©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Quaternary Structure of Casein Kinase 2
CHARACTERIZATION OF MULTIPLE OLIGOMERIC STATES AND RELATION WITH ITS CATALYTIC ACTIVITY (*)

(Received for publication, October 12, 1994; and in revised form, January 19, 1995)

Emmanuelle Valero Salvatore De Bonis (1) Odile Filhol Richard H. Wade (1) Joerg Langowski (2) Edmond M. Chambaz Claude Cochet (§)

From the  (1)Commissariat á l'Energie Atomique, Biochimie des Régulations Cellulaires Endocrines, INSERM Unit 244, Département de Biologie Moléculaire et Structurale, CEN.G, F-38054 Grenoble Cedex 9, France, the Laboratoire de Microscopie Electronique Structurale, Institut de Biologie Structurale, F-38027 Grenoble, France, and (2)EMBL, C/O ILL, 156X, F-38042 Grenoble Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The structure-activity relationship of casein kinase 2 (CK2) was examined with regard to its previously reported property to self-aggregate in vitro. Sedimentation velocity and electron microscopy studies showed that the purified kinase exhibited four major, different oligomeric forms in aqueous solution. This self-polymerization was a reproducible and fully reversible process, highly dependent upon the ionic strength of the medium, suggesting that electrostatic interactions are mostly involved. At high salt concentrations (e.g. 0.5 M NaCl), CK2 appears as spherical moieties with a 18.7 ± 1.6 nm average diameter, roughly corresponding to the alpha(2)beta(2) protomer, as deduced by measurements of the Stokes radius and by light scattering studies. At lower ionic strength (e.g. 0.2 M NaCl), the protomers associate to form ring-like structures with a diameter (averaging 36.6 ± 2.1 nm) and Stokes radius indicating that they are most likely made of four circularly associated alpha(2)beta(2) protomers. At 0.1 M NaCl, two additional polymeric structures were visualized: thin filaments (16.4 ± 1.4 nm average), as long as 1 to 5 µm, and thick and shorter filaments (28.5 ± 1.6 nm average). Examination of the molecular organization of CK2 under different catalytic conditions revealed that the ring-like structure is the favored conformation adopted by the enzyme in the presence of saturating concentrations of substrates and cofactors. During catalysis, well-known cofactors like MgCl(2) or spermine are the main factors governing the stabilization of the active ring-like structure. On the other hand, inhibitory high salt concentrations promote the dissociation of the active ring-like structure into protomers. Such observations suggest a strong correlation between the ring-like conformation of the enzyme and optimal specific activity. Thus, CK2 may be considered as an associating-dissociating enzyme, and this remarkable property supports the hypothesis of a cooperative and allosteric regulation of the kinase in response to appropriate regulatory ligands possibly taking place in intact cells.


INTRODUCTION

Casein kinase 2 (CK2) (^1)is a ubiquitous serine-threonine protein kinase present in both soluble and nuclear extracts of eukaryotic cells(1, 2, 3, 4) . The enzyme is transiently stimulated in cells following treatment with various growth factors or serum stimulation(5, 6, 7, 8) , and it has been reported to accumulate in the nuclei when quiescent cells are stimulated to proliferate(9) .

CK2 exhibits several distinctive properties: it uses either ATP or GTP as the phosphate donor to phosphorylate serine or threonine residues in protein substrates. It is selectively inhibited by heparin(10, 11) , and it can be activated by naturally occurring polycationic compounds such as polyamines(12, 13) . Moreover, the kinase needs high concentrations of MgCl(2) (20 mM) for optimal catalytic activity(24) .

In most animal species, CK2 has been isolated as a heterotetramer composed of three dissimilar subunits, i.e. alpha and alpha` subunits of 35-44 kDa and beta subunits of 24-29 kDa which associate to form alpha(2)beta(2), alphaalpha`beta(2), or alpha`(2)beta(2) native structures(14, 15) . Although the respective roles of each subunit in the kinase activity and regulation remain poorly understood, it has been shown that the alpha and alpha` subunits that are the products of different genes bear the catalytic site of the enzyme(16, 17, 18, 19) . The beta subunit which is the target of kinase self-phosphorylation may be considered as regulatory component since it confers optimal activity to the holoenzyme (17) and may influence its substrate specificity(20) .

Biochemical studies with the purified enzyme have shown that the dissociation of the tetrameric structure of CK2 into its alpha and beta subunits requires rather drastic denaturing conditions(17) . Moreover, the tetrameric structure of CK2 has usually been examined under high salt conditions because it was observed early on that the enzyme aggregates at low salt concentrations(11, 12) . Two reports have shown that the aggregation of CK2 from Drosophila(21) or from bovine heart (22) is an ordered process resulting in the generation of filamentary structures. This in vitro self-polymerization property appears of interest in view of its possible role in the regulation of CK2 activity in the intact cell.

To examine the structure-activity relationship of CK2, we have recently expressed its subunits in the baculovirusdirected insect cell expression system. This approach provides a functional recombinant holoenzyme, as well as its isolated alpha and beta subunits(23) .

The present study reports that self-polymerization of recombinant Drosophila CK2 generates in vitro three different and well-defined polymeric forms. A characterization by electron microscopy and sucrose density gradient sedimentation analysis as well as by light scattering and gel filtration revealed that the generation of each form was a fully ordered and reversible process which was mostly dependent upon the ionic strength of the medium. In suboptimal conditions for catalysis, CK2 is heterogeneous and mainly composed of short thick filaments. By contrast, under optimal catalytic conditions, the enzyme exhibits a ring-like structure. These observations provide a correlation between the ring-like structure and high CK2 specific activity and strongly suggest that assembly as a specific quaternary structure is the form in which the kinase expresses its optimal activity.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP (3,000 Ci/mmol) was purchased from Amersham. The peptide substrate (RRREEETEEE) for CK2 was obtained from Neosystem Laboratory (Strasbourg, France). Spermine was obtained from Sigma.

Preparation of Recombinant Casein Kinase 2

Recombinant oligomeric CK2 from Drosophila melanogaster was overexpressed in Sf9 cells, purified to homogeneity as described previously(23) , and stored at -80 °C in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 2% glycerol, 0.1% Triton X-100, and 1 M NaCl.

Velocity Sedimentation

Linear 5-25% (w/v) sucrose gradients were prepared in TD buffer: 10 mM Tris-HCl, pH 7.5, 1 mM DTT, supplemented with various components, as indicated in the figure legends. For each experiment, samples were incubated at 4 °C in 0.1 ml of TD buffer for 2 h prior to loading. Gradients were centrifuged at 4 °C for 3.5 h at 200,000 times g and fractionated by pipetting 200-µl aliquots. For each ionic condition, identical gradients were run using aldolase (8 S), catalase (11.2 S), and alpha(2)-macroglobulin (19 S) as sedimentation markers which were localized by the Bradford protein assay.

Polyacrylamide Gel Electrophoresis

Proteins were solubilized and boiled in Laemmli's sample buffer and analyzed by 12% SDS-PAGE. Proteins were revealed by silver staining.

Casein Kinase 2 Assay

CK2 activity was assayed using either casein or the synthetic peptide substrate (RRREEETEEE), as described previously(24, 23) .

Electron Microscopy

Recombinant CK2 (15 µg) was incubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, and different NaCl concentrations. Samples were then sprayed onto a fresh mica surface using a vaporizer and rotary-shadowed under vacuum with tantalum/tungsten at an incidence angle of 10-15° to the horizontal. Replicas were coated with a 20-nm-thick supporting film of carbon. Electron micrographs were obtained under standard bright field imaging conditions using a Zeiss 10C microscope operating at 100 keV. Size determination was performed on at least 50 independent objects.

Gel Filtration

A Sephadex S-300 gel filtration column was calibrated by using porcine thyroglobulin (M(r) = 669,000, Stokes radius = 85.0 Å), ferritin (M(r) = 440,000, Stokes radius = 61.0 Å), aldolase (M(r)= 158,000, Stokes radius = 48.1 Å), bovine serum albumin (M(r) = 67,000, Stokes radius = 35.5 Å) as standards. The void volume (V(0)) was estimated by elution of blue dextran 2000. K values for standards were calculated from their elution volumes (V(e)) by the equation: K = (V(e) - V(0))/(V(t) - V(0)) where V(t) = the total column volume. CK2 (30 µg), preincubated for 2 h at 4 °C in 0.4 M or 0.2 M NaCl, was applied to the column equilibrated in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 2% glycerol containing either 0.4 or 0.2 M NaCl.

Dynamic Light Scattering (DLS)

Recombinant CK2 (107 µg) was preincubated for 2 h at 4 °C in 500 µl of 10 mM Tris-HCl, pH 7.5, containing either 0.4 M NaCl or 0.2 M NaCl. DLS measurements were performed at 14.5 °C using a system consisting of a BI 2030 AT 4N-bit autocorrelator with real-time channels and multiple sample time option (Brookhaven Instruments), a stepping motor goniometer (Amtec, Nice, France), and a Spectra-Physics 2025 argon ion laser. The incident laser power was 500 milliwatts at 488 nm. DLS autocorrelation functions were collected through an angular range of 40° to 90°. The data were analyzed by either a maximum entropy procedure which directly yields the particle size distribution (25) or a non linear least-squares fit to a squared sum of exponentials. The diffusion coefficients D(t) were obtained through D(t) = (K^2), K being the scattering vector and the relaxation time. D(t) was corrected to 20 °C by D(t) = D(t)bullet(293/T)bullet(/), T being the absolute temperature, and the viscosity of water at T.

Determination of CK2 Specific Activity

The kinase assay contains a reaction volume of 100 µl containing 10 mM Tris-HCl, pH 7.5, 1.2 mg/ml casein, 10 µM ATP, 1 mM MgCl(2), variable [-P]ATP concentrations to obtain appropriate -P incorporation, and 0.2 M NaCl. Various amounts of recombinant CK2 and recombinant alpha subunit were preincubated in the kinase assay medium at room temperature for 2 min, and the reactions were started by addition of magnesium. For each condition, a time course study was performed to check the linearity of the reaction. Materials used in these experiments were siliconized, then saturated with a solution containing 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 2% glycerol, and 1 mg/ml bovine serum albumin prior to use.


RESULTS

Molecular Forms of CK2 in Aqueous Solution under Low and High Salt Concentrations

Homogeneous recombinant Drosophila CK2 was analyzed by velocity sedimentation through a sucrose gradient containing either 0.1 M or 1 M NaCl (Fig. 1). Aliquots of the collected fractions were analyzed for CK2 activity (Fig. 1A) and by SDS-PAGE (Fig. 1, B and C). In the presence of 1 M NaCl, the enzyme sedimented as a single peak with a 6 S sedimentation coefficient which is the value expected for the alpha(2)beta(2) holoenzyme, further referred to as the CK2 protomer. By contrast, at 0.1 M NaCl, the enzyme exhibited a polydisperse distribution. As illustrated in Fig. 1A, a continuum of species having sedimentation coefficients ranging from 13.6 S to 47 S could be visualized. Examination of fractions showing CK2 activity by SDS-PAGE shows that both alpha and beta subunits are present with a similar stoichiometry all along the sucrose gradients (Fig. 1, B and C). Therefore, the sedimentation behavior of the recombinant CK2 is strongly dependent upon the ionic strength of the medium. In addition, the heavy forms of CK2 occurring at low ionic strength contain both the alpha and the beta subunits of the kinase, with a stoichiometry identical with that observed for the protomer (alpha(2)beta(2)). This suggests that these heavy forms are generated by protomer association.


Figure 1: Sedimentation profiles of recombinant CK2 on sucrose density gradients. Purified recombinant Drosophila CK2 (15 µg) was incubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, containing either 1 M or 0.1 M NaCl and sedimented through 5-25% sucrose gradient under the same salt conditions. Gradients were fractionated. A, CK2 activity in a 1 M NaCl gradient (circle) and in a 0.1 M NaCl gradient (bullet). Positions of alpha(2)-macroglobulin (19 S), catalase (11.2 S), and aldolase (8 S) which were sedimented in parallel gradients of identical compositions (except DTT for alpha(2)-macroglobulin) are indicated. B and C, protein contained in the fractions were precipitated by 10% trichloroacetic acid, analyzed by 12% SDS-PAGE, and revealed by silver staining. B, 1 M NaCl; C, 0.1 M NaCl.



The recombinant CK2 was then analyzed by electron microscopy using replicas produced by low-angle rotary shadowing with tantalum/tungsten. Electron micrographs of CK2 incubated in buffer containing either 1 M or 0.1 M NaCl are shown in Fig. 2, A and B, respectively. As expected from the sedimentation data, a homogeneous population made of roughly circular structures with an average diameter of 18.7 ± 1.6 nm was observed at high salt concentrations, consistent with the CK2 protomer (Fig. 2A). By contrast, four different structural organizations of the protein could be visualized in 0.1 M NaCl (Fig. 2B). 1) Some rare 18.7 ± 1.6 nm diameter particles (Fig. 2B, panel 1). 2) Roughly round structures with an average diameter of 36.6 ± 2.1 nm and usually showing in their center a pronounced decrease of the metal density (Fig. 2B, panel 1). This structure will be referred to as the ``ring-like'' structure. 3) Thin filaments with a uniform average width of 16.4 ± 1.4 nm and variable lengths (up to 5 µm) (Fig. 2B, panel 2). 4) Thick filaments of about 28.5 ± 1.6 nm width (Fig. 2B, panel 3). These structures are all derived from different associations of the alpha(2)beta(2) protomer, which self-assembles when the ionic strength of the medium is lowered.


Figure 2: Electron microscopy of recombinant CK2 in 0.1 M and 1 M NaCl. Purified recombinant Drosophila CK2 (15 µg) was incubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, containing either 1 M or 0.1 M NaCl. Samples were then prepared for electron microscopy as described under ``Experimental Procedures.'' Bars = 100 nm. A, recombinant CK2 in 1 M NaCl. B, recombinant CK2 in 0.1 M NaCl. C, gallery of selected images of different molecular forms of CK2 in 0.1 M NaCl.



The behavior of the isolated alpha subunit was also examined by electron microscopy under high and low salt conditions. It always appeared on electron micrographs as an homogeneous population of spherical structures (data not shown) indicating that the beta subunit is required in the CK2 self-polymerization process.

Molecular Forms of CK2 as a Function of Ionic Strength

To examine in more detail how the self-polymerization of the kinase takes place as a function of ionic strength, velocity sedimentation and electron microscopy analysis of CK2 were carried out in parallel, under different salt concentrations. In 0.4 M NaCl, the enzyme sedimented as a single peak with a sedimentation coefficient of 6 S (Fig. 3B, panel e). Electron microscopy imaging showed that under these conditions the enzyme appears as a homogeneous population of condensed spherical structures, with a size fitting well with an alpha(2)beta(2) protomeric form of the protein (Fig. 3A, panel a). Decrease of the NaCl concentration to 0.3 M resulted in the occurrence of a new peak of CK2 activity with a sedimentation coefficient of 13.6 S (Fig. 3B, panel f). In addition to the presence of protomers, the corresponding electron micrographs disclosed a population of ring-like structures (Fig. 3A, panel b). The occurrence of ring-like structures may reflect the beginning of a transition suggesting that 0.3 M NaCl represents a threshold salt concentration below which ring-like structure formation is favored. Indeed, in 0.2 M NaCl, the enzyme sedimented as a homogeneous peak with a 13.6 S sedimentation coefficient (Fig. 3B, panel g). Electron micrographs showed that the ring-like structure represented the most prominent form of the enzyme at this salt concentration (Fig. 3A, panel c). Lowering the NaCl concentration to 0.1 M resulted in an enzyme sedimentation pattern further in the sucrose gradient reflecting a polydisperse distribution from 15 to 44 S and a pelleted form (Fig. 3B, panel h). Electron microscopy examination of these two populations revealed that the corresponding molecular forms of CK2 were characterized by the occurrence of thick and thin filaments (Fig. 3A, panel d).


Figure 3: Electron microscopy and velocity sedimentation of CK2 as a function of ionic conditions. Purified recombinant Drosophila CK2 (15 µg) was preincubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, and 0.4 M (a), 0.3 M (b), 0.2 M (c), 0.1 M (d) NaCl, and prepared for electron microscopy, as described under ``Experimental Procedures.'' In parallel, purified recombinant Drosophila CK2 (1.5 µg) was preincubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, and 0.4 M (e), 0.3 M (f), 0.2 M (g), 0.1 M (h) NaCl, and sedimented through a 5-25% sucrose gradient under the same salt conditions. Gradients were fractionated and kinase activity was measured in each fraction. Bars = 100 nm.



To cross-check these results, electron microscopy analysis was carried out directly on isolated fractions recovered following gradient centrifugation and representing the different self-assembled CK2 populations. For these experiments, a glycerol gradient equivalent to the 5-25% sucrose gradient was used since sucrose was not compatible with the shadowing procedure used to prepare the specimens for microscopy. In total agreement with the results illustrated in Fig. 3, we observed that the enzyme sedimenting at 13.6 S corresponded mostly to the ring-like structure (Fig. 4). Thick filaments were the most abundant in the large intermediary peak (15 to 44 S), and long thin filaments were mainly detected in the fractions sedimenting at the bottom of the tube. Unorganized aggregates were also observed at the bottom of the tube.


Figure 4: Electron microscopic analysis of the different molecular forms of CK2 separated on a glycerol gradient. Recombinant Drosophila CK2 (75 µg) was preincubated 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, and 0.1 M NaCl, then sedimented under the same salt condition on a 8-41% glycerol gradient. The gradient was fractionated and the kinase activity was measured in each fraction. Then selected fractions were used for electron microscopy as described under ``Experimental Procedures.''



We found that all the polymeric structures of the enzyme could be dissociated with NaCl concentrations higher than 0.4 M NaCl (not shown). Thus, the self-polymerization of CK2 is readily reversible in the appropriate ionic strength environment.

Relationship between the Different Molecular Forms of CK2

We further characterized the structure of the protomer and the ring-like structure by gel filtration and dynamic light scattering (DLS) analysis under two ionic strength conditions (0.4 M and 0.2 M NaCl, respectively). The major physical characteristics obtained for the different CK2 molecular forms are given in Table 1. Apparent molecular masses of 669 kDa and 158 kDa were determined by gel filtration analysis for the ring-like structure and the protomer, respectively. From the Stokes radius calculated from the DLS data, and the sedimentation coefficients from the sucrose gradient centrifugation, it was possible to determine molecular sizes of 524 kDa and 129 kDa for the ring-like and the protomeric structures, respectively. With both techniques, the ratio between the apparent molecular masses of these two forms of the enzyme was very close to 4. A similar observation could be made for the Stokes radius determined by the two independent techniques (Table 1). Higher magnification electron micrographs showing CK2 ring-like structures is illustrated in Fig. 5(panel Aa). Together with dimensional criteria (i.e. an average diameter of 36.6 ± 2.1 nm for these particles as compared to 18.7 ± 1.6 nm for the protomer), these data suggest that the ring-like structural organization of CK2 is formed by the circular association of alpha(2)beta(2) protomers. The data are compatible with the idea that the ring-like structure represents a tetramer of the kinase protomers, i.e. (alpha(2)beta(2))(4) interacting side by side on a plane.




Figure 5: Structural relationship between the different molecular forms of CK2. Recombinant Drosophila CK2 (15 µg) was preincubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, and 0.1 M NaCl and prepared for electron microscopy, as described under ``Experimental Procedures.'' A, gallery of selected images of CK2 polymers. B, drawings of the structures shown in A to assist interpretation of the photomicrographs. Bars = 100 nm.



The morphology of the thick filaments suggests that they could result from a linear association of ring-like moieties (Fig. 5, panels Ab and Ac). If so, the ring-like structures must compact a little during association since the average width of the thick filaments (28.5 ± 1.6 nm) was slightly smaller than the diameter of the isolated ring-like structures (36.6 ± 2.1 nm). With regard to the thin filament organization, it may result from a linear association of protomers. In some cases, thin filaments appeared as if they were generated from thick filaments either by splitting (see Fig. 5, panel Ad) or by internal molecular rearrangement (see Fig. 2C, panel 4). On the other hand, thick filaments may result from the side by side association of thin filaments. We have no experimental evidence at the moment to clarify further the relationship between these different filamentary arrangements.

Molecular Forms of CK2 and Catalytic Activity

The four different types of CK2 structures isolated following ultracentrifugation on sucrose gradients were detected by their protein kinase activity (Fig. 3). We found that all the CK2 molecular forms assayed in the different sucrose gradient-collected fractions exhibited similar specific protein kinase activities. However, it should be stressed that the four different conformations of CK2 were isolated under a resting state (i.e. in the absence of any substrate), whereas the protein kinase activity was then determined in an assay medium under optimal catalytic conditions. This prompted us to examine the molecular forms of the kinase when placed under catalytic conditions.

Molecular Organization of CK2 under Different Catalytic Conditions

Oligomeric preparations of CK2 were analyzed on sucrose gradients containing 0.1 M NaCl in the absence or in the presence of 10 µM ATP, 150 µM peptide substrate and with either 1 mM or 20 mM MgCl(2) (Fig. 6). As previously shown in Fig. 1, in the absence of substrates, the enzyme sedimented as a mixture of three major different polymerized structures: the ring-like structure, the thick, and the thin filaments. In the presence of substrates and 1 mM MgCl(2) (i.e. in nonoptimal catalytic conditions), the CK2 polymerization pattern was changed. The number of thick filaments decreased dramatically whereas there were more ring-like structures. However, the CK2 population remained heterogeneous under these catalytic conditions. In contrast, in the presence of substrates and 20 mM MgCl(2) (i.e. under optimal catalytic conditions), the enzyme sedimented strikingly as a sharp symmetrical peak with a sedimentation behavior (13.6 S) corresponding to that of the ring-like structure. Control experiments disclosed that during centrifugation, the peptide substrate present in the sucrose gradient was readily phosphorylated (not shown). These results strongly suggest that the ring-like structure of the kinase corresponds to the favored molecular organization of the protomers when the enzyme is optimally active during the catalytic reaction.


Figure 6: Velocity sedimentation of CK2 under different catalytic conditions. Recombinant Drosophila CK2 (1.5 µg) was preincubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 M NaCl, then sedimented on a 5%:25% sucrose gradient in the same salt condition () or containing 150 µM peptide substrate, 10 µM ATP, 30 mM NaCl, and 1 mM (box) or 20 mM (bullet) MgCl(2). Gradients were fractionated and CK2 activity was measured in each fraction with casein as described under ``Experimental Procedures.'' Positions of protomer, ring structures, and polymers are indicated.



Effects of Spermine and MgCl(2)on the Molecular Organization of CK2

Naturally occurring polyamines such as spermine are potent activators of CK2(12, 13) . We have previously demonstrated that this activation occurs at least partly through a direct interaction between spermine and the beta subunit of CK2(26) . We therefore analyzed the sedimentation behavior of CK2 in the presence of 1 mM spermine or 20 mM MgCl(2) (Fig. 7). As expected, CK2 sedimented as a mixture of various interconverting oligomers. By contrast, in the presence of either 1 mM spermine or 20 mM MgCl(2), the enzyme sedimented as a sharp peak (13.6 S) corresponding to the ring-like structure. This experiment shows that two potent activators of CK2 activity are able to dissociate the high molecular weight oligomers and to stabilize the enzyme in its ring-like structure. It should be stressed that this effect requires neither the presence of the peptide substrate nor ATP. In addition, this experiment clearly establishes the occurrence of the ring-like structure under conditions where CK2 has been shown to bind the polyamine and to be optimally activated by the polycation(12, 13) . Binding of spermine to the beta subunit of CK2 thus appears sufficient to induce a change in the quaternary structure of the enzyme corresponding to a catalytically active conformation.


Figure 7: Velocity sedimentation of CK2 in the presence of spermine or MgCl(2). Recombinant Drosophila CK2 (1.5 µg) was preincubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 M NaCl in the absence (bullet) or the presence of 1 mM spermine (box) or 20 mM MgCl(2) () and sedimented on 5%:25% sucrose gradients under the same conditions. Gradients were fractionated, and CK2 activity was measured in each fraction with casein as described under ``Experimental Procedures.'' Positions of protomer and ring structures are indicated.



Change in CK2 Specific Activity upon Enzyme Dilution

The concentration of a dissociating enzyme influences the equilibrium between the monomer and the oligomer populations, and it is expected that dilution should have an effect on the specific activity of the kinase by changing the ratio of active and inactive species. Fig. 8illustrates a dilution experiment in which the enzyme concentration was varied over 3 orders of magnitude under ionic conditions (0.2 M NaCl) which favored the ring-like structure (Fig. 3). Maximal specific activity (135 nmol of P/min/mg of CK2) was obtained for a CK2 concentration (i.e. 5 µg/ml; 38 nM) at which the enzyme exhibits a ring-like structure, as determined by sucrose gradient centrifugation (Fig. 3). Decreasing the enzyme concentration to 0.02 µg/ml (0.15 nM) induced a striking drop (9-fold) in its specific activity. Similarly, increasing the enzyme concentration from 38 nM to 380 nM led to a 2-fold decrease in its specific activity. Linearity versus time of the protein kinase reaction could not be obtained at higher enzyme concentrations. A similar experiment was performed with the isolated alpha subunit. The catalytic subunit does not form oligomeric structures (data not shown), and no change in its specific activity could be detected upon dilution (Fig. 8).


Figure 8: Changes in CK2 specific activity upon dilution of the enzyme. Specific activity of recombinant Drosophila CK2 (bullet) or the alpha (circle) subunit was determined as described under ``Experimental Procedures'' at different concentrations. For each condition, a time course study was performed to check the linearity of the reaction.



From these experiments, it is suggested that changing the enzyme concentration leads to changes in the equilibrium between the different oligomeric forms of the kinase in correlation with striking changes in its specific activity.


DISCUSSION

Using recombinant CK2, the present study confirms the well known property of the purified enzyme to self-aggregate in subphysiological ionic strength conditions(11, 12) . In this respect, the behavior of the baculovirus-directed recombinant Drosophila CK2 used in the present work appears similar to that of its native counterpart from insect (21) or bovine (22) sources. Our observations are also consistent with previous reports(21, 22) , showing that the aggregation process is an ordered and fully reversible phenomenon leading to filamentary polymeric forms of the kinase. Electron microscopic examination combined with velocity sedimentation, DLS, and gel filtration analysis, disclosed that there are four major forms. As has been reported many times, the purified enzyme remains stable in a high salt environment (e.g. 0.5 M NaCl) as a protomer made of two tightly bound alpha and beta subunits with an alpha(2)beta(2) stoichiometry(14, 15) . When the ionic strength is lowered to 0.2 M NaCl, this protomeric kinase can associate to form ring-like structures with a shape, size, and Stokes radius compatible with a circular association of four protomers (alpha(2)beta(2))(4). At 0.1 M NaCl, CK2 is a mixture of: (i) alpha(2)beta(2) protomers, (ii) ring-like structures, (iii) long thin filaments, and (iv) thick filaments. Remarkably, raising the ionic strength of the medium (e.g. to 0.4 M NaCl) resulted in the disappearance of these polymeric organizations and the total recovery of the enzyme in its protomeric form. As mentioned previously by others (21, 22) , this obviously suggests that electrostatic interactions are mostly concerned in the self-organization of CK2 into polymeric structures. On the other hand, the intramolecular association between the alpha and the beta subunits in the alpha(2)beta(2) protomer is different in nature and requires drastic conditions to be ruptured(17) .

The isolated catalytic (alpha) subunit of the kinase does not self-polymerize (data not shown). This observation strongly suggests that the beta subunit in the protomer plays a crucial role in the initiation of the self-association process and the stabilization of the various characterized polymeric forms of the enzyme. This would be in line with the suggestion by Glover (21) that the beta subunit, which is self-phosphorylatable in the native enzyme, may trigger inter-protomer association due to an enzyme-substrate interaction.

While the present study indicates that the CK2 ring-like structures are probably an association of four alpha(2)beta(2) protomers and that thin filaments are likely to be made of linearly associated protomers, it is not yet possible to clearly understand how the filamentary structures are inter-related. According to their average width, the thick filaments could be made of a linear association of ring-like structures or result from side by side association of two thin filaments. On the other hand, thin filaments might result from splitting of thick ones. However, we have regularly observed that thin filaments are on the average much longer than the thicker ones. Careful kinetic studies taking into account the ionic strength of the medium as well as the protein concentration and temperature remain to be carried out to clarify the relationship between the filamentary structures. Putative models representing the different molecular forms of CK2 which would be compatible with our observation are proposed in Table 2.



With regard to this remarkable self-polymerization property, CK2 may be considered an associating-dissociating enzyme. An important feature of dissociating enzymes with regard to this regulation is that the association-dissociation process can be modulated in the presence of their substrates or in response to appropriate regulatory ligands(27) . Examination of the molecular organization of CK2 under different catalytic conditions revealed that the ring-like structure was the only conformation recovered in sucrose gradients containing saturating concentrations of substrates and cofactors, i.e. under optimal catalytic conditions. The fact that the (alpha(2)beta(2))(4) moiety is indeed an active state of the kinase is supported by the fact that the peptide substrate present in the enzyme environment was extensively phosphorylated during the sucrose gradient sedimentation. Our data clearly establish that the active ring-like structure of CK2 is stabilized by known activating agents such as MgCl(2) or polyamines. For instance, spermine at submillimolar concentration binds to CK2(26) , induces the formation of the catalytically active ring-like structure, and prevents filament formation. The fact that MgCl(2) at high concentration had the same effect is in agreement with data showing that spermine can substitute for high MgCl(2) concentrations in supporting optimal kinase activity(28) . From these data it is expected that the beta subunit, which is required for the polyamine interaction(26) , may play a crucial role in governing the molecular organization and the activity of the enzyme.

The present study provides several clues suggesting a strong correlation between the ring conformation of the enzyme and a high specific activity. 1) Two well-known activators such as polyamines and MgCl(2) promote the dissociation of CK2 thick filaments and stabilize the ring-like structure. 2) Striking changes in CK2 activity as a function of the ionic conditions have often been reported(28, 29) . Maximal catalytic activity was detected around 0.2 M NaCl as the enzyme adopts mostly a ring-like structure. Increasing the salt concentration to 0.3 M NaCl strongly inhibited the kinase activity, and this inhibition was correlated with the dissociation of the ring-like structure into protomers. These observations suggest that CK2 filaments and protomers are relatively inactive molecular forms of the kinase, whereas the ring-like structure represents the most active form of the enzyme.

Early studies by Glover (21) have shown that polymerization of CK2 depends on the enzyme concentration. Our data are in agreement with his observations. Our dilution experiments showed that in 0.2 M NaCl, CK2 at 38 nM had a maximal specific activity (i.e. 135 nmol of P/min/mg of CK2) (Fig. 5). Analysis of the enzyme by sucrose gradient sedimentation showed that in this range of concentration the protein mostly adopted a ring-like structure. Decreasing the enzyme concentration promoted the dissociation of this structure into protomers, and this was concomitant with a striking drop of the specific activity of the kinase. Increasing the enzyme concentration favored the association of the ring-like structures into filaments and could explain the observed decrease of the kinase specific activity.

One may speculate on the functional advantages of the ring-like structure conformation of the kinase with regard to its activity. Reversible interactions between subunits in the ring-like structure may permit a large flexibility in functional regulations such as the following. 1) The ring-like structure in quadrupling the subunits may enhance the substrate binding surface for maximal catalytic activity. 2) Because the advantage of a polymeric state has long been understood as the basis for cooperativity and allosteric regulation, the ring-like structure of CK2 may generate an enzyme species more responsive to regulation by allosteric effectors.

The data presented in this work show that the catalytic function of CK2 is strongly influenced by its quaternary structure. Although these data have been obtained in vitro, they raise new possibilities with regard to the regulation of the kinase activity in the living cell. At present, it is difficult to determine the molecular organization of the kinase in the intact cell. It has been reported that CK2 might form multimolecular complexes with specific intracellular components such as HSP90(30) , cytoskeleton, double-stranded DNA(31) , or the nuclear protein p53(32) . It is of interest to mention that the beta subunit of the kinase which appears to be required for the self-polymerization of the enzyme is also required for the CK2-p53 interaction(32) . Altogether these observations suggest that in the intact cell, CK2 may be present (or targeted) into different subcellular localizations through interaction with cellular components, resulting in the control of its oligomeric organization, and consequently in the targeting of its activity from one substrate to another in response to intracellular specific signals.

As a new approach in the study of CK2 regulation, we propose that the polymerization behavior of the enzyme could be used to supplement the usual enzyme activity assay as a means for identifying and characterizing possible physiological regulators of this ubiquitous and pleiotropic protein kinase.


FOOTNOTES

*
This work was supported by the INSERM (Unité 244), the Commissariat á l'Energie Atomique (DSV/DBMS/BRCE), the Association pour la Recherche contre le Cancer, the Fédération Nationale des Centres de Lutte contre le Cancer, the Fondation pour la Recherche Médicale, and the Ligue Nationale Française contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 33-76-88-42-04; Fax: 33-76-88-50-58.

(^1)
The abbreviations used are: CK2, casein kinase 2; DTT, dithiothreitol; DLS, dynamic light scattering; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Christian Closse in electron microscopy. We are indebted to Sonia Lidy for editing the manuscript.


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