©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Identification of a Polyamine Binding Domain on the Regulatory Subunit of the Protein Kinase Casein Kinase 2 by Photoaffinity Labeling (*)

(Received for publication, March 23, 1995)

Didier Leroy (1), Nathalie Schmid (2), Jean-Paul Behr (2), Odile Filhol (1), Serge Pares (3), Jérome Garin , Jean-Jacques Bourgarit (1), Edmond M. Chambaz (1), Claude Cochet (1)(§)

From the  (1)Commissariat l'Energie Atomique, Biochimie des Régulations Cellulaires Endocrines, INSERM Unit 244, Departement de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires/Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France, (2)Chimie génétique, CNRS Unit 1386, Faculté de Pharmacie, F-67401, Illkirch Cedex, France, Chimie des protéines DBMS, Commissariat l'Energie Atomique, CEN/G, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France, and the (3)Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale 41 rue des Martyrs, F-38027 Grenoble Cedex 1, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphorylation of many protein substrates by the protein kinase casein kinase 2 (CK2) is stimulated severalfold in the presence of polyamines such as spermine. Previous experiments have shown that CK2 is a polyamine binding protein and that the regulatory subunit is required for this binding activity.

To delineate the spermine binding site of CK2, we have applied a photoaffinity labeling method using a tritiated photoactivable analog of spermine, [H]sperminediazonium.

The photoaffinity labeled subunit was cleaved with cyanogen bromide, and two labeled peptides were separated by high performance liquid chromatography. The major one was the peptide TEQAAEM and the minor one was a 22-amino acid peptide comprising residues Ile to Met. Thr and His were identified as the labeled amino acids of the Thr-Met and Ile-Met peptides, respectively.

In the same manner, we succeeded in determining the residue Leu as an subunit residue covalently bound to the probe.

The photoaffinity labeling method described here enabled the first elucidation, by direct microsequencing, of a polyamine binding site on CK2 for which we propose a provisional structural model.

These observations suggest a possible mechanism for CK2 activation by polyamines at the molecular level.


INTRODUCTION

Protein kinase casein kinase 2 (CK2)()is a ubiquitous serine-threonine protein kinase found in both the cytoplasm and the nucleus of eucaryotic cells(1, 2) . CK2 from most sources has been purified as a heterotetramer composed of three dissimilar subunits, i.e. and ` subunits of 35-44 kDa and a subunit of 24-29 kDa, which associate to form native , `, ` structures(3, 4) . It has been shown that the and ` subunits are the products of different genes and bear the catalytic site of the enzyme(5) . The subunit, which is the target of the kinase self phosphorylation, is a regulatory subunit that is required for optimal CK2 activity of the subunit (6, 7, 8) and may influence the substrate specificity of the kinase(9) . A number of protein substrates are phosphorylated by CK2 including several nuclear oncoproteins such as Myc(10) , Myb(2, 11) , Max(12) , Fos(13) , the adenovirus E1A protein(13) , the human papillomavirus E7 protein(14, 15) , the SV40 large T antigen(2, 16) , and the nuclear p53 protein (17) . We have shown that CK2 and p53 associate in a tight molecular complex that involves the subunit of the kinase(18) . Considerable interest in CK2 has arisen recently because its activity was found transiently stimulated following treatment with several growth factors (19, 20, 21) . Similar activation occurred following serum stimulation(22) , and the enzyme was reported to accumulate in nuclei of actively growing cells(23) . However, the functional significance and the regulation of this protein kinase in the intact cell are still poorly understood. As yet, no intracellular messenger involved in CK2 regulation has been recognized.

CK2 is markedly activated in vitro by polycationic structures including polyamines, spermine being the most potent(24) . A special interest is raised by polyamines since they are ubiquitous cellular components that are required for normal cell growth (25) and that have been shown to modulate a number of biological activities involved in the process of cellular signaling. The thyrotropin-stimulated adenylate cyclase from beef thyroid plasma membrane (26) and a phosphatidylinositol kinase located in the plasma membranes from A431 cells (27) are stimulated by polyamines. It has also been reported that the catalytic activity of an erythrocyte multifunctional proteinase (28) and protein phosphatases 1 and 2A (29) are stimulated by polyamines. Polyamines have been shown likewise to activate DNA polymerases, DNA gyrase, DNA methylases, and the eucaryotic type I (30) and type II topoisomerases(31) . We have previously suggested that intracellular polyamines may express part of their biological action through an effect upon CK2. Although an effect through the protein substrate conformation may contribute to the activation of the phosphotransferase activity of the kinase by polyamines(32) , we have demonstrated a direct interaction between these polycations and the enzyme(33) . CK2 binds spermine with high affinity, and the subunit plays an essential role in this interaction. Moreover, the interaction of the protein p53 with CK2 is driven by the subunit, and this interaction is strongly inhibited in the presence of spermine(18) , suggesting that p53 and spermine share an overlapping binding domain on the CK2 subunit.

Although hydrogen bonds as well as ionic and Van Der Waals' forces have been reported as the driving forces by which polyamines interact with nucleic acids, proteins, and phospholipids(34, 35, 36, 37, 38, 39) , nothing is known concerning the molecular mechanism by which polyamines enhance CK2 activity. As a prerequisite, the identification of the kinase domain(s) involved and the nature of this interaction should help understanding of the regulation of this protein kinase. The present study was undertaken with the aim of identifying accurately the spermine binding site at the primary structure level of CK2. Photoaffinity labeling with aryldiazonium salts has previously been used to label several proteins (for review, see (40) ) as well as nucleic acid binding sites(41, 42) . The present study made use of such a tritiated photoactivable analog of spermine to map the polyamine binding domain of the recombinant Drosophila CK2. Subsequent analysis revealed a preferential labeling of the subunit. Controlled proteolysis and microsequencing analysis of the isolated labeled peptide resulted in the identification of threonine 72 as the main residue covalently bound to the photoaffinity probe. As a working hypothesis, we propose a structural model for the major polyamine binding site of CK2 where both glutamic acid residues 73 and 77 could be crucial determinants for the interaction.


EXPERIMENTAL PROCEDURES

Synthesis of the Photoaffinity Probe

The nonradioactive sperminediazonium salt was prepared as described previously(43) . The photoaffinity labeling probe [H]sperminediazonium (Fig. SI) was synthesized as follows. p-Amino-m-diiodoaniline was obtained by reduction of m-diiodo-p-nitroaniline with NaBHS (Lancette's reagent) in Tetrahydro Furan under reflux for 16 h. The residue was chromatographed on a Silica gel column 4% MeOH-containing CHCL. R (CHCL, 5% MeOH, 1% AcOH) = 0.8, yield 63%. H NMR (CDCl) d (ppm): 3.3 (bs, 2H, NH); 4.1 (bs, 2H, NH); 7.1 (s, 2H, Har). Tetra-N-t-butoxycarbonylspermine carboxylic acid (43) was activated with N-hydroxysuccinimide (1.1 eq) in the presence of dicyclohexilcarbodiimide (1 eq) in CHCl. After filtration, the resulting activated ester solution was reacted with p-amino-m-diiodoaniline (2.2 eq) under reflux for 12 h. Silica gel chromatography (1% MeOH in CHCl) yielded 78% of tetra-N-t-butoxycarbonyl-o,o`-diiodoanilinospermine. R (CHCl, 5% MeOH, 1% AcOH) = 0.55. MS (fast atomic bombardment): 988 (M+). Analysis (CHINO = 988.7).

Calculated: C 44.94% H 6.32% N 8.50%

Found: C 45.16% H 6.52% N 8.22%


Figure SI: Scheme I.



H NMR of a trifluoroacetic acid-deprotected sample (DO) d (ppm): 1.7-2.2 (m, 8H, 4 CH bN); 3-3.3 (m, 1OH, 5 CHN); 4.05 (t, J = 6 Hz, 1H, CHN); 7.9 (s, 2H, Har). [H]anilinospermine was synthesized at the Commissariat l'Energie Atomique (Saclay, France) by catalytic dehalogenation of the o,o`-diiodo derivative in the presence of tritium gas.()Small aliquots of [H]sperminediazonium were obtained after diazotation with isopentylnitrite and removal of the t-butoxycarbonyl protecting groups in CFCOH, essentially as described previously(43) . The concentration (323 = 2.25 10 dmmolcm) and specific radioactivity of the probe were determined to be 350 µM and 15 Ci/mmol, respectively. N-Propyl alcohol required for HPLC was purchased from Carlo Erba. Trifluoroacetic acid and trifluoroethanol were respectively from Carlo Erba and Aldrich.

Expression and Purification of Drosophila CK2

Insect cells (Sf9 cells, 10/ml) were coinfected with EV 55 Dm and EV 55 Dm viruses at a multiplicity of infection of 5-10 as described previously by Filhol et al.(7) . The cell lysate was sonicated for 3 min and centrifuged at 200,000 g for 30 min. The soluble extract was diluted to give a final concentration of 0.2 M NaCl applied onto a phosphocellulose column previously equilibrated with 10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1% glycerol, 0.1% Triton X-100 (buffer A) and recycled 3 times through the column. A 0.2-1.5 M linear NaCl gradient in buffer A was applied. Aliquots of collected fractions were used for CK2 activity. The fractions containing CK2 activity were pooled and concentrated in a filtration cell (Amicon) through a XM 50 membrane to give a final volume of 90 ml. The concentrated solution was diluted 5 times and loaded onto a heparin-Sepharose column previously equilibrated in 10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1% glycerol (buffer B). A 0.4-1.2 M NaCl linear gradient in buffer B was developed. The protein concentration of the collected fractions was assayed by Coomassie Blue staining according to Bradford(44) . Aliquots of fractions containing CK2 were checked by electrophoresis on a SDS 12% polyacrylamide gel, and the corresponding fractions were pooled and concentrated in a filtration cell (Amicon) through a PM 30 membrane. The concentrated solution was diluted 10 times and loaded onto a DEAE cellulose column previously equilibrated in buffer B. A 0-1 M linear NaCl gradient in buffer B was applied. Aliquots of the eluted fractions were analyzed by Coomassie Blue staining according to Bradford (44) and electrophoresis on a SDS 12% polyacrylamide gel. The fractions containing CK2 were pooled and stored in 1 M NaCl until used.

Photoaffinity Labeling

Characterization of the photoaffinity reaction was performed by incubation of 1-2 µg of purified recombinant CK2 (7.1-14.2 pmols) with a 20-fold molar excess of [H]sperminediazonium (9 µM) in 10 mM Tris-HCl, pH 7.4, for 2 min in the dark at 4 °C. UV irradiation (330 nm) was carried out with a Hanao 4-watt lamp at a distance of 2 cm from the top of the opened microtube containing the sample. The CK2 catalytic activity was assayed as described previously(7) .

Determination of the polyamine binding site were performed similarly. A CK2 amount of 300 µg (2.14 nmol) was incubated with a 10-fold molar excess of [H]sperminediazonium (21.4 nmol) prior to chemical proteolysis.

Separation of the and Labeled Subunits

The photolabeling mixture was loaded on a 0.1%-SDS 12% polyacrylamide gel, and electrophoresis was performed during 1.5 h at 150 V according to Laemmli(45) . The separated subunits were electrophoretically transferred onto an Immobilon P transfer membrane (Millipore, Saint Quentin, Yvelines, France), which was then dried for 2 h at 37 °C and subjected to autoradiography for 12 h, using Hyperfilm-H (Amersham Corp.).

The membrane slices corresponding to the and the subunits were analyzed for radioactivity by scintillation counting.

For the determination of the spermine binding site, both the and the subunits were detected by Coomassie Blue staining (0.2% Coomassie Brilliant Blue R-250, 50% methanol, 10% acetic acid) and 50% methanol, 10% acetic acid destaining.

Chemical Proteolysis and Chromatographic Separation of Peptides

The gel pieces corresponding respectively to the and the subunits were washed twice in 1 ml of Milli Q water (Millipore) and once in 200 µl of 70% formic acid (BDH Aristar). The gel slices were then immersed in 200-300 µl of 100 mg/ml CNBr, 70% formic acid for 15 h in the dark under an argon atmosphere. The CNBr solution was diluted 5-fold with ultrapure water, and the resulting peptides were concentrated using a Speed Vac (Savant Instruments, Inc, Hicksville, NY) to a final volume of 60 µl. The peptides were loaded onto a Merck LiChrospher RP column (4.5 mm 13 cm) previously equilibrated in sonicated ultrapure water containing 0.1% trifluoroacetic acid, 4% trifluoroethanol. The column was developed with a linear 0-60% gradient of propyl alcohol at a flow rate of 0.5 ml/min, and 0.5-ml fractions were collected. The HPLC system used was a Beckman programmable solvent module 126 connected to a Beckman diode array detector module 168.

Peptide-containing fractions were spotted onto a glass fiber disk coated with Polybrene, and amino acid sequence was performed.

Modelling

Modelling of the spermine binding site was performed on Evans and Sutherland computers.

The helix containing the photolabeled site in the subunit was built with the program O(46) . The [H]sperminediazonium molecule was built with the program MAD (package Oxford Molecular, 1993). Both helix and [H]sperminediazonium structures were refined by the program Xplor(47) .


RESULTS

Characterization of the Photoaffinity Labeling Probe as a Spermine-like CK2 Ligand

The [H]sperminediazonium molecule bears two different chemical groups, i.e. the reactive diazonium on one hand and the spermine on the other hand. It was thus required to check whether this spermine analog exhibited the same characteristics as spermine toward CK2. A rapid gel filtration technique was used in the dark to estimate the binding affinity of recombinant Drosophila CK2 for [H]sperminediazonium. Plotting the data, according to Scatchard(48) , disclosed an apparent dissociation constant of 6 µM for the spermine analog, a value very close to that previously determined for spermine itself (Table 1). When the same experiment was performed under daylight conditions, it was found that nonradioactive sperminediazonium was 10 times more efficient than spermine in displacing [H]spermine bound to CK2. In fact, under light irradiation, the two ligand molecules are not equivalent because, unlike spermine, the interaction of a sperminediazonium molecule with CK2 results in covalent and irreversible binding, therefore preventing the noncovalent binding of spermine at the same site. To compare the potency of the affinity probe to that of spermine as an activator of CK2, increasing sperminediazonium concentrations were introduced in the dark in the usual CK2 activity assay with casein as the protein substrate. Half-maximal activation of the enzyme was obtained with 150 µM of sperminediazonium as compared with 120 µM for spermine (Table 1).



It thus appeared that, before light activation, spermine diazonium exhibits the major properties of spermine toward CK2, with regard to binding as well as stimulating activities. These observations validate the use of the photoactivable analog as a probe to characterize the spermine binding domain in the kinase molecule.

Covalent Photoaffinity Labeling of CK2 with [H]Sperminediazonium

Recombinant CK2 was incubated with [H]sperminediazonium (50 µM), and the mixture was subjected to irradiation for various periods of times. Covalent labeling of CK2 was found to rapidly increase with irradiation time to reach a plateau within 2 min (not shown). When the kinase was submitted to irradiation in the absence of the photoaffinity probe, no significant change in its catalytic activity was observed following 0.5-15 min irradiation (Table 1). It was therefore assumed that this treatment did not drastically alter the structural organization of the enzyme, and a 2 min irradiation time was used in all further affinity labeling experiments.

CK2 was then incubated with increasing concentrations of [H]sperminediazonium; the mixture was submitted to a 2-min irradiation, and the subunits of the enzyme were separated by SDS-polyacrylamide gel electrophoresis. Autoradiography of the corresponding gels showed that the radioactivity was found associated with both the and the subunit of the protein kinase (Fig. 1). Quantitation of the radioactivity associated with the two subunits showed a typical saturation curve exhibiting a maximal covalent labeling for a [H]sperminediazonium concentration of 50 µM and a half-maximum effective concentration of about 10 µM. This value is in good agreement with the CK2 binding constant of 11 µM previously determined for spermine(33) . As a control experiment, covalent labeling of each kinase subunit was examined by irradiation of the enzyme for 2 min in the presence of [H]sperminediazonium that has been previously photolyzed by UV irradiation for various periods of time. Under these conditions, a large decrease in the reactivity of the preirradiated probe was observed, indicating the requirement of a photoactivated reaction for the diazonium group in the covalent labeling process (Fig. 2).


Figure 1: Photoincorporation of [H]sperminediazonium into CK2. CK2 (20 pmol) was incubated with increasing concentrations of [H]sperminediazonium for 2 min in the dark at 4 °C. The resulting complex was irradiated for 2 min at 330 nm as described under ``Experimental Procedures.'' Laemmli buffer was added, and samples were loaded onto a 12% polyacrylamide gel. Following electrophoresis, both () and () subunits were transferred onto a Western blotting membrane (Immobilon P), and the associated radioactivity was visualized by autoradiography and quantified by liquid scintillation counting.




Figure 2: Photolabeling of CK2 with prephotolyzed [H]sperminediazonium. [H]Sperminediazonium (10 µM) was photolyzed for different times before being incubated with CK2 for 2 min in the dark at 4 °C followed by 2 min of irradiation at 330 nm as described under ``Experimental Procedures.'' Following electrophoresis on a 12% polyacrylamide gel, both () and () subunits were transferred on a Western blotting membrane (Immobilon P), and the associated radioactivity was analyzed by liquid scintillation counting.



When the photoaffinity labeling was carried out in the presence of increasing concentrations of unlabeled sperminediazonium, a decrease in the radioactivity associated with the subunit and to a lesser extent with the subunit was observed (Fig. 3A). Similarly, the presence of increasing concentrations of spermine during the photolabeling reaction led to a progressive decrease of the radioactivity covalently associated with the and subunits of the kinase (Fig. 3B). Under these experimental conditions, it may appear that sperminediazonium exhibits a higher binding affinity. However, it should be emphasized that after light irradiation, the two ligand molecules are not equivalent with respect to their interaction with CK2. The light-induced covalent binding of sperminediazonium is responsible for a ``trapping effect'' as it was described previously for example with the photoaffinity labeling of the regulatory subunit of protein kinase A(49) . Spermine binds to the enzyme by the way of low energy forces leading to an equilibrium state in which the spermine site number remains constant. Sperminediazonium behavior is similar to that of spermine during incubation in the dark. However the situation becomes different under irradiation conditions (Fig. 3) since a spermine binding site disappears on CK2 for each CK2-diazonium covalent bond created. Therefore the spermine concentrations required to block the binding of [H]sperminediazonium were much higher than those necessary when the incubations were carried out in the dark (Fig. 3B).


Figure 3: Inhibition of CK2 radioactive photoaffinity labeling by spermine and by the nonradioactive photoaffinity probe. After 2 min of incubation at 4 °C in the dark, the samples were irradiated for 2 min at 330 nm as described under ``Experimental Procedures.'' Laemmli buffer was added, and samples were loaded onto a 12% polyacrylamide gel. Following electrophoresis, both the () and the () subunits were transferred onto a Western blotting membrane (Immobilon P), and the associated radioactivity was assayed by liquid scintillation counting. A, [H]sperminediazonium (4.6 µM) was incubated with CK2 (0.4 µM) in the absence or presence of increasing concentrations of sperminediazonium. B, [H]sperminediazonium (4.6 µM) was incubated with CK2 (0.4 µM) in the absence or presence of increasing concentrations of spermine.



From the experiment described in Fig. 3, it may thus be concluded that the and subunits are both target sites of covalent labeling with [H]sperminediazonium. However, previous observations (33) have clearly shown that only oligomeric CK2 is stimulated by spermine and that the kinase activity of the isolated subunit is unaffected by the presence of polyamine. Furthermore, a specific binding of [H]spermine could be detected with the oligomeric CK2, and no detectable binding was observed with the isolated catalytic subunit(33) . Scatchard analysis disclosed the existence of two binding sites for [H]spermine (33) with binding parameters similar to those determined for [H]sperminediazonium, therefore suggesting that the affinity labeling obtained with [H]sperminediazonium corresponds to the spermine binding site of CK2.

Following the photoaffinity labeling of the tetrameric form of CK2, it was determined that the labeling of the subunit represents 60-70% of the subunit labeling. However this proportion decreased to 18% when the photoaffinity labeling was performed on a mixture of equimolar quantities of nonassociated and subunits. This strongly suggests that in the native tetrameric enzyme, the labeling site corresponds to a region of the subunit that is in close proximity with the spermine binding site of the subunit (data not shown).

We therefore decided to investigate first the domain of the subunit, which is involved in the polyamine binding and covalently labeled with the reactive spermine probe. We thought it may be of interest to secondly delineate the subunit-labeled sequence to get more structural information concerning the - subunit interaction in the tetrameric form of the kinase.

Characterization of the Sperminediazonium Binding Domains in the CK2 Subunit Sequences

CK2 was photoaffinity labeled in the presence of a 10-fold molar excess of [H]sperminediazonium probe. After electrophoresis, the separated affinity labeled subunits were subjected to a chemical proteolysis by cyanogen bromide. The resulting peptides were extracted from the gel and separated by a C8 reverse phase HPLC, and the peptide-associated radioactivity was measured in the collected fractions. The peptide mixture generated from the subunit yielded three major radioactive peaks (peak A (retention time, 4 min), peak B (retention time, 6 min), and peak C (retention time, 22-23 min)), which coeluted with three absorbency peaks at 250 nm (Fig. 4A). Following polyacrylamide gel electrophoresis analysis, the two radiolabeled peptides corresponding to peaks B and C were chosen to be sequenced as described under ``Experimental Procedures'' because of their smaller apparent size, comparatively to the peak A peptide. The identification of the resultant PTH derivatives revealed the sequence X-Glu-Gln-Ala-Ala-Glu-X (Fig. 4B) for peptide B. Cycles 1 and 7 did not yield any identifiable PTH derivatives, but these missing residues were identified respectively as Thr and Met in the derived sequence of the subunit of the Drosophila CK2. The absence of any identifiable PTH derivative in the first position together with the observation that most of the radioactivity was released at cycle 1 was consistent with Thr being modified and provided corroborative evidence that this threonyl residue was the target site of the photoaffinity labeling. Methionine chemically modified by the cyanogen bromide treatment was not detectable by microsequencing. The absence of any PTH derivatives in further cycles was in good agreement with a size of seven amino acid residues for the identified peptide.


Figure 4: Reversed-phase HPLC separation and microsequencing of subunit peptides generated by cyanogen bromide cleavage. CK2 (715 pmol) was photoaffinity labeled by [H]sperminediazonium (7.15 nmol). The and subunits were separated by electrophoresis on a 12% polyacrylamide gel. The subunit was cleaved by cyanogen bromide as described under ``Experimental Procedures.'' The cleavage solution (CNBr 100 mg/ml; formic acid 70%) was diluted 5 times in ultrapure water, and resulting peptides were concentrated in a final volume of 50 µl. Peptides were loaded onto a Merck LiChrospher RP column (4.5 mm 13 cm) equilibrated in sonicated ultrapure water, 0.1% trifluoroacetic acid, 4% trifluoroethanol. A, the separation was performed by a linear gradient up to 60% n-propyl alcohol, 0.1% trifluoroacetic acid, 4% trifluoroethanol at a flow rate of 0.3 ml/min. An aliquot (30 µl) of each collected fraction was analyzed for radioactivity by liquid scintillation counting. B, the major photolabeled peptide isolated from the subunit (peakB) was sequenced as described under ``Experimental Procedures,'' and the fractions containing the extracted PTH amino acid were analyzed for radioactivity by liquid scintillation counting. C, the minor photolabeled peptide isolated from the subunit (peakC) was sequenced and analyzed as described above.



Microsequencing of peptide C revealed the sequence X-Glu-X-X-Gln-Thr-Gly-Asp-Phe-Gly-X-X-Pro-X-Val-Tyr (Fig. 4C). The missing residues X, X, X, X, X, and X were identified, respectively, as Ile, Lys, Tyr, His, Cys, and Arg in the derived sequence of the subunit of the Drosophila CK2. The absence of any detectable PTH derivative at cycle 11 together with the presence of a major peak of radioactivity released at this position provided evidence that the histidyl residue 108 was a photoaffinity-labeled site in the subunit.

The same strategy was applied to identify the photoaffinity labeled site on the subunit (Fig. 5A). Microsequencing analysis of the major radiolabeled peptide (retention time, 5 min) revealed the sequence X-Ala-Ser-X(Fig. 5B). Cycle 4 failed to yield any detectable PTH derivative, but the corresponding residue was identified as Met in the derived sequence of the CK2 subunit. Radioactivity was mainly released at cycle 1, suggesting Leu as the amino acid residue covalently bound to the probe.


Figure 5: Reversed-phase HPLC separation and microsequencing of subunit peptides generated by cyanogen bromide cleavage. CK2 (715 pmol) was photoaffinity labeled by [H]sperminediazonium (7.15 nmol), and both and subunits were separated by electrophoresis on a 12% polyacrylamide gel. The subunit was cleaved by cyanogen bromide The sample was then processed as described in Fig. 4. A, subunit peptides were separated by reversed-phase HPLC. B, the major photolabeled peptide isolated from the subunit was sequenced as described under ``Experimental Procedures,'' and the fractions containing the extracted PTH amino acid were analyzed for radioactivity by liquid scintillation counting.



Modeling of the Polyamine Binding Domain of the CK2 Subunit

A three-dimensional representation of an hypothetical helix lying between the amino acid residues Met and Met corresponding to the major labeled site on the subunit was built to investigate how this domain would allow electrostatic interactions with the sperminediazonium molecule. Because of the lack of crystallographic data concerning [H]sperminediazonium, the package Oxford Molecular was used to build and refine the L conformation of the probe molecule. A covalent bond was drawn between the carbon atom at the para position of the probe phenyl group and the oxygen atom of the threonine 72 side chain. Two of the four nitrogen atoms of the [H]sperminediazonium molecule were located spatially in order to generate electrostatic interactions with the carboxylic groups of the glutamic residues 73 and 77. The distances between the atoms involved in these interactions were settled to 2.6 Å.

The final step of energy minimization was performed on the hypothetic model using the Xplor program and yielded a large negative value corresponding to one of the most stabilized conformation.

The resulting proposed molecular model is illustrated in Fig. 6. Interestingly, the electrostatic interactions between the positive nitrogen atoms of the spermine moiety and the negative carboxylic groups of the glutamic residues 73 and 77 are remarkably conserved.


Figure 6: Ribbon representation of the proposed structure of the major polyamine binding site domain in the CK2 subunit. The sperminediazonium molecule was built with the drug design program MAD (Oxford Molecular). A first structural refining was performed with the same program by an energy minimizing step. A covalent bond was drawn between the para position of the phenyl group and the oxygen atom of the photolabeled threonine 72 residue. Both glutamic acids 73 and 77 belonging to the predicted helix in the regulatory subunit were disposed in order to interact with two of the four positive nitrogen atoms of the sperminediazonium. The final refining step performed on the model exhibited a large negative value indicating a high stability of this photolabeled site.




DISCUSSION

The aim of this work was to identify the polyamine binding site along the primary structure of the Drosophila CK2. Determination of the amino acid residues involved in the binding of spermine was carried out by a photoaffinity labeling method using a tritiated spermine analog, i.e. [H]sperminediazonium. This reagent has been previously used to identify spermine binding sites on DNA(41, 42) . This labeled probe was first shown to mimic spermine in exhibiting similar binding and activating properties toward CK2. These observations indicated that the probe behaved as a spermine analog and met the criteria to be used for the determination of a polyamine binding site. We have determined that an irradiation time of 2 min was sufficient to reach the highest photoaffinity labeling of CK2. Proteolysis of the photolabeled kinase led to the identification of two radiolabeled peptides on the regulatory subunit. The first one was the seven-amino acid sequence Thr-Glu-Gln-Ala-Ala-Glu-Met in which the amino acid residue covalently bound to [H]sperminediazonium was identified as threonine 72. Based on the photoaffinity labeling of this amino acid residue and on the predicted secondary structure of the surrounding sequence, we propose as a working hypothesis a structural model that may account for a spermine binding domain. This three-dimensional representation exhibits a stable conformation of the photolabeled peptide in which both glutamic acid 73 and 77 are able to generate electrostatic interactions with two of the four positives charges of the [H]sperminediazonium molecule. According to this model, both glutamic acid residues 73 and 77 would be two amino acid residues involved in the binding of spermine to the CK2 subunit. It may be noticed that the two remaining free positive charges of spermine should probably interact with two other acidic amino acid residues of the acidic stretch comprising residues Asp-Asp of the subunit. Analysis of the subunit sequence lying between residues 55 and 80 by the Chou and Fasman method yielded a predicted secondary structure exhibiting an helix-loop- helix motif with a large amphipatic behavior for the second helix. This would suggest that both helices could contribute to the formation of the spermine binding domain. This hypothesis would be in line with the recent report of an acidic peptide bearing an helix conformation when associated with spermine and exhibiting the sequence: , EQAAE 2(50) .

The affinity labeling experiment described in Fig. 1disclosed that both the and the subunit of the kinase were labeled by [H]sperminediazonium. The labeling of the subunit could be interpreted as reflecting a second spermine binding site present on this subunit. However, previous observations have demonstrated that the [H]spermine binding activity of oligomeric CK2 required the presence of the subunit of the enzyme. No binding activity could be detected with the isolated subunit (33) . Furthermore, spermine activation of the oligomeric protein kinase activity required the presence of the subunit of the kinase, the catalytic activity of the isolated subunit being completely insensitive to the polyamine(7) . In addition, a weak labeling of the subunit was observed in the presence of equimolar nonassociated subunit comparatively with that obtained when the subunit is inserted in the tetrameric form of the enzyme. In the absence of knowledge concerning the CK2 three-dimensional structure, it is tentatively concluded that the labeling of the subunit observed in the present study may possibly be explained by the close vicinity of the catalytic subunit sequence Leu-Ala-Ser-Met with the spermine binding domain of the subunit. This proximity would allow the diazonium reactive moiety of the spermine analog to react with the residue Leu of the subunit.

The major aim of this study was to gain information concerning the major spermine binding domain on the subunit of CK2. A spermine binding site was identified at the primary structure level in the acidic stretch located between amino acid residues 55 and 80. Our results are in agreement with observations by others showing that an acidic N-terminal cluster of 50 residues is responsible for an intrinsic negative regulation of CK2 basal activity and for an efficient autophosphorylation of the subunit and is possibly implicated in the response to polybasic effectors like polyamines(51) . Recently we have generated a mutant form of human CK2 in which the glutamic acid residues 60, 61, and 63 of the subunit have been replaced by three alanine residues. These mutations led to an active enzyme exhibiting a basal specific activity 3 times that of wild-type CK2, in agreement with the observations of Boldyreff et al.(52) . In addition, the stimulation of this CK2 mutant by spermine was found to be impaired.()This is consistent with our hypothesis that in addition to the glutamic acid residues 73 and 77, one or two of the glutamic residues 60, 61, and 63 may participate to the electrostatic interactions with two of the four positive charges of spermine.

The data presented here provide chemical evidence for the localization of a major spermine binding domain of CK2 on its subunit. Detailed mapping of the residues involved in the interaction will require site-directed point mutagenesis in this domain. This study is in progress in our laboratory, it is hoped that it will shed new light on the possible role of the polyamine interaction in the regulation of CK2 activity in the intact cell.


FOOTNOTES

*
This work was supported by the INSERM (Unité 244), the Commissariat l'Energie Atomique (Direction des Sciences du Vivant/Departement de Biologie Moléculaire et Structurale/Biochimie des Régulations Cellulaires Endocrines), the Association pour la Recherche sur le Cancer, the Fédération Nationale des Centres de Lutte contre le Cancer, the Fondation pour la Recherche Médicale, and the Ligue Nationale Franaise 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 should be addressed. Tel.: 33 76 88 42 04; Fax: 33 76 88 50 58.

The abbreviations used are: CK2, protein kinase CK2; PTH, phenylthiohydantoin; HPLC, high performance liquid chromatography.

M. Goeldner and C. Hirth, unpublished results.

D. Leroy and J. K. Hériché unpublished results.


ACKNOWLEDGEMENTS

We thank S. Lidy for secretarial work.


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