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

Abstract CK2 is an essential, ubiquitous, and highly pleiotropic protein kinase whose catalytic subunits (α and α′) and holoenzyme (composed by two catalytic and two regulatory β-subunits) are both constitutively active, a property that is suspected to contribute to its pathogenic potential. Extensive interactions between the N-terminal 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 (Tyr26 → Phe, Glu180 → Ala, Tyr182 → Phe) and deletions (Δ2–6, Δ2–12, Δ2–18, Δ2–24, Δ2–30) expected to affect these interactions are more or less detrimental to catalytic activity of the α-subunit of human CK2, the deleted mutants Δ2–24 and Δ2–30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants Δ2–12, Δ2–18, Δ2–24, and Y182F is mainly accounted for by dramatic increases in theK m values for ATP, whereas a drop inK cat with K m values almost unchanged was found with mutants Y26F and E180A. Holoenzyme reconstitution restored the activity of mutants Δ2–12, Δ2–18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, Δ2–24 and Δ2–30. 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 β-subunit through a different mechanism.