MODES OF REGULATION OF CASEIN KINASE-II

Casein kinase II is unique when compared to other protein kinases; it utilizes GTP with almost the same effectiveness as ATP and exists as an active holoenzyme which does not need to be activated by dissociation of regulatory subunits or unfolding of regulatory domains. In vitro, the activity of casein kinase II is inhibited by acidic compounds and stimulated by basic compounds. Casein kinase II activity is inhibited by 2,3-bisphosphoglycerate and stimulated by polyamines at levels which are physiological in red cells. To examine the effects of autophosphorylation of the beta subunit on activity, two mutants of the Drosophila beta subunit have been constructed in which Ser-4 or Ser-(2-4) are changed to alanine residues. Analysis of autophosphorylation with wild-type and mutant recombinant holoenzymes reveals Ser-2 and Ser-3 as the major autophosphorylation sites. Autophosphorylation does not affect the phosphorylation of casein, but reduces the rate of phosphorylation of glycogen synthase by 30%, elongation factor I by 50-70%, and calmodulin by 20-40%. The data indicate that autophosphorylation of the beta subunit can negatively regulate the phosphotransferase activity of casein kinase II with physiological substrates. To examine regulation of casein kinase II activity by the beta subunit, recombinant alpha and beta subunits from human and Drosophila were expressed in Escherichia coli. Upon formation of the holoenzyme, the beta subunit stimulated the catalytic activity 4- to 5-fold. The catalytic alpha subunit contains the eleven conserved subdomains characteristic of all protein kinases. Val-66 in subdomain II and Trp-176 in subdomain VII of the human alpha subunit were substituted with alanine and phenylalanine, the residues in the corresponding positions of more than 95% of all known protein kinase sequences. These mutations in residues unique to casein kinase II reduced the utilization of GTP and reduced or eliminated stimulation by the beta subunit without dissociation of the tetrameric structure. Thus, stimulation of catalytic activity by the beta subunit is correlated with the ATP/ GTP utilization, and formation of the holoenzyme is not sufficient for the stimulation of catalytic activity; a concomitant conformational change is required.