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Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

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Polypeptides rich in glutamic acid are strong inhibitors purified from isolated nuclei of Xenopus luevis oocytes of casein kinase II. The presence of tyrosine in these peptides greatly enhances their inhibitory capacity. Using casein as a substrate, copolyglu:tyr (41) has an Z5,, value of 20 nM, 250 fold lower than that of polyglutamic acid which is 5 PM. A similar large difference is observed when a synthetic peptide is used as substrate. The inhibition of copolyglu:tyr is competitive with casein and can be completely reversed by high ionic strength. The relative inhibitory capacity of the polypeptides tested, in descending order, is copolyglu:tyr (4: 1) > copolyglu:tyr (1: 1) > polyglu > copolyglu:phe (4: 1) > copolyglu:ala (64) > copolyglu:leu (4:l). The high affinity for tyrosine-containing acid peptides is shared by rat liver and yeast casein kinase II so that it seems to be a general property of these. enzymes.

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