ACTIVITY OF RECOMBINANT ALPHA-SUBUNITS AND BETA-SUBUNITS OF CASEIN KINASE-II FROM XENOPUS-LAEVIS

Casein kinase II (CKII) is a ubiquitous protein kinase, found predomin antly in cell nuclei, which has two subunits in a tetrameric alpha2bet a2 or alphaalpha'beta2 conformation. The catalytic center is present i n the alpha subunit which is active by itself while beta is a regulato ry subunit that can greatly enhance the activity of alpha. The cDNA ge nes of Xenopus laevis coding for the alpha and beta subunits of CKII h ave been expressed in Escherichia coli and extensively purified. The r ecombinant subunits reconstitute a fully active holoenzyme when incuba ted in stoichiometric amounts, Mutations that change serines in positi ons 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significan tly affect the capacity of beta to activate alpha. A fusion protein co mposed of glutathione transferase linked to the X. laevis CKII beta su bunit can also activate alpha. This fusion protein binds to glutathion e-agarose beads and can mediate the binding of the alpha subunit to th is matrix. Conversely, the alpha subunit was found to bind to glass fi ber filters in an active form that can still be activated by beta to a n extent similar to that seen in solution. Using peptides containing t yrosine and glutamic acid as inhibitors of the activity of the isolate d alpha subunit and of the holoenzyme, the effect of beta on the speci ficity of inhibition was studied. The results obtained demonstrate tha t the beta subunit participates in the specific recognition of tyrosin e by the holoenzyme and in the discrimination in the position of the t yrosyl residues with respect fo the acidic amino acids.