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. The same acidic stretch also heavily influences the autophosphorylation of the beta subunit although the autophosphorylation site (Ser 2) is more than 50 amino acids apart in the primary sequence. This strongly suggests that in addition to amino acids 5 and 6 distant amino acids within the sequence 55-64 are also involved in the process of autophosphorylation, possibly by means of a loop formation. Deletion mutants where the last 45 or more amino acids are missing are not able to assemble with the alpha subunit. Amino acids 171-181 are involved in interaction with the alpha subunit and residues 181-208 seem to play a role during formation of the holoenzyme.