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

Citation
A. Krehan et al., INTERACTION SITES BETWEEN CATALYTIC AND REGULATORY SUBUNITS IN HUMAN PROTEIN-KINASE CK2 HOLOENZYMES AS INDICATED BY CHEMICAL CROSS-LINKING AND IMMUNOLOGICAL INVESTIGATIONS, Biochemistry, 35(15), 1996, pp. 4966-4975
Citations number
60
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
35
Issue
15
Year of publication
1996
Pages
4966 - 4975
Database
ISI
SICI code
0006-2960(1996)35:15<4966:ISBCAR>2.0.ZU;2-C
Abstract
Protein kinase CK2, a heterotetramer composed of two catalytic subunit s (alpha and/or alpha') and two regulatory subunits (beta), has been e xamined for intermolecular contact sites by methods that allow investi gation of the native, unaltered proteins. Antibodies were raised again st 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 t he sequence specific antibodies identified a tight interaction between positions beta 55-70 and alpha 65-80 (alpha'66-81) of subunits beta a nd alpha (alpha'), respectively. This was corroborated by cross-linkin g of subunits with peptides alpha 65-80 and beta 55-70 by a peptide-ba sed enzyme-linked immunosorbent assay in which peptides bound to wells via C-10 spacer arms are probed for complexing individual subunits an d by immunoprecipitation with antibodies anti-alpha 65-80 and anti-bet a 55-70, resulting in precipitation but not coprecipitation of subunit s. 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 bas ic (alpha or alpha') domain involved in activity and stability control and in substrate and cosubstrate binding (kinase domain II/III), resp ectively. By contrast, a number of suspected contact sites were found to be rather loose and not essential for enzyme control as concluded f rom precipitation behavior of respective antibodies and the toleration of attached antibodies when active holoenzymes were being reconstitut ed. At subunit beta, these include the terminal positions beta 2-14 an d beta 204-213, the positions beta 97-105 and beta 140-156, and, surpr isingly, also beta 171-186 which has been shown by deletion mutation a nd peptide replacement studies to represent a positively affecting int eraction site. At subunits alpha and alpha', these are the C-terminal positions alpha 329-343 and alpha'336-350. Binding of antibodies to th e positions alpha 15-27 (alpha'16-28) and position alpha 151-166 (alph a'152-167), on the other hand, inhibits activity.