Protein kinase CK2 structure-function relationship: effects of the beta subunit on reconstitution and activity.
Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features as the native enzyme. The alpha subunit alone, although catalytically active by itself, has different biochemical and biophysical properties than the holoenzyme, e.g., it is extremely salt sensitive, already 50 mM monovalent salt can lead to a 50% inhibition of the catalytic activity. Furthermore, it is readily inactivated through urea, protease, and heat treatment. In contrast, the holoenzyme, either reconstituted or native, is much more stable when similar negative insults prevail. The beta subunit has at least three functions: (a) it is necessary for maximum activity of the enzyme under physiological salt conditions, (b) it protects the alpha subunit against denaturing agents or conditions, and (c) it alters the substrate specificity of the alpha subunit. By site-directed mutagenesis, certain functions of the beta subunit could be assigned to specific amino acids or domains. Twenty one mutants of the beta subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations of certain acidic amino acids within this stretch to alanine lead to a hyperactive CK2 holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Boldyreff
B
B
Institut für Humangenetik, Universität des Saarlandes, Homburg, Germany.
Meggio
F
F
Pinna
L A
LA
Issinger
O G
OG
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D010455:Peptides; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases