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 a n active holoenzyme which does not need to be activated by dissociatio n 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 whi ch are physiological in red cells. To examine the effects of autophosp horylation of the beta subunit on activity, two mutants of the Drosoph ila beta subunit have been constructed in which Ser-4 or Ser-(2-4) are changed to alanine residues. Analysis of autophosphorylation with wil d-type and mutant recombinant holoenzymes reveals Ser-2 and Ser-3 as t he major autophosphorylation sites. Autophosphorylation does not affec t the phosphorylation of casein, but reduces the rate of phosphorylati on of glycogen synthase by 30%, elongation factor I by 50-70%, and cal modulin by 20-40%. The data indicate that autophosphorylation of the b eta 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 a nd beta subunits from human and Drosophila were expressed in Escherich ia coli. Upon formation of the holoenzyme, the beta subunit stimulated the catalytic activity 4- to 5-fold. The catalytic alpha subunit cont ains the eleven conserved subdomains characteristic of all protein kin ases. Val-66 in subdomain II and Trp-176 in subdomain VII of the human alpha subunit were substituted with alanine and phenylalanine, the re sidues in the corresponding positions of more than 95% of all known pr otein kinase sequences. These mutations in residues unique to casein k inase II reduced the utilization of GTP and reduced or eliminated stim ulation by the beta subunit without dissociation of the tetrameric str ucture. Thus, stimulation of catalytic activity by the beta subunit is correlated with the ATP/ GTP utilization, and formation of the holoen zyme is not sufficient for the stimulation of catalytic activity; a co ncomitant conformational change is required.