INTERACTION SITES BETWEEN CATALYTIC AND REGULATORY SUBUNITS IN HUMAN PROTEIN KINASE CK2 HOLOENZYMES AS INDICATED BY CHEMICAL CROSS-LINKING AND IMMUNOLOGICAL INVESTIGATIONS

摘要

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 beta 55-70 and alpha 65-80 (alpha'66-81) of subunits beta and alpha (alpha'), respectively. This was corroborated by cross-linking of subunits with peptides alpha 65-80 and beta 55-70 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-alpha 65-80 and anti-beta 55-70, 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 beta 55-70 and alpha 65-80 (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 reconstituted. At subunit beta, these include the terminal positions beta 2-14 and beta 204-213, the positions beta 97-105 and beta 140-156, and, surprisingly, also beta 171-186 which has 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 alpha 329-343 and alpha'336-350. Binding of antibodies to the positions alpha 15-27 (alpha'16-28) and position alpha 151-166 (alpha'152-167), on the other hand, inhibits activity.