RECONSTITUTION OF NORMAL AND HYPERACTIVATED FORMS OF CASEIN KINASE-2 BY VARIABLY MUTATED BETA-SUBUNITS

Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants betaDELTA1-4 and betaA5,6) or bear variable deletions in their C-terminal part (mutants betaDELTA209-215, betaDELTA194-215, betaDELTA181-215, betaDELTA171-215, betaDELTA150-215) or have undergone Ala substitutions for the acidic and basic residues which are concentrated in the sequences 55-70 and 171-180, respectively. All these mutants have been examined for their ability to functionally replace the wild type beta-subunit. All substitutions and the deletions DELTA1-4, DELTA194-215, and DELTA209-215 are compatible with effective binding of the catalytic alpha-subunit, as judged by sucrose density gradient analysis, stimulation of catalytic activity, and protection against thermal denaturation. Deletions DELTA171-215 and DELTA150-215, however, give rise to truncated molecules which are unable to associate with the alpha-subunit. The intermediate deletion DELTA181-215 is still compatible with association, albeit the reconstituted holoenzyme exhibits an altered sedimentation coefficient. The holoenzymes reconstituted with substituted mutants betaA55,57, betaA55-57, and, to a lesser extent, betaA59-61, betaA63,64, and betaA5,6 display a basal activity which is higher (up to 4-fold) than that of the wild type holoenzyme. These data map two functional domains of the beta-subunit: (1) a C-terminal segment which is essential for the association with the alpha-subunit, whose crucial element appears to be the overall conformation of the 171-180 sequence and (ii) an acidic N-terminal region including a number of carboxylic residues (notably Asp-55 and Glu-57) responsible for an intrinsic negative regulation of CK2 basal activity and possibly implicated in the responsiveness to various effectors.