Assembly of protein kinase CK2:: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2β

Protein kinase CK2 is a tetrameric enzyme comprised of two regulatory subunits (CK2 beta) and two catalytic subunits (CK2 alpha and/or CK2 alpha '). The crystal structure of dimeric CK2 beta demonstrated that a zinc ringer mediates CK2 beta dimerization, therefore we constructed a mutant in which cysteine residues 109 and 114 were mutated to serine. Our objectives were to examine the effects of disrupting the zinc finger of the regulatory CK2 beta subunit on CK2 tetramer assembly. Examination of this zinc-finger-deficient mutant of CK2 beta using a yeast two-hybrid assay demonstrates that the mutant fails to form CK2 beta homodimers. In order to extend these studies, weco- transfected COS-7 cells with epitope-tagged constructs and performed co-immunoprecipitation assays. The results from these studies demonstrate that the mutant fails to form CK2 beta homodimers and fails to interact with catalytic CK2 subunits. Furthermore, we demonstrate that the mutant CK2 beta is not appreciably phosphorylated in cells. Using in vitro binding assays, we demonstrated that the mutant CK2 beta protein fails to interact with glutathione S-transferase-CK2 alpha '. Finally, we demonstrate that the mutant is translated at an equivalent rate to wild-type CK2 beta, but is degraded much more rapidly. Overall, our results are consistent with the model that beta-beta dimerization precedes incorporation of catalytic subunits into tetrameric CK2 complexes, and that beta-beta dimerization is a prerequisite for the stable incorporation of catalytic subunits into CK2 complexes.