PHOSPHORYLATION AND ACTIVATION OF PROTEIN-KINASE CK2 BY P34(CDC2) ARE INDEPENDENT EVENTS

Recombinant isolated P-subunit of protein kinase CK2 is readily phosphorylated by p34(cdc2)/cyclin B kinase at Ser209 with favourable kinetic constants (K-m = 1.7 mu M, V-max = 20 nmol . min(-1) . mg(-1)). Two synthetic peptides reproducing the 170-215 and the 206-215 C-terminal fragments of the beta-subunit are also phosphorylated though with tenfold higher K-m values (19.5 and 28.0 mu M, respectively). In contrast, both the beta-subunit associated with the alpha-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34(cdc2). These data suggest that the Ser209 beta-subunit phosphorylation observed in intact cells occurs prior to beta-subunit incorporation into the holoenzyme. The isolated CK2 alpha-subunit is not phosphorylated to any appreciable extent by p34(cdc2) kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34(cdc2)/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the beta-subunit and it is paralleled by a 40% decrease of p34(cdc2)/cyclin B catalytic activity. Similar to beta-subunit, p34(cdc2)/cyclin B also protects the alpha-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34(cdc2)/cyclin B, albeit less dramatically than the isolated alpha-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated beta-subunit lacking the p34(cdc2) phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the beta or the alpha-subunit. These data indicate that in vitro CK2 alpha-subunit interacts with and is activated by p34(cdc2)/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2.