Abstract
CK2 is a ubiquitous, constitutively active, highly pleiotropic, acidophilic Ser/Thr protein kinase whose holoenzyme is composed of two catalytic (α and/or α’) subunits and a dimer of a non-catalytic β subunit. Abnormally high CK2 level/activity is often associated with malignancy and a variety of cancer cells have been shown to rely on it to escape apoptosis. To gain information about the actual “druggability” of CK2 and to dissect CK2 dependent cellular processes that are instrumental to the establishment and progression of neoplasia we have exploited the CRISPR/Cas9 genome editing technology to generate viable clones of C2C12 myoblasts devoid of either both the CK2 catalytic subunits or its regulatory β-subunit. Suppression of both CK2 catalytic subunits promotes the disappearance of the β-subunit as well, through its accelerated proteasomal degradation. A quantitative proteomics analysis of CK2α/α’(−/−) versus wild type cells shows that knocking out both CK2 catalytic subunits causes a rearrangement of the proteomics profile, with substantially altered level ( > 50%) of 240 proteins, 126 of which are up-regulated, while the other are down-regulated. A functional analysis reveals that up- and down-regulated proteins tend to be segregated into distinct sub-cellular compartments and play different biological roles, consistent with a global rewiring underwent by the cell to cope with the lack of CK2.
Highlights
Phenomena associated with cancer[9,12,15]: it counteracts the efficacy of anticancer drugs, it strengthens the multi drug resistance (MDR) phenotype, it activates the chaperone machinery protecting the onco-kinome, and it sustains neo-vascularization
While in the case of the individual catalytic subunits this proved feasible with a variety of cells, including cancer cells, up to date the knockout of either both the catalytic subunits or the regulatory βsubunit was only successful with the C2C12 myoblast cell line
This is documented both by western blots of the individual subunits (Fig. 1A) and by monitoring the phosphorylation of Ser[129] of Akt, notoriously affected by CK223 (Fig. 1B). pS129 largely disappears in cells where both subunits are suppressed, at variance with the phosphorylation of Akt T308 and S473, which is not catalysed by CK2 and is comparable in wild type (WT) and CK2 null cells (Fig. 1B)
Summary
Phenomena associated with cancer[9,12,15]: it counteracts the efficacy of anticancer drugs, it strengthens the multi drug resistance (MDR) phenotype, it activates the chaperone machinery protecting the onco-kinome, and it sustains neo-vascularization On top of this it plays a global role as an anti-apoptotic and pro-survival agent[4,16]. A proteomics analysis of these clones discloses substantial alterations that reflect their adaptation to live in the absence of CK2 activity and may shed light on cancer hallmarks that critically depend on CK2 These cells will provide a valuable tool for discriminating between on- and off-target effects of currently used CK2 inhibitors and to explore the susceptibility of cells devoid of CK2 to undergo transformation upon transfection with oncogenes and with tumour suppressor genes
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