The role of protein kinase CK2 in metal ion toxicity

Abstract

Protein Kinase CK2 is an enzyme discovered in the 1950s. Since then we have gained knowledge in many aspects of its biological function, such as cell proliferation and anti-apoptosis. This study, by means of gene deletion mutants and unbiased genome-wide screening of the model organism Saccharomyces cerevisiae, has uncovered a new dimension of CK2's functionality, i.e. its role in regulating metal toxicity. This discovery is then further extended to mammalian cells. The findings described in Chapter 3, by using the deletion mutants of CK2 subunits (CKA1, CKA2, CKB1 and CKB2) in S. cerevisiae, revealed that deletion of CKA2, the yeast orthologue of mammalian CK2I±', leads to a pronounced resistant phenotype against Zn2+ and Al3+, whilst deletion of CKB1 or CKB2 results in tolerance to Cr6+ and As3+. These data demonstrate that CK2 is involved in regulating metal toxicity, and individual CK2 subunits have distinct roles in this regulation. Furthermore, the results add to the growing evidence that show monomeric CK2 subunits can function discretely, apart from the tetrameric holoenzyme, which contains both catalytic and regulatory subunits. Further investigation using double deletion mutants and inductively coupled plasma mass spectrometry (ICP-MS) uncovered the basis for the resistant phenotype of cka2I" in response to the exposure to Al3+ or Zn2+. Double deletion of CKA2 with either CKB1 or CKB2 and the inhibition of CK2 function by TBB (4,5,6,7-Tetrabromo-2-azabenzimidazole) in cka2I" revealed that the resistant phenotype of cka2I" under Al3+ exposure was not affected, suggesting that CKA2 is the solely responsible CK2 gene for Al3+ toxicity. ICP-MS analysis demonstrated that deletion of CKA2 resulted in 52% reduction in Al3+ content compared to the wildtype at 4 h, 85% reduction at 8 h and a 65% reduction at 12 and 16 h post treatment. This clearly shows that Cka2p is involved in Al3+ uptake in a manner independent of the other CK2 subunits present in the cell. In contrast to this finding, the resistant phenotype of cka2I" under Zn2+ exposure was found to be related to the function of the remaining CK2 subunits (Cka1p, Ckb1p and Ckb2p). The resistant growth phenotype was markedly reduced when CKA2 was deleted together with either of the regulatory subunits (CKB1 or CKB2). Further, the resistant phenotype of cka2I" was completely abolished when CK2 function was inhibited by TBB. ICP-MS quantification showed that the level of Zn2+ found in cka2I" was very similar to the wildtype at all time-points tested, indicating that, rather than conferring resistance by excluding Zn2+ from the cell, the remaining CK2 subunits were participating in intracellular sequestration of Zn2+, possibly by interacting with vacuolar Zn2+ transport proteins Zrc1p and Cot1p. The monomeric regulatory subunits of CK2 are involved in As3+ toxicity. While deletion of CKB1 or CKB2 conferred resistance to As3+, deletion of both these genes resulted in sensitivity. This decrease in viability under As3+ treatment when both regulatory subunits are deleted indicates that the regulatory subunits have functions outside the tetramer. The genome-wide deletion screen data shown in Chapter 4 reveal the molecular mechanisms related to As3+ toxicity, uptake and detoxification by identification of tolerant and sensitive genes. Of the genes identified in the screen, their deletion coupled with the deletion of CK2 genes showed that Ckb1p or Ckb2p likely interacts with the genes in the Hog1 (MAPK) pathway. ICP-MS validated that deletion of CKB1 and CKB2 resulted in lower intracellular levels of As3+. Similarly, deletion of CKB1 or CKB2 leads to resistance against Cr6+ due to the reduced intracellular level of the metal ion. The genome wide screen demonstrates oxidative stress as a major cause for Cr6+ toxicity. The increased growth of CKB1 and CKB2 deletion mutants under Cr6+ exposure was found to also be correlated with reduced oxidative stress. Finally, the experimental findings in mammalian neuronal cells with the CK2 inhibitor TBB demonstrated that CK2 activity under the exposure of metal ions such as Al3+ and Zn2+ is disadvantageous to the cell's viability. Further analysis with specific siRNA against the individual CK2 subunits reveals that such negative effect of CK2 to the cell's viability under metal stress is largely a result of the CK2I±' subunit. The viability of both Neuro2a and SH-SY5Y cells, when exposed to IC25 and IC50 doses of Zn2+ or Al3+, was significantly increased when Ck2I±' expression was knocked down (p < 0.001). This finding demonstrates CK2 is involved in the regulation of metal toxicity in mammalian cells, just like what is described previously in S. cerevisiae. Overall, the work contained in this thesis establishes a new role for protein kinase CK2 in metal ion toxicity and demonstrates that individual subunits of CK2 have discrete functions. The findings, which have already been published in peer-reviewed journals, have enhanced our knowledge and understanding of CK2's biological roles, and have provided useful data which have been used to update the yeast genomic functionality in Saccharomyces Genome Database (https://www.yeastgenome.org/). Due to the links between metals, neurodegenerative diseases and cancers, the discovery of the linkage of CK2 with metal toxicity here opens an exciting vista for tackling major health problems such as cancers and neurodegenerative disorders.

Date of Award	2018
Original language	English