The physiological significance of the casein kinase 2 (CK2)-mediated phosphorylation of type II CAMP-dependent protein kinase (PKAIIalpha) and free type II regulatory (R) subunit (RIIalpha) on their activities was mainly investigated in vitro. In these experiments, [gamma-P-32]GTP was used as a phosphate donor for the CK2-mediated phosphorylation of free RIIalpha and PKAIIalpha (bovine heart) in vitro. It was found that: (i) CK2 phosphorylated only threonine (Thr)-residues of free RIIalpha and phosphorylated preferentially Thr-residues of the R subunit (RIIalpha) of PKAIIalpha (PKA RIIalpha) in vitro; (ii) this phosphorylation was selectively inhibited by quercetin (an CK2 inhibitor): and (iii) the phosphorylation of free RIIalpha by CK2 resulted in the reduction of its suppressive effect on the activity (phosphorylation of histone H2B) of the catalytic (C) subunit and in the reduction of its ability to form a complex with the C subunit in vitro. As expected. the activity of PKAIIalpha was approx. 3.5-fold enhanced after its R subunit was fully phosphorylated by CK2 in vitro. CAMP synergistically stimulated the activity of PKAIIalpha phosphorylated by CK2 in vitro. These results strongly suggest that CK2 may be a protein kinase responsible for the activation of PKAIIalpha through specific phosphorylation of its R subunit at the cellular level. (C) 2003 Elsevier Inc. All rights reserved.