The protein kinase CK2 (CK2) family encompasses a small number of acidophilic serine/threonine kinases that phosphorylate substrates involved in numerous biological processes including apoptosis, cell proliferation, and the DNA damage response. CK2 has also been implicated in many human malignancies and other disorders including Alzheimer′s and Parkinson’s diseases, and COVID-19. Interestingly, no single mechanism describes how CK2 is regulated, including activation by external proteins or domains, phosphorylation, or dimerization. Furthermore, the kinase has an elongated activation loop that locks the kinase into an active conformation, leading CK2 to be labelled a constitutively active kinase. This presents an interesting paradox that remains unanswered: how can a constitutively active kinase regulate biological processes that require careful control? Here, we highlight a selection of studies where CK2 activity is regulated at the substrate level, and discuss them based on the regulatory mechanism. Overall, this review describes numerous biological processes where CK2 activity is regulated, highlighting how a constitutively active kinase can still control numerous cellular activities. It is also evident that more research is required to fully elucidate the mechanisms that regulate CK2 and what causes aberrant CK2 signaling in disease.

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Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

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