(Received for publication, October 22, 1996, and in revised form, June 11, 1997)
From the Laboratoire de Biochimie des Régulations Cellulaires Endocrines, Département de Biologie Moléculaire et Structurale, INSERM Unité 244, CEA Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France
The means by which the cell regulates protein
kinase CK2 remain obscure. However, natural polyamines, cellular
compounds required for cell proliferation, have been reported to
strongly stimulate CK2-mediated phosphorylation of a number of
substrates. Using spermine analogs, we have shown that polyamines
directly interact with the CK2 subunit, and the chemical features
of the highly acidic binding site
(Asp51-Tyr80) have been determined. In
the present study, we show that the isolated
subunit region
extending from residue Asp51 to Pro110 exhibits
a specific and efficient polyamine binding activity similar to that of
the entire
subunit. Moreover, the replacement of Glu60,
Glu61, and Glu63 of the
subunit by 3 alanine residues leads to a loss of the spermine-induced stimulation of
CK2 activity which correlates with a decrease in spermine binding
affinity. Thermal stability studies indicate that the binding of
spermine induces a 4 °C decrease of the Tm value
for the holoenzyme. This was confirmed by circular dichroism analyses,
which show that the 6 °C negative shift of the CK2
Tm value provoked by spermine binding, reflects a
conformational change in the kinase. Together, these observations
strongly suggest that this newly defined polyamine-binding domain is
involved in the intrasteric regulation of CK2 activity.
Protein kinase CK2
(CK2)1 is a serine/threonine
protein kinase present in the cell cytoplasm and nucleus of eukaryotic
organisms from yeast to man (1, 2). The kinase is made of the
association of three dissimilar subunits, i.e. the catalytic
subunits and
of 35-44 kDa (3) and the
subunit of 24-29
kDa, to generate native structures exhibiting the stoichiometry
2
2,
2
2,
and
2. The
subunit contains two
autophosphorylation sites located at Ser2 and
Ser3 and is termed the regulatory subunit because of its
potential to stimulate the activity of the
subunit in the tetramer
by 5-10-fold (4-6). Among the numerous substrates of CK2, a large number corresponds to transcription factors and oncoproteins such as
Myc (7), Myb (2, 8), Fos (9), and the anti-oncogene p53 (10). Other
proteins localized in the cytoplasm or associated to membranes were
also identified as CK2 substrates. The disruption of the genes encoding
the CK2 catalytic subunits
and
led to a lethal phenotype in
yeasts, underscoring CK2's essential role in cell proliferation
(11).
To date, no intracellular messenger has been characterized as a crucial
regulator of CK2. However, in vitro experiments that show
significant stimulation of the CK2 catalytic activity in the presence
of naturally occurring polyamines (12), provides strong evidence that
such regulation exists. The stimulation by polyamines is preferentially
observed with selected substrates, such as casein or the transcription
factor MyoD, and is strictly dependent on the presence of the subunit (13) and on low magnesium concentrations (14). Further evidence
supporting a role for polyamines as physiological CK2 stimulators
includes the observation that extraphysiological magnesium
concentrations (20-30 mM) are required for maximal CK2
catalytic activity. Indeed, most of the protein kinases, including the
CK2
subunit alone, are fully active in the presence of 4-5 times
lower magnesium concentrations. It is thought that polyamines may act
in synergy with this physiological magnesium concentration to confer a
maximal activity to the tetrameric form of CK2. However, the precise
molecular mechanism leading to the catalytic stimulation remains to be
determined.
Polyamines are ubiquitous cellular components that are indispensable for cell proliferation and differentiation (15, 16). Polybasic compounds including natural polyamines have been shown to modulate the catalytic activities of a broad range of proteins such as insulin receptor, vitamin D receptor, GTPase activity of G proteins, protein phosphatase 2A, and DNA-topoisomerases I and II (17-22). Considering the crucial roles played in the cell by most of these polyamine-stimulated enzymes, the understanding of the mechanism by which polyamines exert their effects is urgently required.
We have previously described a partial characterization of the
mechanism by which polyamines stimulate CK2 activity. It has been
initially observed that the kinase binds different polyamines and that
the binding activity is localized to the subunit (13). Subsequently, application of a photoaffinity labeling strategy, with a
photoactivated analog of spermine, led to the identification of the
polyamine-binding site within a highly acidic stretch situated between
amino acid residues Asp51 and Tyr80 of the CK2
subunit (23). The characterization of the polyamine-binding site
strongly suggested the involvement of four acidic amino acid residues
in the interaction of the kinase with a spermine molecule. Furthermore,
from the study of the interaction of several spermine analogs with the
kinase, it has been hypothesized that the CK2 polyamine-binding site
could fold into a pocket
shape.2
Overall, these in vitro studies point to the likelihood that the interaction of polyamines with this polyamine-binding site may induce conformational changes responsible for the stimulation of CK2 activity. A detailed analysis of this binding site is presented in this study, along with an evaluation of a potential model for a general mechanism of CK2 stimulation by polybasic ligands. In particular, we ask the following questions: what are the amino acid residues involved in the CK2 polyamine-binding site? Does this binding site behave like an autonomous and functional polyamine-binding domain when expressed alone? To what extent does the binding of polyamines to this domain provoke conformational changes in the CK2 holoenzyme?
Mutagenesis of the CK2 Subunit
The mutagenesis of the CK2 subunit was performed according
to the rapid for site-directed mutagenesis described in Ref. 24. The
plasmid pSG5
HA, containing the cDNA of the chicken
subunit
fused to the nucleotidic sequence coding for the HA epitope, was used
as template for the PCR reactions performed with the following
oligonucleotides: 1, TGGAATTCGGATCCGCCGCCACCATGAGCTCTTCCGAGGAGGTGTCGT; 2, ATGGATCCCCTAGAGGCTAGCATAATCAGGAACATCATAGCGGATGGTCTTCACG; 3, GTGAAATTTGTGATGC; M, CAGCCAGCGCTGCGTCAGGCTCGAGGTCG. The M
oligonucleotide confer the replacement of the
subunit residues
Glu60, Glu61, and Glu63 by 3 alanine residues. The mutated PCR product was digested by endonuclease
BamHI and cloned in the corresponding site of the plasmid
pSG5 polylinker. The resulting recombinant plasmid pSG5
3HA was used
in transient transfections to express the mutated subunit
Ala60,
Ala61,Ala63HA.
Expression of the CK2 and
Subunits in COS Cells
COS 7 cells were cotransfected according to the DEAE-dextran
method by the vectors pSG5 and pSG5
HA or pSG5
and pSG5
3HA (20 µg each) allowing the coexpression of the wild type
and
HA
subunits or the coexpression of the wild type
and the mutated
Ala60,Ala61,Ala63HA subunits, respectively. Transfection of COS 7 cells by the vector pSG5 was performed as control. The transfected cells were grown during 48 h in 6 plates of 10 cm of diameter and
lysed with 6 × 1 ml of lysis buffer (50 mM Tris-HCl,
pH 7.4, 1 mM dithiothreitol, 0, 1% Tween 20, 1 M NaCl). The cell lysate (6 ml) was centrifuged for 10 min
at 15,000 × g. The supernatant was recovered and used
as soluble cell extract.
Immunoprecipitation of the Wild Type and Mutated CK2
The soluble cell extract was diluted 5 times in
immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 0.1% Tween 20) and incubated for 1 h at 4 °C with 15 µl of the monoclonal antibody 12CA5 directed
against the HA epitope of the wild type and mutated CK2 HA subunits.
Protein A-Sepharose, previously equilibrated in the immunoprecipitation
buffer containing 10 mg/ml BSA, was incubated with the
immunoprecipitation mixture for 1 h at 4 °C. Then, the
immunocomplexes were recovered by centrifugation (2 min at 10,000 × g) and washed 4 times in the immunoprecipitation buffer
adjusted to 500 mM NaCl. Two final washes were performed in
the CK2 assay buffer (10 mM Tris-HCl, pH 7.4, 0.1% BSA)
and the immunocomplexes were aliquoted (20 µl) prior to the CK2
activity assay. The immunocomplexes were incubated with 10 mM Tris-HCl, pH 7.4, in the absence or presence of
increasing spermine concentrations and the kinase reaction was
initiated by the addition of the radioactive solution (0.3 mg/ml
casein, 10 µM ATP, 1 mM MgCl2, 1 µCi of [
-32P]ATP) in a final volume of 80 µl.
After 5 min, the kinase reaction was stopped by addition of 2 ml of
12.5% trichloroacetic acid. The following steps of the assay were
performed as described in Ref. 25. An immunoprecipitation performed
with the lysate of COS cells transfected with the vector pSG5 was also
analyzed for CK2 activity and was used for blank values of the kinase
assay.
The polyamine binding assay was achieved by the incubation at 4 °C for 5 min of the immunocomplexes with buffer B (10 mM Tris-HCl, pH 7.4) containing 0.4 µM [3H]spermine (0.5 µCi/assay) in the absence or presence of increasing spermine concentrations in a final volume of 80 µl. The final NaCl concentration was adjusted to 25 mM. The immunocomplexes were then washed three times with buffer B and the binding of [3H]spermine was analyzed by liquid scintillation counting. Polyamine binding assays performed on the immunoprecipitates prepared from cell lysates of COS cells transfected with the vector pSG5 were used for blank values.
Genetic Construction, Expression, and Purification of Fusion Proteins
The subunit domain encompassing amino acid residues
Asp51 to Pro110 and the entire
subunit were
fused to the maltose-binding protein (26). The nucleotidic sequences
encoding the
subunit domain or the entire
subunit were
amplified by PCR from the chicken
cDNA (a generous gift of Dr
E. Nigg), respectively, with the following oligonucleotides:
domain, 5
-CGGATCCGTTGACATGATCCTCGACCTCGAG;
domain,
3
-CAAGCTTGTTAGGGGCAGTACCCGAAATCGCC;
,
5
-ATTGGATCCATGAGCAGCTCCGAGGAGGTG;
,
3
-AGAAGCTTGTCAGCGGATGGTCTTCACGGG. The nucleotide sequence encoding the
3 subunit was amplified from the recombinant plasmid pSG5
3HA with
the oligonucleotides
5
and B 3
. The PCR amplifications led to
fragments carrying a BamHI site located at their 5
extremities and an in-frame stop codon followed by a HindIII
site at their 3
extremities. Following digestion of the fragments by
the corresponding endonucleases, PCR products were cloned in the
polylinker of the pMal C2 vector (protein fusion and purification
system, New England BioLabs). The resulting recombinant vectors were
used to transform Escherichia coli strain BL21. The cultures
were induced during 2 h with 0.3 mM isopropyl
thiogalactopyranoside. The cell pellets were resuspended in cold lysis
buffer (10 mM phosphate, 30 mM NaCl, 0, 25%
Tween 20, 10 mM EDTA, 10 mM EGTA, pH 7.0).
After a thermal shock (
70 °C to +20 °C) and a 3 × 2-min
sonication, the lysates were adjusted to 0.5 M NaCl and
subjected to centrifugation at 9,000 × g for 20 min.
The supernatants were mixed with amylose resin (New England BioLabs) at
4 °C during 1 h and the fusion proteins were eluted with 10 mM maltose added to the column buffer (10 mM
phosphate, 0.5 M NaCl, 1 mM sodium azide, 1 mM EGTA, pH 7.0). The recombinant proteins MBP
51-110,
MBP
1-215, and MBP
3 were finally concentrated on a Centricon cell
up to 2 mg/ml.
Binding of Spermine
Recombinant proteins (0.5-2 µg of CK2 or the fusion proteins
MBP-51-110, MBP-
1-215) were incubated at 4 °C for 5 min with 0.5 µM [3H]spermine (106 cpm)
in the absence or presence of different concentrations of non
radioactive polyamines in a final assay volume of 80 µl of Tris
buffer (10 mM Tris-HCl, pH 7.4). The NaCl concentration was settled at 25 mM in the binding experiments. The mixtures
were then rapidly centrifuged at 4 °C, according to Penefsky (27), through small Sephadex G-50 superfine column previously equilibrated in
Tris buffer containing 1 mg/ml BSA. Bound [3H]spermine
was determined by radioactive counting of the excluded volume, after
subtraction of the blank values obtained in the absence of recombinant
protein. Blank values represent less than 0.2% of the input
radioactivity.
Analysis of the Thermal Denaturation of CK2
CK2 Activity AssayCK2 was incubated for 10 min in the absence or presence of 500 µM spermine or 20 mM MgCl2 at different temperatures ranging from 30 to 65 °C in a test tube heater (Stuart Scientific) before being added to the phosphorylation medium of the kinase assay at a final concentration of 4.8 nM. The CK2 activity assay was performed as described previously (6) and the amount of phosphorylated peptide was determined by liquid scintillation counting.
Polyamine Binding AssayCK2 was incubated for 10 min at different temperatures ranging from 30 to 70 °C in a test tube heater (Stuart Scientific) before being added to the medium of the polyamine binding assay at a final concentration of 71 nM. The assay was performed as described above and the amount of [3H]spermine bound to CK2 was determined by liquid scintillation counting.
Circular Dichroism AnalysesCK2 (2.14 µM) was incubated at different temperatures for 5 min in TDG buffer (10 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 2% glycerol) in the absence or presence of 500 µM spermine in a final volume of 100 µl. CD analyses were performed on a CD6 dichrograph (Jobin Yvon, Lyon, France). The temperature of the quartz cell was maintained by a water bath monitored through a Spectralink which is connected to the dichrograph. Data were collected for each 0.5 nm between 200 and 270 nm with an acquisition time of 4 s. Control analyses were performed similarly but in the absence of CK2 and the data were deduced from those obtained in the presence of the kinase.
Previous experiments have
suggested the involvement of negatively charged amino acid residues in
the polyamine-binding site of the CK2 subunit. Thus, we predicted
that the acidic residues located in the polyacidic region encompassing
residues Asp51 to Tyr80 represent good
candidates for site-directed mutagenesis. Amino acid residues
Glu60, Glu61, and Glu63 of the CK2
HA subunit were replaced by alanine residues. The resulting mutated
cDNA (
Ala60,Ala61,Ala63) was cotransfected with the cDNA of
the human
subunit in COS cells. Cotransfection of both cDNAs of
and
HA subunits was carried out as control. Both wild type and
mutated CK2 holoenzymes were immunoprecipitated and the kinase activity
was analyzed versus spermine concentrations in basal
magnesium conditions of 1 mM. As shown in Fig.
1, A and B, the CK2
activity of the mutated form was 4 times higher than the wild type form
activity in the absence of spermine. Beyond 50 µM
spermine, both activities were equivalent and reached a plateau at 250 µM spermine. The resulting stimulation factor calculated as the ratio between the CK2 activities measured in the absence or
presence of 500 µM spermine, was 4 times weaker for the
mutated kinase.
When the immunoprecipitates of both wild type and mutated CK2 were incubated with tritiated spermine, the total binding of polyamine was 2.3 times lower with the mutated CK2, as compared with the wild type enzyme (data not shown). Scatchard plot analyses showed that both CK2 forms exhibit two binding systems (Fig. 1, C and D). Calculation of the affinity constants for the highest affinity system gave a Kd of 1.4 µM for the wild type CK2 (Fig. 1C) and 4.3 µM for the mutated kinase (Fig. 1D). For both forms of CK2, the stoichiometries of bound spermine were equivalent. Thus, the 3-fold lower affinity of the mutated CK2 for spermine strongly suggests the involvement of at least one of the three mutated glutamic acids in the CK2 polyamine-binding site.
We have already observed the apparent divergence between the concentration of spermine required to stimulate kinase activity by 50% and the calculated KD values for the binding of spermine to CK2 (28). Such feature was attributed to nonspecific interactions between polyamines and casein in the activity assay (29).
Determination of an Autonomous and Functional Polyamine-binding Domain in the CK2Previous experiments using a
photoactivable spermine analog have shown the labeling of two subunit peptides at amino acid residues Thr72 and
His108 (23). In addition, the mutational analysis described
above suggests the involvement of at least one of the three glutamic acid residues 60, 61, and 63, located in the polyacidic region of the
CK2
subunit, in polyamine binding. Together, these observations define a domain lying from Asp51, the first amino acid
residue of the
polyacidic region, to Pro110, the first
secondary structure breaker following His108. The
corresponding sequence was fused to the maltose-binding protein and the
overexpressed fusion protein MBP
51-110 was purified in one step on
amylose resin to roughly 90% purity. The entire
subunit was fused
to MBP and was purified similarly (Fig.
2).
Spermine binding analyses were performed using the fusion proteins
MBP51-110, MBP
1-215, and MBP
lacZ as control. No spermine binding activity could be detected with the fusion protein MBP
lacZ (data not shown). In contrast, a spermine binding activity was observed
for the MBP
51-110 and MBP
1-215 fusion proteins and half-maximal
binding values were, respectively, 1.7 and 1.0 µM. The
data analyzed by Scatchard plot showed the presence of one single
binding system exhibiting affinity constants of 0.5 and 0.6 µM for the fusion proteins MBP
51-110 and
MBP
1-215, respectively (Fig. 2, A and B).
From the same plots, it was calculated that 0.8 and 0.6 mol of spermine
were bound per mol of fusion proteins MBP
51-110 and MBP
1-215,
respectively. The calculated binding affinities fit with the values
obtained in the experiments described in Fig. 1 using recombinant wild
type CK2.
The ligand binding specificities of both fusion proteins were analyzed
and compared with those of the recombinant wild type CK2. Recombinant
CK2 or the fusion proteins were incubated with [3H]spermine in the presence of spermine, spermidine,
putrescine, polylysine (10 µM), and magnesium (10 mM). Spermine was found to be the strongest competitor for
the interaction, since the presence of 10 µM spermine
decreases the bound [3H]spermine by 60 and 80%, for CK2
and the fusion proteins MBP51-110 and MBP
1-215, respectively
(Fig. 3). A 30-50% loss of spermine binding was detected for all three proteins in the presence of spermidine. Putrescine was found to be the weakest competitor decreasing the interaction of spermine by only 10-20%. Polylysine is
much less efficient at competing with spermine for binding full-length
CK2 (20% displacement) while on both fusion proteins polylysine
provokes a 60-80% displacement. Finally, magnesium was shown to
antagonize the binding of spermine by 40-70% on all three proteins.
Thus, we conclude that the fusion proteins MBP
51-110, MBP
1-215,
and the oligomeric CK2 exhibit comparable ligand binding specificities.
Effect of Spermine on the Thermal Stability of CK2
The
thermal stability of CK2 was analyzed by the measurement of CK2
activity after preincubation of the enzyme at different temperatures in
the absence or presence of 500 µM spermine or 20 mM magnesium. As shown on Fig.
4A, a maximal activity of CK2 alone was determined for preincubation temperatures ranging between 30 and 45 °C. A striking decrease of CK2 activity was detected after
preincubation of the enzyme at temperatures between 45 and 55 °C,
and the catalytic activity was completely lost at temperatures above
55 °C. Half-maximal activity was obtained at 48 °C. In the presence of 500 µM spermine or 20 mM
magnesium, a maximal CK2 activity was detected for preincubation
temperatures ranging between 30 and 40 °C. This activity strongly
decreased between 40 and 50 °C. Preincubation temperatures higher
than 50 °C led to a total loss of CK2 activity. Under these
conditions, half-maximal activities were obtained at 44 °C. A small
variation in ionic strength of the buffer (equivalent to 50
mM NaCl) was observed after inclusion of 20 mM
MgCl2. However, this variation could not account for the
change in the thermal stability of the kinase. Similarly, resistivity
determination indicated that the spermine-induced change in the thermal
stability of CK2 could not be attributed to variation in the ionic
strength of the medium. Therefore, the data provide strong evidence for
a specific role of spermine in the change of CK2 thermed stability.
The spermine binding activity of CK2 preincubated at different temperatures was also measured as described under "Experimental Procedures." A maximal binding of spermine (10.0-13.0 pmol) was obtained for CK2 preincubated between 30 and 45 °C (Fig. 4B). A strong decrease of the spermine binding efficiency was observed between 45 and 55 °C and a residual binding activity (15%) was observed after preincubation of the enzyme at temperatures above 55 °C. Half-maximal binding of spermine was reached at 48 °C.
Changes in CK2 Conformation upon Spermine BindingTo further
investigate the effects of spermine on CK2 conformation, a
spectroscopic method was used to reliably monitor the structural
modifications that accompany the binding of this polyamine to the CK2
holoenzyme. Far UV circular dichroism spectra were recorded for CK2 at
different temperatures in the absence or presence of 500 µM spermine. Fig.
5A shows primary and secondary
maxima of the ellipticity at 209 and 223 nm, respectively. A loss of ellipticity was observed as temperatures increased from 20 to 90 °C.
This observation is in agreement with a modulation of the CK2
conformation from a well defined structural state into a random conformation. Addition of 500 µM spermine led to a
decrease of ellipticity as compared with the corresponding conditions
in the absence of spermine. Changes in the spectrum were observed
primary in the ellipticity at 209 nm and secondary at 221 nm (Fig.
5B).
To compare the thermal stability of CK2 in the absence and presence of
500 µM spermine, the maximal ellipticities measured at
209 nm were represented as a function of the temperature (Fig. 6). Both sigmoidal curves were
characterized by a maximal ellipticity corresponding to the CK2 folded
state and a minimal ellipticity, which was attributed to the random
conformation of the denaturated CK2. Half-denaturation constants of 42 and 48 °C were determined for CK2 incubated in the presence and
absence of spermine, respectively. Thus the binding of spermine induced
a significant difference (6 °C) in the thermal stability of CK2,
which may reflect a fundamental change in the conformation of the
enzyme.
Thermal stability of wild type CK2 and spermine binding-deficient
mutant (Ala60,Ala61,Ala63) was also compared at different temperatures (Fig. 7). A maximal CK2
activity was obtained for CK2 preincubated between 30 and 40 °C. A
decrease of CK2 activity was detected after preincubation of the enzyme
at temperatures between 40 and 55 °C, and the catalytic activity was
completely lost at temperatures above 55 °C. Half-maximal activity
was obtained at 47 °C. In contrast, a sharp and constant decrease of
the catalytic activity of the mutant kinase was observed for
temperatures above 30 °C. Under these conditions, half-maximal
activities were obtained at 41 °C. Thus the mutant kinase exhibits a
much lower thermal stability and the data provide additional evidence
for the implication of the acidic region of the
subunit in the
polyamine-induced change of CK2 conformation.
The biochemical analyses, previously used to demonstrate an
interaction of CK2 with polyamines, were expanded in this work to
investigate the mechanism by which these ligands stimulate the kinase.
Particular attention was paid to the delineation of a polyamine-binding
domain and to the detection of conformational changes induced upon
binding of polyamines to this domain. We have shown that site-directed
mutagenesis of the three glutamic acid residues (Glu60,
Glu61, and Glu63) in the acidic stretch of the
CK2 subunit lead to a decrease in the spermine binding affinity of
the enzyme which is associated with a loss of spermine-induced
stimulation of CK2 activity. These results are in fair agreement with
the observations of Boldyreff et al. (30, 31) who showed an
hyperactivated form of a CK2 holoenzyme containing mutations of acidic
residues 55-57 and 59-64 of the
subunit. In our study, the effect
of spermine on the CK2 activity was analyzed at physiological magnesium
concentration (1 mM). Under these conditions, the basal
activity of the mutated form of CK2 was found 4 times higher than the
activity of the wild type enzyme. Consequently, spermine is able to
stimulate the basal activity of the wild type enzyme by 8-fold, while
the mutated kinase was stimulated only 2-fold. This difference
correlates with the lowered spermine binding affinity observed for the
mutant CK2. Interestingly, for spermine concentrations above 50 µM, the kinase activity of the wild type or mutated CK2
were similar. These data support the notion that mutations of specific
residues in the acidic region of the CK2
subunit eliminate a
catalytic autoinhibition which is responsible, in physiological
conditions, for the low basal activity of the wild type CK2. Thus, this
effect would represent an intrasteric regulation of CK2 involving its
subunit.
Previous observations have shown that a photoactivable spermine analog
interacts with specific residues in a highly acidic stretch lying
between amino acid residues 51 and 80. These data together with the
observation that mutations of acidic residues in this region affect
both the polyamine binding affinity and the polyamine-induced
stimulation of the enzyme point to the likelihood that this region
represents an important CK2 regulatory domain. This contention is
supported by the experiments showing that a fusion protein involving
the subunit region Asp51 to Pro110 exhibits
an efficient and specific polyamine binding activity similar to that
observed for a fusion protein containing the full-length
subunit.
As compared with CK2 holoenzyme, both fusion proteins exhibit a 2.5 higher affinity for spermine. A possible interpretation for this
difference would be that the conformation of the
subunit changes
when it is associated with the catalytic subunit. The second
interpretation which is not exclusive of the first, is that the higher
polyamine binding affinity of the free
subunit is due to the
absence of interactions between its spermine-binding domain and the
subunit catalytic site (see below). Thus the polyamine-binding domain
of the CK2
subunit is still functional when it is isolated from the
full-length protein, a situation already described for the well known
SH2, SH3, and PH domains. For instance, SH2 and SH3 domains isolated
from the proteins phosphatidylinositol 3
-kinase-associated protein p85
and phosphatidylinositol 3
-kinase exhibit full binding efficiencies
for tyrosine-phosphorylated peptides and proline-rich peptides,
respectively (32, 33). Similarly, pleckstrin homology (PH) domains
isolated from the proteins phospholipase C-
1 and pleckstrin were
shown to specifically bind inositol phosphates (34).
In a series of experiments designed to test the possibility that the binding of polyamines could induce changes in the CK2 conformation, it was observed that the thermal stability of the enzyme was affected by the presence of polyamines and magnesium. A difference of 4 °C in the Tm values was observed for the holoenzyme incubated in the absence or presence of spermine or magnesium. This is large enough to correspond to a loss of CK2 stability upon spermine or magnesium binding. By analogy, a shift of 7 °C was recorded for the Tm of the T4 lysozyme containing an engineered disulfide bond as compared with the Tm of the wild type T4 lysozyme (35, 36). In this study, the difference in thermal stability was interpreted as reflecting a change in the conformation of the mutated protein, providing a protection against a conformationally-related irreversible inactivation.
The effect of spermine on the thermal stability of CK2 was also readily monitored over a wide range of temperatures by using far UV circular dichroism analyses. Raising the temperature induces a decrease in the ellipticity at 209 and 221 nm. Upon spermine binding a striking change in the ellipticity recorded at 209 nm was observed and a difference of 6 °C in the half-denaturation constants for CK2 incubated in the absence or presence of spermine was determined. Again this difference, which fits with the change in the Tm values obtained in the study of the thermal inactivation of CK2 activity, is a strong indication that the observed loss of CK2 stability reflects a spermine-induced change in the enzyme conformation. The modifications in the circular dichroism spectra observed in the presence of spermine may arise from a decrease of the helical feature of the polyamine-binding domain complexed with spermine.
Based on enzymatic and spectroscopic data, we propose a possible
mechanism for the regulation of CK2 activity by polyamines. The CK2
holoenzyme is drawn as a globular sphere representing the association
of the catalytic subunit with the
regulatory subunit (Fig.
8). In addition to the C-terminal domain,
which is involved in the tight interaction between the two subunits (30), the complex is stabilized by four electrostatic interactions between the positively charged region of the
catalytic site and the
highly negative stretch of the polyamine-binding domain of the
subunit. The resulting closed conformation is maintained in the
presence of 1 mM magnesium and is responsible for the
steric obstruction of the catalytic site and the limited access of
protein substrates. Addition of spermine or increasing the magnesium
concentration leads to the release of the polyamine-binding domain,
resulting in an open conformation. The efficiency of the substrate
binding to the catalytic pocket is consequently enhanced and the
overall kinase activity is stimulated. This model takes into account
the thermal denaturation studies, which show that, in the absence of
spermine or high magnesium concentrations, the enzyme exists in a
stable form (corresponding to the closed conformation), whereas, upon
spermine or magnesium binding, the kinase adopts a less stable form
possibly reflecting the open conformation.
This model is also supported by the experiments performed on a CK2
holoenzyme containing specific mutations in the polyamine-binding domain. The prediction is that a decrease in the number of
electrostatic interactions between the subunit polyamine-binding
domain and the
subunit catalytic site would lead to a weaker
interaction resulting in a more efficient conversion to the open
conformation, and an increased catalytic activity at low magnesium
concentrations. As a result, the mutant was found to have a lowered
thermal stability and a difference of 5 °C in the half-denaturation
constants was observed for the wild type and mutant kinase. The
polyamine-induced change in the CK2 conformation is also supported by
our previous observations that both the maximal velocity of the
catalytic reaction and the affinity of the kinase for its substrate
increase in the presence of spermine (37). According to our model, the
accessibility of the catalytic site, which depends greatly on the
kinase conformation, should be also influenced by the size of the
protein substrate. This is in keeping with our observations that the
optimal phosphorylation of a substrate such as casein by either the
free catalytic subunit or the CK2 holoenzyme requires very different
magnesium concentrations (7 and 20 mM, respectively). In
contrast, an optimal phosphorylation of the small peptide substrate
RRRDDDSEEE by both forms of CK2 is observed at the same magnesium
concentration (7 mM). Moreover, the phosphorylation of this
peptide substrate by the CK2 holoenzyme was found to be totally
insensitive to polyamines at physiological magnesium
concentrations.3
In summary, CK2 appears to be the target of an intrasteric regulation
that involves an autoinhibition of its catalytic subunit by the regulatory subunit. The driving force for this inhibition requires the
acidic region of the
subunit, which is able to bind polyamines. The
binding of polybasic ligands to this domain releases an intrasteric
inhibition and allows access of the catalytic cleft to large protein
substrates. These experimental results predict that the region of the
subunit that blocks the catalytic site has a relatively large
freedom of movement. In this respect, CK2 belongs to the growing list
of modular protein kinases such as protein kinase C, myosin light chain
kinase, and calmodulin-dependent protein kinase which
catalytic sites are autoinhibited by regulatory domains (38-40).
Whether the intrasteric regulation of CK2 is relevant to the enzyme
in vivo remains to be explored.
We thank Dr. Susan M. Gasser for comments on the manuscript.