Protein kinase CK2, a heterotetramer composed of two catalytic subunits (α and/or α') and two regulatory subunits (β), has been examined for intermolecular contact sites by methods that allow investigation of the native, unaltered proteins. Antibodies were raised against a series of 11 subunit peptides, affinity purified, and ensured for site specific binding by peptide competition. Chemical crosslinking of CK2 subunits with a hydrophilic carbodiimide and analysis of fused subunits and of CNBr-digested fusion products by immunoblotting with the sequence specific antibodies identified a tight interaction between positions β55-70 and α65-80 (α'66-81) of subunits β and α (α'), respectively. This was corroborated by cross- linking of subunits with peptides α65-80 and β55-70 by a peptide-based enzyme-linked immunosorbent assay in which peptides bound to wells via C-10 spacer arms are probed for complexing individual subunits and by immunoprecipitation with antibodies anti-α65-80 and anti-β55-70, resulting in precipitation but not coprecipitation of subunits. This α-β (α'-β) interaction site obviously is also of functional importance since subunits with attached antibodies cannot reconstitute to the fully active holoenzyme. Indeed, sites β55-70 and α65-80 (α'66-81) correspond to an acidic (β) and a basic (α or α') domain involved in activity and stability control and in substrate and cosubstrate binding (kinase domain II/III), respectively. By contrast, a number of suspected contact sites were found to be rather loose and not essential for enzyme control as concluded from precipitation behavior of respective antibodies and the toleration of attached antibodies when active holoenzymes were being reconstituted. At subunit β, these include the terminal positions β2-14 and β204-213, the positions β97-105 and β40- 156, and, surprisingly, also β171-186 which has been shown by deletion mutation and peptide replacement studies to represent a positively affecting interaction site. At subunits α and α', these are the C-terminal positions α329-343 and α'336-350. Binding of antibodies to the positions α15-27 (α'16-28) and position α151-166 (α'152-167), on the other hand, inhibits activity.
|Publication status||Published - 16 Apr 1996|