Interaction between CK2α and CK2β, the Subunits of Protein Kinase CK2: Thermodynamic Contributions of Key Residues on the CK2α Surface

Abstract

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.

Inhibition of CK2α Down-Regulates Hedgehog/Gli Signaling Leading to a Reduction of a Stem-Like Side Population in Human Lung Cancer Cells

“…To further validate the role played by CK2α in the Hh pathway, we used two small-molecule CK2α inhibitors: TBB (4,5,6,7-tetrabromobenzotriazole), a well-known inhibitor of CK2α [36], and CX-4945 (5-(3-chlorophenylamino)benzo[c] [2], [6]naphthyridine-8-carboxylic acid), a first-in-class, selective, oral inhibitor of CK2α under investigation in Phase 1 clinical trials [37].…”

“…CK2 has a heterotetrameric structure consisting of two catalytic subunits (42-kDa α or 38-kDa α’) and the regulatory subunit (28-kDa β), forming the configurations α2β2, αα’β2 and α’2β2. CK2 is a multifunctional protein kinase [2], that has been shown to be involved in nearly every aspect of cell proliferation and survival [3], [4], [5]. The level of CK2α expression is tightly regulated in normal cells [6], and increased CK2α level and activity has been consistently observed in a variety of human cancers [7], [8], [9].…”

Structure of the Human Protein Kinase CK2 Catalytic Subunit CK2α′ and Interaction Thermodynamics with the Regulatory Subunit CK2β

“…47,49 Such a function has been applied with limited success in a recent study on hsCK2α/hsCK2β interaction: 50 while the heat capacity ΔC p could be well reproduced and the standard Gibbs enthalpy ΔG°could be roughly computationally reproduced, the division of the latter into an enthalpic contribution (ΔH°) and an enthalpic contribution (TΔS°) was completely different from experimentally determined data. The mainly hydrophobic character of the interaction surfaces led, in particular, to a positive theoretical ΔH°, in strong contrast to the large negative enthalpic term measured for the first time by Raaf et al but also visible in Fig.…”

“…1c) (and its associated effects on the solvent structure) was nominated. 25,50 The results of this study do not allow a strict validation of this hypothesis, but they are at least partly consistent with it: in hsCK2α′, the β4/β5 loop is already preformed so that no equivalent motion of the β4/ β5 loop is necessary for hsCK2β binding; simultaneously, heat production as a consequence of hsCK2α′ Cys336Ser /hsCK2β association is much lower than in the case of hsCK2α 1-335 (Fig. 2b), and, even more important, this loss of negative enthalpy is only partly compensated for by entropy, resulting eventually in a reduced affinity of hsCK2α′ Cys336Ser for hsCK2β compared to hsCK2α 1-335 .…”

CK2¿, over-expressed in human malignant pleural mesothelioma, regulates the Hedgehog signaling pathway in mesothelioma cells

“…CK2 has a heterotetrameric structure consisting of two catalytic subunits (42-kDa α or 38-kDa α') and the regulatory subunit (28-kDa β), forming the configurations α2β2, αα'β2 and α'2β2. CK2 is a multifunctional protein kinase [2], that has been shown to be involved in nearly every aspect of cell proliferation and survival [3][4][5]. The level of CK2α expression is tightly regulated in normal cells [6], and increased CK2α level and activity has been consistently observed in a variety of human cancers [7][8][9].…”