Phosphorylation of Calmodulin by the Catalytic Subunit of Casein Kinase II Is Inhibited by the Regulatory Subunit

Casein kinase II (CKII) is composed of a catalytic subunit (α) and a regulatory subunit (β) that combine to form an α2β2 holoenzyme. The α-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme, and the addition of purified β subunit stimulates its activity against casein (C. Cochet and E. M. Chambaz, 1983, J. Biol. Chem. 258, 1403-1406). Here we report a kinetic analysis of the phosphorylation of various protein and peptide substrates by the a subunit and the holneazyme of Drosophila melanogaster CKII. We demonstrate that the α subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (Rh) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as α-casein and the synthetic peptide RRREEETEEE. The Km of the α subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the α subunit alone. While the Km values of the α subunit and the holoenzyme for calmodulin are similar, the Vmax of the α subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the α subunit contains the necessary determinants for CKII substrate specificity, the β subunit can either inhibit or activate it, in a substrate-dependent manner. Finally, we also demonstrate that polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the a subunit as well.