Assembly of protein kinase CK2: investigation of complex formation between catalytic and regulatory subunits using a zinc-finger-deficient mutant of CK2beta.
Protein kinase CK2 is a tetrameric enzyme comprised of two regulatory subunits (CK2beta) and two catalytic subunits (CK2alpha and/or CK2alpha'). The crystal structure of dimeric CK2beta demonstrated that a zinc finger mediates CK2beta 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 CK2beta subunit on CK2 tetramer assembly. Examination of this zinc-finger-deficient mutant of CK2beta using a yeast two-hybrid assay demonstrates that the mutant fails to form CK2beta homodimers. In order to extend these studies, we co-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 CK2beta homodimers and fails to interact with catalytic CK2 subunits. Furthermore, we demonstrate that the mutant CK2beta is not appreciably phosphorylated in cells. Using in vitro binding assays, we demonstrated that the mutant CK2beta protein fails to interact with glutathione S-transferase-CK2alpha'. Finally, we demonstrate that the mutant is translated at an equivalent rate to wild-type CK2beta, 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.
Canton
D A
DA
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Zhang
C
C
Litchfield
D W
DW
D000818:Animals; D019556:COS Cells; D047390:Casein Kinase II; D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers; D001616:beta-Galactosidase / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017027:Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D001616:beta-Galactosidase