caspase cleavage. To investigate the global role of phosphorylation in the regulation of caspase signaling, a novel sequence-based protein identification targeting strategy, (sPITS) was employed. Intriguingly, the constitutively active and oncogenic protein kinase CK2 shares an overlapping requirement with caspases for an acidic consensus sequence, therefore, a comprehensive identification of overlapping CK2/caspase targets was carried out. A number of proteins involved in cell survival were identified, including pro-caspase-3, which was shown to be phosphorylated by CK2 preventing its caspase-dependent activation in cells. Validation of the phosphorylation-dependent protection of pro-caspase-3, as well as identification of numerous candidate CK2/caspase targets in the present studies, support a role for phosphorylation as a global mechanism of regulation of caspase signaling pathways.;Keywords. Protein kinase CK2, tumorigenesis, phosphorylation, kinase inhibitors, chemo-proteomics, bioinformatics, apoptosis, caspases, mass spectrometry, 2D gel electrophoresis;In the present studies, an unbiased evaluation of CK2 inhibitors TBB, TBBz and DMAT was carried out to elucidate the mechanism of action, as well as inhibitor specificity of these compounds. Utilizing a chemo-proteomic approach in conjunction with inhibitor-resistant mutant studies, isoforms CK2alpha and CK2alpha' were identified as bona fide targets of TBB, TBBz and DMAT in cells. However, a number of putative off-target inhibitor interactions were identified, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme Quinone Reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors, as well as drug specificity, which will assist in the development of more specific next-generation CK2 inhibitors. |
