Unique Activation Mechanism of Protein Kinase CK2 THE N-TERMINAL SEGMENT IS ESSENTIAL FOR CONSTITUTIVE ACTIVITY OF THE CATALYTIC SUBUNIT BUT NOT OF THE HOLOENZYME*

CK2 is an essential, ubiquitous, and highly pleiotropic protein kinase whose catalytic subunits ( (cid:1) and (cid:1) (cid:1) ) and holoenzyme (composed by two catalytic and two regulatory (cid:2) -subunits) are both constitutively active, a property that is suspected to contribute to its pathogenic potential. Extensive interactions between the N-termi-nal segment and the activation loop are suspected to underlie the high constitutive activity of the isolated catalytic subunit. Here we show that a number of point mutations (Tyr 26 3 Phe, Glu 180 3 Ala, Tyr 182 3 Phe) and deletions ( (cid:3) 2–6, (cid:3) 2–12, (cid:3) 2–18, (cid:3) 2–24, (cid:3) 2–30) expected to affect these interactions are more or less detrimental to catalytic activity of the (cid:1) -subunit of human CK2, the deleted mutants (cid:3) 2–24 and (cid:3) 2–30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants (cid:3) 2–12, (cid:3) 2–18, (cid:3) 2–24, and Y182F is mainly accounted for by dramatic increases in the K m values for ATP, whereas a drop in K cat with K m values almost unchanged was found with mutants Y26F and E180A. Holoenzyme reconstitution restored the activity of mutants (cid:3) 2–12, (cid:3) 2–18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, (cid:3) 2–24 and (cid:3) 2–30. These data demonstrate that specific interactions between the N-terminal segment and the activation loop are essential to provide a fully active conformation to the catalytic subunits of CK2; they also show that these interactions become dispensable upon formation of the holoenzyme, whose constitutive activity is conferred by the (cid:2) -subunit through a different mechanism. purified to near homogeneity as described previously (23). Purified proteins were dialyzed against 25 m M Tris, pH 7.5, and 50% of glycerol and stored at (cid:3) 20 °C. Protein Determination— Proteins were determined by the method of Bradford (24) using bovine serum albumin as standard. Phosphorylation Assay— Phosphorylation experiments were rou-tinely performed at 30 °C for 10 min in a medium (30 (cid:4) l final volume) containing 50 m M Tris-HCl, pH 7.5, 12 m M MgCl 2 . The presence of 100 m M NaCl and the concentration of the kinase are detailed in the legends of the figures and tables. The reaction was started by addition of 50 (cid:4) M [ (cid:3) - 32 P]ATP (specific activity 300–1000 cpm/pmol). 32 P incorporation was evaluated by the phosphocellulose paper procedure (25) using the synthetic peptide RRRADDSDDDDD as specific substrate. In the case of protein phosphorylation and CK2 autophosphorylation experiments the reaction was blocked by addition of Laemmli solution followed by SDS-PAGE (26), and the gel was subjected to direct scanning on an Instant Imager (Canberra Packard). Kinetic constants were determined by double-reciprocal plots constructed from initial rate measurements fitted to the Michaelis-Menten equation.