Interaction sites between catalytic and regulatory subunits in human protein kinase CK2 holoenzymes as indicated by chemical cross-linking and immunological investigations.

Abstract

Protein kinase CK2, a heterotetramer composed of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta), 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 cross-linking 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 beta55-70 and alpha65-80 (alpha'66-81) of subunits beta and alpha (alpha'), respectively. This was corroborated by cross-linking of subunits with peptides alpha65-80 and beta55-70 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 immunoprecipitation with antibodies anti-alpha65-80 and anti-beta55-70, resulting in precipitation but not coprecipitation of subunits. This alpha-beta (alpha'-beta) interaction site obviously is also of functional importance since subunits with attached antibodies cannot reconstitute to the fully active holoenzyme. Indeed, sites beta55-70 and alpha65-80 (alpha'66-81) correspond to an acidic (beta) and a basic (alpha or alpha') 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 constituted. At subunit beta, these include the terminal positions beta2-14 and beta204-213, the positions beta97-105 and beta140-156, and, surprisingly, also beta171-186 which have been shown by deletion mutation and peptide replacement studies to represent a positively affecting interaction site. At subunits alpha and alpha', these are the C-terminal positions alpha329 -343 and alpha'336-350. Binding of antibodies to the positions alpha15-27 (alpha'16-28) and position alpha151-166(alpha'152-167), on the other hand, inhibits activity.