(Received for publication, March 23, 1995)
From the
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
To delineate the spermine
binding site of CK2, we have applied a photoaffinity labeling method
using a tritiated photoactivable analog of spermine,
[
The photoaffinity
labeled
In the
same manner, we succeeded in determining the residue Leu
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.
Protein kinase casein kinase 2 (CK2)
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
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
Calculated: C 44.94% H 6.32% N 8.50%
Found: C 45.16% H 6.52% N 8.22%
Figure SI:
Scheme I.
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
[
The membrane slices
corresponding to the
For the determination of
the spermine binding site, both the
Peptide-containing
fractions were spotted onto a glass fiber disk coated with Polybrene,
and amino acid sequence was performed.
The
CK2 was then incubated with increasing concentrations
of [
Figure 1:
Photoincorporation of
[
Figure 2:
Photolabeling of CK2 with prephotolyzed
[
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
Following the photoaffinity labeling of the
tetrameric form of CK2, it was determined that the labeling of the
We
therefore decided to investigate first the domain of the
Figure 4:
Reversed-phase HPLC separation and
microsequencing of
The same
strategy was applied to identify the photoaffinity labeled site on the
Figure 5:
Reversed-phase HPLC separation and
microsequencing of
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
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. [
The
affinity labeling experiment described in Fig. 1disclosed that
both the
The major aim of
this study was to gain information concerning the major spermine
binding domain on the
The data presented here provide chemical evidence for the
localization of a major spermine binding domain of CK2 on its
We thank S. Lidy for secretarial work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit is
required for this binding activity.
H]sperminediazonium.
subunit was cleaved with cyanogen bromide, and two
labeled peptides were separated by high performance liquid
chromatography. The major one was the peptide
T
EQAAEM
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.
as an
subunit residue covalently bound to the probe.
(
)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.
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.
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.
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
NaBH
S
(Lancette's reagent) in Tetrahydro
Furan under reflux for 16 h. The residue was chromatographed on a
Silica gel column 4% MeOH-containing CH
CL
. R
(CH
CL
, 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 CH
Cl
. 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 CH
Cl
)
yielded 78% of
tetra-N-t-butoxycarbonyl-o,o`-diiodoanilinospermine. R
(CH
Cl
, 5% MeOH,
1% AcOH) = 0.55. MS (fast atomic bombardment): 988 (M+).
Analysis (C
H
I
N
O
= 988.7).
H NMR of a trifluoroacetic acid-deprotected sample
(D
O) d (ppm): 1.7-2.2 (m, 8H, 4
CH
bN); 3-3.3 (m, 1OH, 5
CH
N); 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
CF
CO
H, essentially as described
previously(43) . The concentration (
323 = 2.25
10
dm
mol
cm
)
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) .
H]sperminediazonium (21.4 nmol) prior to
chemical proteolysis.
Separation of the
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- and
Labeled
Subunits
H (Amersham Corp.).
and the
subunits were analyzed for
radioactivity by scintillation counting.
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.
Modelling
Modelling of the spermine binding site
was performed on Evans and Sutherland computers.
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) .
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
[
Recombinant
CK2 was incubated with [H]Sperminediazonium
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.
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).
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.
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).
(
) 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.
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).
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.
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.
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.
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
A three-dimensional representation of an hypothetical
Subunit
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.
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.
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) .
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.