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.