The catalytic subunits of protein kinase CK2, expression, covalent modification, and regulatory interactions

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Bosc, Denis G.
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Biochemical and genetic studies have demonstrated that protein kinase CK2 (CK2) is one component of the regulatory protein kinase network implicated in cell proliferation and cell cycle progression. Its ubiquitous distribution, lethal effects of gene disruption and high degree of conservation suggest an important role for CK2 in eukaryotic cells, although its exact role in cells remains poorly understood. CK2 is a protein serine/threonine kinase which is composed of two catalytic subunits (CK2_ and/or CK2_') and two regulatory subunits (CK2B). The CK2_ and CK2_' catalytic isozymes are encoded by different genes, are highly conserved between many species of animals and are structurally similar, but differ dramatically at their respective carboxyl terminal domains (CTDs). This observation, coupled with reports that CK2_ and CK2_' display different cell cycle dependent intracellular distributions and phosphorylation has led us to hypothesize that functional differences exist between CK2_ and CK2_'. In order totest this hypothesis we have: (i) examined the expression levels of these isozymes during cell cycle progression, (ii) used synthetic peptides and fusion proteins to identify the mitotic sites of phosphorylation within CK2_, and (iii) used the yeast two-hybrid system to identify potential regulatory protein partners. We have found that, although expression levels of CK2_ and CK2_ ' are relatively similar in cells progressing through the cell cycle, there is a dramatic increase in protein levels of both isozymes after stimulation of cells to enter the cell cycle. Moreover, there are increased levels of CK2B in mitotically arrested cells. These results suggest that egulation of the protein levels of the subunits of CK2 within cells is important for entry and exit of the cell cycle. We have identified Thr344, Thr360, Ser362, and Ser370 as the mitotic sites of phosphorylation on CK2_. Identification of these sites is an important step in defining the role of CKII and its phosphorylation during cell division. We have also identified a novel protein, Phu, which interacts specifically with CK2_, but not with CK2_' . The work presented in this thesis demonstrates that regulated protein levels of CK2_ and CK2_' are important for cell cycle entry, and that differences between CK2_ and CK2_ ' are reflected in isoform specific post-translational modifications and protein:protein interactions.