Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

At the first glance CK2α, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2α the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2α structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2α structures. In the latter a surprising plasticity of important ATP-binding elements – the interdomain hinge region and the glycine-rich loop – was discovered. In fully active CK2α the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the γ-phospho group from its functional position. In addition to this partially inactive state human CK2α was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2β: if CK2β blocks this site, the fully active conformation of CK2α is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.     

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation.     

Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2α belongs to the CMGC family of the eukaryotic protein kinases. CK2 is an important survival and stability factor in eukaryotic cells: its catalytic activity is elevated in a wide variety of tumors while its down-regulation can lead to apoptosis. Thus, CK2 is a valuable target for drug development and for chemical biology approaches of cell biological research, and small organic inhibitors addressing CK2 are of considerable interest. We describe here the complex structure between a C-terminal deletion mutant of human CK2α and the ATP-competitive inhibitor emodin (1,3,8-trihydroxy-6-methylanthraquinone, International Union of Pure and Applied Chemistry name: 1,3,8-trihydroxy-6-methylanthracene-9,10-dione) and compare it with a previously published complex structure of emodin and maize CK2α. With a resolution of 1.5 Å, the human CK2α/emodin structure has a much better resolution than its maize counterpart (2.6 Å). Even more important, in spite of a sequence identity of more than 77% between human and maize CK2α, the two structures deviate significantly in the orientation, in which emodin is trapped by the enzyme, and in the local conformations around the ligand binding site: maize CK2α shows its largest adaptations in the ATP-binding loop, whereas human CK2α shows its largest adaptations in the hinge region connecting the two main domains of the protein kinase core. These observations emphasize the importance of local plasticity for ligand binding and demonstrate that two orthologues of an enzyme can behave quite different in this respect.     

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2?? and its paralogous isoform CK2????

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open β4/β5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2β. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.     

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation.     

Structural and functional determinants of protein kinase CK2��: facts and open questions

Ser/Thr protein kinase CK2 is involved in several fundamental processes that regulate the cell life, such as cell cycle progression, gene expression, cell growth, and differentiation and embryogenesis. In various cancers, CK2 shows a markedly elevated activity that has been associated with conditions that favor the onset of the tumor phenotype. This prompts to numerous studies aimed at the identification of compounds that are able to inhibit the catalytic activity of this oncogenic kinase, in particular, of ATP-competitive inhibitors. The many available crystal structures indicate that this enzyme owns some regions of remarkable flexibility which were associated to important functional properties. Of particular relevance is the flexibility, unique among protein kinases, of the hinge region and the following helix αD. This study attempts to unveil the structural bases of this characteristic of CK2. We also analyze some controversial issues concerning the functional interpretation of structural data on maize and human CK2 and try to recognize what is reasonably established and what is still unclear about this enzyme. This analysis can be useful also to outline some principles at the basis of the development of effective ATP-competitive CK2 inhibitors.     

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK.     

First Inactive Conformation of CK2α, the Catalytic Subunit of Protein Kinase CK2

The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2β, noncatalytic subunit of CK2). CK2α belongs to the superfamily of eukaryotic protein kinases (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix αC and in the activation segment, leading to a characteristic arrangement of catalytic key elements. For CK2α, however, no strict physiological control of activity is known. Accordingly, CK2α was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N-terminal segment on one side, and helix αC and the activation segment on the other side. We report here the structure of a C-terminally truncated variant of human CK2α in which the enzyme adopts a decidedly inactive conformation for the first time. In this CK2α structure, those regulatory key regions still are in their active positions. Yet the glycine-rich ATP-binding loop, which is normally part of the canonical anti-parallel β-sheet, has collapsed into the ATP-binding site so that ATP is excluded from binding; specifically, the side chain of Arg47 occupies the ribose region of the ATP site and Tyr50, the space required by the triphospho moiety. We discuss some factors that may support or disfavor this inactive conformation, among them coordination of small molecules at a remote cavity at the CK2α/CK2β interaction region and binding of a CK2β dimer. The latter stabilizes the glycine-rich loop in the extended active conformation known from the majority of CK2α structures. Thus, the novel inactive conformation for the first time provides a structural basis for the stimulatory impact of CK2β on CK2α.     

Molecular simulation uncovers the conformational space of the λ Cro dimer in solution

The significant variation among solved structures of the λ Cro dimer suggests its flexibility. However, contacts in the crystal lattice could have stabilized a conformation which is unrepresentative of its dominant solution form. Here we report on the conformational space of the Cro dimer in solution using replica exchange molecular dynamics in explicit solvent. The simulated ensemble shows remarkable correlation with available x-ray structures. Network analysis and a free energy surface reveal the predominance of closed and semi-open dimers, with a modest barrier separating these two states. The fully open conformation lies higher in free energy, indicating that it requires stabilization by DNA or crystal contacts. Most NMR models are found to be unstable conformations in solution. Intersubunit salt bridging between Arg⁴ and Glu⁵³ during simulation stabilizes closed conformations. Because a semi-open state is among the low-energy conformations sampled in simulation, we propose that Cro-DNA binding may not entail a large conformational change relative to the dominant dimer forms in solution.     

Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation.     

Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.     

Probing flexibility and "induced-fit" phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations

Aldose reductase is a promising target for the treatment of diabetic complications, and as such, has become the focus of various drug design projects. As revealed by a survey of available crystal structures, the protein shows pronounced induced-fit effects upon ligand binding. Although helping to explain the enzyme's substrate promiscuity, phenomena of this kind are still responsible for significant complications in structure-based design efforts directed to aldose reductase. Accordingly, a deeper understanding of the principles governing conformational alterations in this enzyme would be of utmost practical importance. As a first step in addressing this issue, molecular dynamics (MD) simulations have been carried out. The ultrahigh resolution crystal structure of aldose reductase complexed with inhibitor IDD594 served as ideal starting point for a set of different simulations of nanosecond time scale: the native complexed state with bound inhibitor, the uncomplexed state (after removal of the inhibitor) at standard temperature, and the uncomplexed state at elevated temperature. The reference simulation of the complex exhibits extraordinary stability of the overall fold, whereas two distinct conformational substates are found for the binding-site region. In contrast, already at standard temperature pronounced changes are observed in the binding region during the simulation of the uncomplexed state. Leu300, for example, closes the access to the pocket opened by IDD594. On the other hand, conformations around the catalytic site are highly conserved, with the His110-Tyr48-NADP+ orientation being stabilized by a water molecule. Detailed analysis of the trajectories allows to reveal a set of distinct conformational substates that may prove useful as alternative structural templates in virtual screening for new aldose reductase inhibitors.     

A Correspondence Between Solution-State Dynamics of an Individual Protein and the Sequence and Conformational Diversity of its Family

Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using “Backrub” motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics.     

Structural flexibility in Trypanosoma brucei enolase revealed by X-ray crystallography and molecular dynamics

Enolase is a validated drug target in Trypanosoma brucei. To better characterize its properties and guide drug design efforts, we have determined six new crystal structures of the enzyme, in various ligation states and conformations, and have carried out complementary molecular dynamics simulations. The results show a striking structural diversity of loops near the catalytic site, for which variation can be interpreted as distinct modes of conformational variability that are explored during the molecular dynamics simulations. Our results show that sulfate may, unexpectedly, induce full closure of catalytic site loops whereas, conversely, binding of inhibitor phosphonoacetohydroxamate may leave open a tunnel from the catalytic site to protein surface offering possibilities for drug development. We also present the first complex of enolase with a novel inhibitor 2-fluoro-2-phosphonoacetohydroxamate. The molecular dynamics results further encourage efforts to design irreversible species-specific inhibitors: they reveal that a parasite enzyme-specific lysine may approach the catalytic site more closely than crystal structures suggest and also cast light on the issue of accessibility of parasite enzyme-specific cysteines to chemically modifying reagents. One of the new sulfate structures contains a novel metal-binding site IV within the catalytic site cleft.     

Protein dynamics and conformational selection in bidirectional signal transduction

Protein conformational dynamics simultaneously allow promiscuity and specificity in binding. The multiple conformations of the free EphA4 ligand-binding domain observed in two new EphA4 crystal structures provide a unique insight into the conformational dynamics of EphA4 and its signaling pathways. The heterogeneous ensemble and loop dynamics explain how the EphA4 receptor is able to bind multiple A- and B-ephrin ligands and small molecules via conformational selection, which helps to fine-tune cellular signal response in both receptor and ligand cells.     

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.     

Protein conformer selection by sequence-dependent packing contacts in crystals of 3-phosphoglycerate kinase

In several crystal structures of 3-phosphoglycerate kinase (PGK), the two domains occupy different relative positions. It is intriguing that the two extreme (open and closed) conformations have never been observed for the enzyme from the same species. Furthermore, in certain cases, these different crystalline conformations represent the enzyme-ligand complex of the same composition, such as the ternary complex containing either the substrate 3-phosphoglycerate (3-PG) and beta,gamma-imido-adenosine-5'-triphosphate (AMP-PNP), an analogue of the substrate MgATP, or 3-PG and the product MgADP. Thus, the protein conformation in the crystal is apparently determined by the origin of the isolated enzyme: PGK from pig muscle has only been crystallized in open conformation, whereas PGK from either Thermotoga maritima or Trypanosoma brucei has only been reported in closed conformations. A systematic analysis of the underlying sequence differences at the crucial hinge regions of the molecule and in the protein-protein contact surfaces in the crystal, in two independent pairs of open and closed states, have revealed that 1) sequential differences around the molecular hinges do not explain the appearance of fundamentally different conformations and 2) the species-specific intermolecular contacts between the nonconserved residues are responsible for stabilizing one conformation over the other in the crystalline state. A direct relationship between the steric position of the contacts in the three-dimensional structure and the conformational state of the protein has been demonstrated.     

Expression and localization of epitope-tagged protein kinase CK2

Protein kinase CK2, formerly known as casein kinase II, is a ubiquitous protein serine/threonine kinase. The enzyme exists in tetrameric complexes composed of two catalytic (CK2α and/or CK2α′) subunits and two subunits (CK2β) that appear to have a role in modulating the activity of the catalytic subunits. With the exception of their unrelated carboxy-terminal domains, the two isozymic forms of mammalian CK2 display extensive sequence identity. Furthermore, CK2α and CK2α′ exhibit remarkable conservation between species, suggesting that they may have unique functions. In the present study, the cDNAs encoding CK2α and CK2α′ were modified by addition of the hemagglutinin tag of the influenza virus at the amino terminus of the respective proteins. The epitope-tagged proteins were transfected into Cos-7 cells and the localization of the expressed proteins determined by indirect immunofluorescence using monoclonal antibodies specific for the epitope tag. The use of transfection favors the formation of homotetrameric complexes (i.e., α₂β₂, α′₂β₂) instead of heterotetrameric complexes (i.e., αα′β₂) that are present in many cells. Epitope-tagged CK2α and CK2α′ displayed kinase activity and the ability to form complexes with CK2β. The results of these studies also indicate definitively that CK2α and CK2α′ are both localized predominantly within the nucleus. Mutation of conserved lysine residues within the ATP binding domains of CK2α and CK2α′ resulted in loss of kinase activity. However, examination of these mutants indicates that kinase activity is not essential for formation of complexes between subunits of CK2 and is not required for nuclear localization of CK2. J. Cell. Biochem. 64: 525–537. © 1997 Wiley-Liss, Inc.