Effects of Autophosphorylation on Casein Kinase II Activity: Evidence from Mutations in the β Subunit

Casein kinase II is a heterotetramer composed of two catalytic (α) and two regulatory (β) subunits. To examine the effects of autophosphorylation of the β subunit on enzyme activity, two mutants of the β subunit from Drosophila were constructed in which either Ser₄ or Ser2_–4 was changed to alanine residues by oligonucleotide-directed mutagenesis and the proteins were expressed in Escherichia coli. The wild-type α and individual β subunits present in inclusion bodies were renatured, and the biochemical properties of the reconstituted holoenzymes were examined. Analysis of autophosphorylation revealed that phosphate incorporation was about 0.8 mol/mol of β subunit for the wild type and Ala₄ mutant; Ser₂ and Ser3 were the major sites of autophosphorylation with some phosphate in Ser₄ as shown by Edman degradation. No autophosphorylation was observed with the Ala_2–4 mutant. Substitution of alanine for serine residues at positions 4 or 2–4 of the β subunits did not influence the reassociation of the α and β subunits to form holoenzyme, or the function of the β subunit in stimulating catalytic activity or in responding to basic compounds. To measure the effects of autophosphorylation on casein kinase II activity, the wild-type and mutant holoenzymes were preincubated in the presence and absence of ATP, and the rate of phosphorylation was measured with various substrates. In the absence of autophosphorylation, the wild-type, Ala₄, and Ala_2–4 forms of the holoenzyme displayed similar rates of phosphorylation of glycogen synthase. After preincubation with ATP, the rate of phosphorylation of glycogen synthase by the wild-type and Ala₄ enzymes was inhibited by 30%. No inhibition was observed with the Ala_2–4 mutant under the same conditions. Similar results were observed with EF-1β and -δ (50-70% inhibition) and calmodulin (20-40% inhibition) with the autophosphorylated wild type and Ala₄ mutant. No effect of autophosphorylation was observed with casein. The data indicate that autophosphorylation of the β subunit can negatively regulate the phosphotransferase activity of casein kinase II with physiological substrates.