Exploring the CK2 Paradox: Restless, Dangerous, Dispensable

Abstract

The history of protein kinase CK2 is crowded with paradoxes and unanticipated findings. Named after a protein (casein) that is not among its physiological substrates, CK2 remained in search of its targets for more than two decades after its discovery in 1954, but it later came to be one of the most pleiotropic protein kinases. Being active in the absence of phosphorylation and/or specific stimuli, it looks unsuitable to participate in signaling cascades, but its "lateral" implication in a variety of signaling pathways is now soundly documented. At variance with many "onco-kinases", CK2 is constitutively active, and no oncogenic CK2 mutant is known; still high CK2 activity correlates to neoplasia. Its pleiotropy and essential role may cast doubts on the actual "druggability" of CK2; however, a CK2 inhibitor is now in Phase II clinical trials for the treatment of cancer, and cell clones viable in the absence of CK2 are providing information about the mechanism by which cancer becomes addicted to high CK2 levels. A phosphoproteomics analysis of these CK2 null cells suggests that CK2 pleiotropy may be less pronounced than expected and supports the idea that the phosphoproteome generated by this kinase is flexible and not rigidly pre-determined.

Keywords: CRISPR/Cas9 technology; cancer; casein kinase 2; non oncogene addiction; phosphoproteomics; protein kinase CK2; signal transduction.

Figure 1

The growing number of proteins phosphorylated by CK2 since its discovery in 1954. Constructed with data from [10,11,12,13]. According to PhosphoSitePlus [10], the number of phosphosites known to be generated by CK2 is presently 640, which belong to almost 400 proteins altogether.

Figure 2

Only a minor proportion of the phosphosites, conforming to the CK2 consensus quantified in C2C12 myoblasts devoid of both CK2 catalytic subunits, is drastically reduced (>50%), as compared to the wild type. A SILAC experiment has been conducted on C2C12 cell lines where both the catalytic CK2 α subunits have been knocked out by the CRISPR/Cas9 technology, as described in Borgo et al. [39]. CK2 null cells have been compared with wild type, both from the proteomics [39] and the phosphoproteomics side (unpublished data). To determine which phosphosites are affected by the absence of CK2, phosphopeptide enrichment has been performed, as already described in [41], and only peptides with alterations in the phosphorylation state of at least 50% have been considered as significantly varied.

Figure 3

The phosphorylation of bona fide CK2 sites is not entirely abrogated in CK2 null cells. Wild-type (WT) and CK2 null (CK2α/α’^(−/−)) cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 10% glycerol, 1 mM EDTA, 150 mM NaCl, and protease and phosphatase inhibitor cocktails. Thirty micrograms of lysate proteins were subjected to 11% SDS-PAGE and analyzed by Western blot with the indicated antibodies. Panel A shows that both the CK2 catalytic subunits are absent in the CK2 null cells. In panel B, the significant residual phosphorylation of two bona fide CK2 sites in these cells can be appreciated. The figure is representative of three independent experiments.