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(Received for publication, October 12, 1994; and in revised form, January 19, 1995) From the
The structure-activity relationship of casein kinase 2 (CK2) was
examined with regard to its previously reported property to
self-aggregate in vitro. Sedimentation velocity and electron
microscopy studies showed that the purified kinase exhibited four
major, different oligomeric forms in aqueous solution. This
self-polymerization was a reproducible and fully reversible process,
highly dependent upon the ionic strength of the medium, suggesting that
electrostatic interactions are mostly involved. At high salt
concentrations (e.g. 0.5 M NaCl), CK2 appears as
spherical moieties with a 18.7 ± 1.6 nm average diameter,
roughly corresponding to the Casein kinase 2 (CK2) ( CK2 exhibits several
distinctive properties: it uses either ATP or GTP as the phosphate
donor to phosphorylate serine or threonine residues in protein
substrates. It is selectively inhibited by
heparin(10, 11) , and it can be activated by naturally
occurring polycationic compounds such as
polyamines(12, 13) . Moreover, the kinase needs high
concentrations of MgCl In most animal species, CK2 has
been isolated as a heterotetramer composed of three dissimilar
subunits, i.e. Biochemical studies with the purified
enzyme have shown that the dissociation of the tetrameric structure of
CK2 into its To examine the structure-activity relationship of CK2, we have
recently expressed its subunits in the baculovirusdirected insect cell
expression system. This approach provides a functional recombinant
holoenzyme, as well as its isolated The present study reports that
self-polymerization of recombinant Drosophila CK2 generates in vitro three different and well-defined polymeric forms. A
characterization by electron microscopy and sucrose density gradient
sedimentation analysis as well as by light scattering and gel
filtration revealed that the generation of each form was a fully
ordered and reversible process which was mostly dependent upon the
ionic strength of the medium. In suboptimal conditions for catalysis,
CK2 is heterogeneous and mainly composed of short thick filaments. By
contrast, under optimal catalytic conditions, the enzyme exhibits a
ring-like structure. These observations provide a correlation between
the ring-like structure and high CK2 specific activity and strongly
suggest that assembly as a specific quaternary structure is the form in
which the kinase expresses its optimal activity.
Figure 1:
Sedimentation profiles of recombinant
CK2 on sucrose density gradients. Purified recombinant Drosophila CK2 (15 µg) was incubated for 2 h at 4 °C in 100 µl of
10 mM Tris-HCl, pH 7.5, 1 mM DTT, containing either 1 M or 0.1 M NaCl and sedimented through 5-25%
sucrose gradient under the same salt conditions. Gradients were
fractionated. A, CK2 activity in a 1 M NaCl gradient
(
The recombinant CK2 was
then analyzed by electron microscopy using replicas produced by
low-angle rotary shadowing with tantalum/tungsten. Electron micrographs
of CK2 incubated in buffer containing either 1 M or 0.1 M NaCl are shown in Fig. 2, A and B,
respectively. As expected from the sedimentation data, a homogeneous
population made of roughly circular structures with an average diameter
of 18.7 ± 1.6 nm was observed at high salt concentrations,
consistent with the CK2 protomer (Fig. 2A). By
contrast, four different structural organizations of the protein could
be visualized in 0.1 M NaCl (Fig. 2B). 1) Some
rare 18.7 ± 1.6 nm diameter particles (Fig. 2B, panel 1). 2) Roughly round structures with an average diameter
of 36.6 ± 2.1 nm and usually showing in their center a
pronounced decrease of the metal density (Fig. 2B, panel 1). This structure will be referred to as the
``ring-like'' structure. 3) Thin filaments with a uniform
average width of 16.4 ± 1.4 nm and variable lengths (up to 5
µm) (Fig. 2B, panel 2). 4) Thick filaments
of about 28.5 ± 1.6 nm width (Fig. 2B, panel
3). These structures are all derived from different associations
of the
Figure 2:
Electron microscopy of recombinant CK2
in 0.1 M and 1 M NaCl. Purified recombinant Drosophila CK2 (15 µg) was incubated for 2 h at 4 °C
in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT,
50% glycerol, containing either 1 M or 0.1 M NaCl.
Samples were then prepared for electron microscopy as described under
``Experimental Procedures.'' Bars = 100 nm. A, recombinant CK2 in 1 M NaCl. B,
recombinant CK2 in 0.1 M NaCl. C, gallery of selected
images of different molecular forms of CK2 in 0.1 M NaCl.
The
behavior of the isolated
Figure 3:
Electron microscopy and velocity
sedimentation of CK2 as a function of ionic conditions. Purified
recombinant Drosophila CK2 (15 µg) was preincubated for 2
h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1
mM DTT, 50% glycerol, and 0.4 M (a), 0.3 M (b), 0.2 M (c), 0.1 M (d) NaCl, and prepared for electron microscopy, as
described under ``Experimental Procedures.'' In parallel,
purified recombinant Drosophila CK2 (1.5 µg) was
preincubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, and 0.4 M (e), 0.3 M (f), 0.2 M (g), 0.1 M (h) NaCl, and sedimented
through a 5-25% sucrose gradient under the same salt conditions.
Gradients were fractionated and kinase activity was measured in each
fraction. Bars = 100 nm.
To
cross-check these results, electron microscopy analysis was carried out
directly on isolated fractions recovered following gradient
centrifugation and representing the different self-assembled CK2
populations. For these experiments, a glycerol gradient equivalent to
the 5-25% sucrose gradient was used since sucrose was not
compatible with the shadowing procedure used to prepare the specimens
for microscopy. In total agreement with the results illustrated in Fig. 3, we observed that the enzyme sedimenting at 13.6 S
corresponded mostly to the ring-like structure (Fig. 4). Thick
filaments were the most abundant in the large intermediary peak (15 to
44 S), and long thin filaments were mainly detected in the fractions
sedimenting at the bottom of the tube. Unorganized aggregates were also
observed at the bottom of the tube.
Figure 4:
Electron microscopic analysis of the
different molecular forms of CK2 separated on a glycerol gradient.
Recombinant Drosophila CK2 (75 µg) was preincubated 2 h at
4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, and 0.1 M NaCl, then sedimented under the same salt
condition on a 8-41% glycerol gradient. The gradient was
fractionated and the kinase activity was measured in each fraction.
Then selected fractions were used for electron microscopy as described
under ``Experimental
Procedures.''
We found that all the polymeric
structures of the enzyme could be dissociated with NaCl concentrations
higher than 0.4 M NaCl (not shown). Thus, the
self-polymerization of CK2 is readily reversible in the appropriate
ionic strength environment.
Figure 5:
Structural relationship between the
different molecular forms of CK2. Recombinant Drosophila CK2
(15 µg) was preincubated for 2 h at 4 °C in 100 µl of 10
mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, and 0.1 M NaCl and prepared for electron microscopy, as described
under ``Experimental Procedures.'' A, gallery of
selected images of CK2 polymers. B, drawings of the structures
shown in A to assist interpretation of the photomicrographs. Bars = 100 nm.
The morphology of the
thick filaments suggests that they could result from a linear
association of ring-like moieties (Fig. 5, panels Ab and Ac). If so, the ring-like structures must compact a
little during association since the average width of the thick
filaments (28.5 ± 1.6 nm) was slightly smaller than the diameter
of the isolated ring-like structures (36.6 ± 2.1 nm). With
regard to the thin filament organization, it may result from a linear
association of protomers. In some cases, thin filaments appeared as if
they were generated from thick filaments either by splitting (see Fig. 5, panel Ad) or by internal molecular
rearrangement (see Fig. 2C, panel 4). On the
other hand, thick filaments may result from the side by side
association of thin filaments. We have no experimental evidence at the
moment to clarify further the relationship between these different
filamentary arrangements.
Figure 6:
Velocity sedimentation of CK2 under
different catalytic conditions. Recombinant Drosophila CK2
(1.5 µg) was preincubated for 2 h at 4 °C in 100 µl of 10
mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 M NaCl,
then sedimented on a 5%:25% sucrose gradient in the same salt condition
(
Figure 7:
Velocity sedimentation of CK2 in the
presence of spermine or MgCl
Figure 8:
Changes in CK2 specific activity upon
dilution of the enzyme. Specific activity of recombinant Drosophila CK2 (
From these experiments, it is suggested that
changing the enzyme concentration leads to changes in the equilibrium
between the different oligomeric forms of the kinase in correlation
with striking changes in its specific activity. Using recombinant CK2, the present study confirms the well
known property of the purified enzyme to self-aggregate in
subphysiological ionic strength conditions(11, 12) .
In this respect, the behavior of the baculovirus-directed recombinant Drosophila CK2 used in the present work appears similar to
that of its native counterpart from insect (21) or bovine (22) sources. Our observations are also consistent with
previous reports(21, 22) , showing that the
aggregation process is an ordered and fully reversible phenomenon
leading to filamentary polymeric forms of the kinase. Electron
microscopic examination combined with velocity sedimentation, DLS, and
gel filtration analysis, disclosed that there are four major forms. As
has been reported many times, the purified enzyme remains stable in a
high salt environment (e.g. 0.5 M NaCl) as a protomer
made of two tightly bound The
isolated catalytic ( While the present study indicates that the CK2 ring-like structures
are probably an association of four
With regard to this
remarkable self-polymerization property, CK2 may be considered an
associating-dissociating enzyme. An important feature of dissociating
enzymes with regard to this regulation is that the
association-dissociation process can be modulated in the presence of
their substrates or in response to appropriate regulatory
ligands(27) . Examination of the molecular organization of CK2
under different catalytic conditions revealed that the ring-like
structure was the only conformation recovered in sucrose gradients
containing saturating concentrations of substrates and cofactors, i.e. under optimal catalytic conditions. The fact that the
( The present study provides several clues
suggesting a strong correlation between the ring conformation of the
enzyme and a high specific activity. 1) Two well-known activators such
as polyamines and MgCl Early studies by Glover (21) have shown that
polymerization of CK2 depends on the enzyme concentration. Our data are
in agreement with his observations. Our dilution experiments showed
that in 0.2 M NaCl, CK2 at 38 nM had a maximal
specific activity (i.e. 135 nmol of One may speculate on the functional advantages of the ring-like
structure conformation of the kinase with regard to its activity.
Reversible interactions between subunits in the ring-like structure may
permit a large flexibility in functional regulations such as the
following. 1) The ring-like structure in quadrupling the subunits may
enhance the substrate binding surface for maximal catalytic activity.
2) Because the advantage of a polymeric state has long been understood
as the basis for cooperativity and allosteric regulation, the ring-like
structure of CK2 may generate an enzyme species more responsive to
regulation by allosteric effectors. The data presented in this work
show that the catalytic function of CK2 is strongly influenced by its
quaternary structure. Although these data have been obtained in
vitro, they raise new possibilities with regard to the regulation
of the kinase activity in the living cell. At present, it is difficult
to determine the molecular organization of the kinase in the intact
cell. It has been reported that CK2 might form multimolecular complexes
with specific intracellular components such as HSP90(30) ,
cytoskeleton, double-stranded DNA(31) , or the nuclear protein
p53(32) . It is of interest to mention that the As a new
approach in the study of CK2 regulation, we propose that the
polymerization behavior of the enzyme could be used to supplement the
usual enzyme activity assay as a means for identifying and
characterizing possible physiological regulators of this ubiquitous and
pleiotropic protein kinase.
Volume 270,
Number 14,
Issue of April 7, 1995 pp. 8345-8352
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CHARACTERIZATION OF MULTIPLE OLIGOMERIC STATES AND RELATION WITH
ITS CATALYTIC ACTIVITY (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
protomer, as deduced by measurements of the Stokes radius and by light
scattering studies. At lower ionic strength (e.g. 0.2 M NaCl), the protomers associate to form ring-like structures with a
diameter (averaging 36.6 ± 2.1 nm) and Stokes radius indicating
that they are most likely made of four circularly associated
protomers. At 0.1 M NaCl,
two additional polymeric structures were visualized: thin filaments
(16.4 ± 1.4 nm average), as long as 1 to 5 µm, and thick and
shorter filaments (28.5 ± 1.6 nm average). Examination of the
molecular organization of CK2 under different catalytic conditions
revealed that the ring-like structure is the favored conformation
adopted by the enzyme in the presence of saturating concentrations of
substrates and cofactors. During catalysis, well-known cofactors like
MgCl or spermine are the main factors governing the
stabilization of the active ring-like structure. On the other hand,
inhibitory high salt concentrations promote the dissociation of the
active ring-like structure into protomers. Such observations suggest a
strong correlation between the ring-like conformation of the enzyme and
optimal specific activity. Thus, CK2 may be considered as an
associating-dissociating enzyme, and this remarkable property supports
the hypothesis of a cooperative and allosteric regulation of the kinase
in response to appropriate regulatory ligands possibly taking place in
intact cells.
)is a ubiquitous
serine-threonine protein kinase present in both soluble and nuclear
extracts of eukaryotic
cells(1, 2, 3, 4) . The enzyme is
transiently stimulated in cells following treatment with various growth
factors or serum
stimulation(5, 6, 7, 8) , and it has
been reported to accumulate in the nuclei when quiescent cells are
stimulated to proliferate(9) . (20 mM) for optimal
catalytic activity(24) . and ` subunits of 35-44 kDa
and subunits of 24-29 kDa which associate to form
, `, or
` native
structures(14, 15) . Although the respective roles of
each subunit in the kinase activity and regulation remain poorly
understood, it has been shown that the and ` subunits that
are the products of different genes bear the catalytic site of the
enzyme(16, 17, 18, 19) . The
subunit which is the target of kinase self-phosphorylation may be
considered as regulatory component since it confers optimal activity to
the holoenzyme (17) and may influence its substrate
specificity(20) . and subunits requires rather drastic
denaturing conditions(17) . Moreover, the tetrameric structure
of CK2 has usually been examined under high salt conditions because it
was observed early on that the enzyme aggregates at low salt
concentrations(11, 12) . Two reports have shown that
the aggregation of CK2 from Drosophila(21) or from
bovine heart (22) is an ordered process resulting in the
generation of filamentary structures. This in vitro self-polymerization property appears of interest in view of its
possible role in the regulation of CK2 activity in the intact cell. and
subunits(23) .
Materials
[
-
P]ATP
(3,000 Ci/mmol) was purchased from Amersham. The peptide substrate
(RRREEETEEE) for CK2 was obtained from Neosystem Laboratory
(Strasbourg, France). Spermine was obtained from Sigma.Preparation of Recombinant Casein Kinase
2
Recombinant oligomeric CK2 from Drosophila melanogaster was overexpressed in Sf9 cells, purified to homogeneity as
described previously(23) , and stored at -80 °C in 10
mM Tris-HCl, pH 7.5, 1 mM DTT, 2% glycerol, 0.1%
Triton X-100, and 1 M NaCl.Velocity Sedimentation
Linear 5-25% (w/v)
sucrose gradients were prepared in TD buffer: 10 mM Tris-HCl,
pH 7.5, 1 mM DTT, supplemented with various components, as
indicated in the figure legends. For each experiment, samples were
incubated at 4 °C in 0.1 ml of TD buffer for 2 h prior to loading.
Gradients were centrifuged at 4 °C for 3.5 h at 200,000 
g and fractionated by pipetting 200-µl aliquots. For each
ionic condition, identical gradients were run using aldolase (8 S),
catalase (11.2 S), and -macroglobulin (19 S) as
sedimentation markers which were localized by the Bradford protein
assay.Polyacrylamide Gel Electrophoresis
Proteins were
solubilized and boiled in Laemmli's sample buffer and analyzed by
12% SDS-PAGE. Proteins were revealed by silver staining.Casein Kinase 2 Assay
CK2 activity was assayed
using either casein or the synthetic peptide substrate (RRREEETEEE), as
described previously(24, 23) .Electron Microscopy
Recombinant CK2 (15 µg)
was incubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, and different
NaCl concentrations. Samples were then sprayed onto a fresh mica
surface using a vaporizer and rotary-shadowed under vacuum with
tantalum/tungsten at an incidence angle of 10-15° to the
horizontal. Replicas were coated with a 20-nm-thick supporting film of
carbon. Electron micrographs were obtained under standard bright field
imaging conditions using a Zeiss 10C microscope operating at 100 keV.
Size determination was performed on at least 50 independent objects.Gel Filtration
A Sephadex S-300 gel filtration
column was calibrated by using porcine thyroglobulin (M
= 669,000, Stokes radius = 85.0 Å), ferritin (M = 440,000, Stokes radius = 61.0
Å), aldolase (M= 158,000, Stokes
radius = 48.1 Å), bovine serum albumin (M = 67,000, Stokes radius = 35.5 Å) as
standards. The void volume (V
) was estimated by
elution of blue dextran 2000. K
values for
standards were calculated from their elution volumes (V
) by the equation: K = (V - V)/(V
- V) where V = the total column volume. CK2 (30 µg), preincubated for 2 h
at 4 °C in 0.4 M or 0.2 M NaCl, was applied to
the column equilibrated in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 2% glycerol containing either 0.4 or 0.2 M NaCl.Dynamic Light Scattering (DLS)
Recombinant CK2
(107 µg) was preincubated for 2 h at 4 °C in 500 µl of 10
mM Tris-HCl, pH 7.5, containing either 0.4 M NaCl or
0.2 M NaCl. DLS measurements were performed at 14.5 °C
using a system consisting of a BI 2030 AT 4
N-bit
autocorrelator with real-time channels and multiple sample time option
(Brookhaven Instruments), a stepping motor goniometer (Amtec, Nice,
France), and a Spectra-Physics 2025 argon ion laser. The incident laser
power was 500 milliwatts at 488 nm. DLS autocorrelation functions were
collected through an angular range of 40° to 90°. The data were
analyzed by either a maximum entropy procedure which directly yields
the particle size distribution (25) or a non linear
least-squares fit to a squared sum of exponentials. The diffusion
coefficients D were obtained through D = (K
)
, K being the
scattering vector and the relaxation time. D was corrected to 20 °C by D
= D
(293/T)(
/
), T being the absolute temperature, and the
viscosity of water at T.Determination of CK2 Specific Activity
The kinase
assay contains a reaction volume of 100 µl containing 10 mM Tris-HCl, pH 7.5, 1.2 mg/ml casein, 10 µM ATP, 1
mM MgCl, variable
[-P]ATP concentrations to obtain
appropriate -P incorporation, and 0.2 M NaCl. Various amounts of recombinant CK2 and recombinant
subunit were preincubated in the kinase assay medium at room
temperature for 2 min, and the reactions were started by addition of
magnesium. For each condition, a time course study was performed to
check the linearity of the reaction. Materials used in these
experiments were siliconized, then saturated with a solution containing
10 mM Tris-HCl, pH 7.5, 1 mM DTT, 2% glycerol, and 1
mg/ml bovine serum albumin prior to use.
Molecular Forms of CK2 in Aqueous Solution under Low
and High Salt Concentrations
Homogeneous recombinant Drosophila CK2 was analyzed by velocity sedimentation through
a sucrose gradient containing either 0.1 M or 1 M NaCl (Fig. 1). Aliquots of the collected fractions were
analyzed for CK2 activity (Fig. 1A) and by SDS-PAGE (Fig. 1, B and C). In the presence of 1 M NaCl, the enzyme sedimented as a single peak with a 6 S
sedimentation coefficient which is the value expected for the
holoenzyme, further referred to as
the CK2 protomer. By contrast, at 0.1 M NaCl, the enzyme
exhibited a polydisperse distribution. As illustrated in Fig. 1A, a continuum of species having sedimentation
coefficients ranging from 13.6 S to 47 S could be visualized.
Examination of fractions showing CK2 activity by SDS-PAGE shows that
both and subunits are present with a similar stoichiometry
all along the sucrose gradients (Fig. 1, B and C). Therefore, the sedimentation behavior of the recombinant
CK2 is strongly dependent upon the ionic strength of the medium. In
addition, the heavy forms of CK2 occurring at low ionic strength
contain both the and the subunits of the kinase, with a
stoichiometry identical with that observed for the protomer
(). This suggests that these heavy
forms are generated by protomer association.
) and in a 0.1 M NaCl gradient (). Positions of
-macroglobulin (19 S), catalase (11.2 S), and aldolase
(8 S) which were sedimented in parallel gradients of identical
compositions (except DTT for -macroglobulin) are
indicated. B and C, protein contained in the
fractions were precipitated by 10% trichloroacetic acid, analyzed by
12% SDS-PAGE, and revealed by silver staining. B, 1 M NaCl; C, 0.1 M NaCl.
protomer, which
self-assembles when the ionic strength of the medium is lowered.
subunit was also examined by electron
microscopy under high and low salt conditions. It always appeared on
electron micrographs as an homogeneous population of spherical
structures (data not shown) indicating that the subunit is
required in the CK2 self-polymerization process.Molecular Forms of CK2 as a Function of Ionic
Strength
To examine in more detail how the self-polymerization
of the kinase takes place as a function of ionic strength, velocity
sedimentation and electron microscopy analysis of CK2 were carried out
in parallel, under different salt concentrations. In 0.4 M NaCl, the enzyme sedimented as a single peak with a sedimentation
coefficient of 6 S (Fig. 3B, panel e).
Electron microscopy imaging showed that under these conditions the
enzyme appears as a homogeneous population of condensed spherical
structures, with a size fitting well with an
protomeric form of the protein (Fig. 3A, panel a). Decrease of the NaCl
concentration to 0.3 M resulted in the occurrence of a new
peak of CK2 activity with a sedimentation coefficient of 13.6 S (Fig. 3B, panel f). In addition to the
presence of protomers, the corresponding electron micrographs disclosed
a population of ring-like structures (Fig. 3A, panel b). The occurrence of ring-like structures may reflect
the beginning of a transition suggesting that 0.3 M NaCl
represents a threshold salt concentration below which ring-like
structure formation is favored. Indeed, in 0.2 M NaCl, the
enzyme sedimented as a homogeneous peak with a 13.6 S sedimentation
coefficient (Fig. 3B, panel g). Electron
micrographs showed that the ring-like structure represented the most
prominent form of the enzyme at this salt concentration (Fig. 3A, panel c). Lowering the NaCl
concentration to 0.1 M resulted in an enzyme sedimentation
pattern further in the sucrose gradient reflecting a polydisperse
distribution from 15 to 44 S and a pelleted form (Fig. 3B, panel h). Electron microscopy
examination of these two populations revealed that the corresponding
molecular forms of CK2 were characterized by the occurrence of thick
and thin filaments (Fig. 3A, panel d).
Relationship between the Different Molecular Forms of
CK2
We further characterized the structure of the protomer and
the ring-like structure by gel filtration and dynamic light scattering
(DLS) analysis under two ionic strength conditions (0.4 M and
0.2 M NaCl, respectively). The major physical characteristics
obtained for the different CK2 molecular forms are given in Table 1. Apparent molecular masses of 669 kDa and 158 kDa were
determined by gel filtration analysis for the ring-like structure and
the protomer, respectively. From the Stokes radius calculated from the
DLS data, and the sedimentation coefficients from the sucrose gradient
centrifugation, it was possible to determine molecular sizes of 524 kDa
and 129 kDa for the ring-like and the protomeric structures,
respectively. With both techniques, the ratio between the apparent
molecular masses of these two forms of the enzyme was very close to 4.
A similar observation could be made for the Stokes radius determined by
the two independent techniques (Table 1). Higher magnification
electron micrographs showing CK2 ring-like structures is illustrated in Fig. 5(panel Aa). Together with dimensional criteria (i.e. an average diameter of 36.6 ± 2.1 nm for these
particles as compared to 18.7 ± 1.6 nm for the protomer), these
data suggest that the ring-like structural organization of CK2 is
formed by the circular association of protomers. The data are compatible with the idea that the
ring-like structure represents a tetramer of the kinase protomers, i.e. ()
interacting side by side on a plane.
Molecular Forms of CK2 and Catalytic Activity
The
four different types of CK2 structures isolated following
ultracentrifugation on sucrose gradients were detected by their protein
kinase activity (Fig. 3). We found that all the CK2 molecular
forms assayed in the different sucrose gradient-collected fractions
exhibited similar specific protein kinase activities. However, it
should be stressed that the four different conformations of CK2 were
isolated under a resting state (i.e. in the absence of any
substrate), whereas the protein kinase activity was then determined in
an assay medium under optimal catalytic conditions. This prompted us to
examine the molecular forms of the kinase when placed under catalytic
conditions.Molecular Organization of CK2 under Different Catalytic
Conditions
Oligomeric preparations of CK2 were analyzed on
sucrose gradients containing 0.1 M NaCl in the absence or in
the presence of 10 µM ATP, 150 µM peptide
substrate and with either 1 mM or 20 mM MgCl (Fig. 6). As previously shown in Fig. 1, in the
absence of substrates, the enzyme sedimented as a mixture of three
major different polymerized structures: the ring-like structure, the
thick, and the thin filaments. In the presence of substrates and 1
mM MgCl (i.e. in nonoptimal catalytic
conditions), the CK2 polymerization pattern was changed. The number of
thick filaments decreased dramatically whereas there were more
ring-like structures. However, the CK2 population remained
heterogeneous under these catalytic conditions. In contrast, in the
presence of substrates and 20 mM MgCl (i.e. under optimal catalytic conditions), the enzyme sedimented
strikingly as a sharp symmetrical peak with a sedimentation behavior
(13.6 S) corresponding to that of the ring-like structure. Control
experiments disclosed that during centrifugation, the peptide substrate
present in the sucrose gradient was readily phosphorylated (not shown).
These results strongly suggest that the ring-like structure of the
kinase corresponds to the favored molecular organization of the
protomers when the enzyme is optimally active during the catalytic
reaction.
) or containing 150 µM peptide substrate, 10
µM ATP, 30 mM NaCl, and 1 mM (
)
or 20 mM () MgCl. Gradients were
fractionated and CK2 activity was measured in each fraction with casein
as described under ``Experimental Procedures.'' Positions of
protomer, ring structures, and polymers are
indicated.
Effects of Spermine and MgCl
Naturally occurring
polyamines such as spermine are potent activators of
CK2(12, 13) . We have previously demonstrated that
this activation occurs at least partly through a direct interaction
between spermine and the on the
Molecular Organization of CK2 subunit of CK2(26) . We
therefore analyzed the sedimentation behavior of CK2 in the presence of
1 mM spermine or 20 mM MgCl (Fig. 7). As expected, CK2 sedimented as a mixture of
various interconverting oligomers. By contrast, in the presence of
either 1 mM spermine or 20 mM MgCl, the
enzyme sedimented as a sharp peak (13.6 S) corresponding to the
ring-like structure. This experiment shows that two potent activators
of CK2 activity are able to dissociate the high molecular weight
oligomers and to stabilize the enzyme in its ring-like structure. It
should be stressed that this effect requires neither the presence of
the peptide substrate nor ATP. In addition, this experiment clearly
establishes the occurrence of the ring-like structure under conditions
where CK2 has been shown to bind the polyamine and to be optimally
activated by the polycation(12, 13) . Binding of
spermine to the subunit of CK2 thus appears sufficient to induce
a change in the quaternary structure of the enzyme corresponding to a
catalytically active conformation.
. Recombinant Drosophila CK2 (1.5 µg) was preincubated for 2 h at 4 °C in 100
µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 M NaCl in the absence () or the presence of 1 mM spermine () or 20 mM MgCl ()
and sedimented on 5%:25% sucrose gradients under the same conditions.
Gradients were fractionated, and CK2 activity was measured in each
fraction with casein as described under ``Experimental
Procedures.'' Positions of protomer and ring structures are
indicated.
Change in CK2 Specific Activity upon Enzyme
Dilution
The concentration of a dissociating enzyme influences
the equilibrium between the monomer and the oligomer populations, and
it is expected that dilution should have an effect on the specific
activity of the kinase by changing the ratio of active and inactive
species. Fig. 8illustrates a dilution experiment in which the
enzyme concentration was varied over 3 orders of magnitude under ionic
conditions (0.2 M NaCl) which favored the ring-like structure (Fig. 3). Maximal specific activity (135 nmol of P/min/mg of CK2) was obtained for a CK2 concentration (i.e. 5 µg/ml; 38 nM) at which the enzyme
exhibits a ring-like structure, as determined by sucrose gradient
centrifugation (Fig. 3). Decreasing the enzyme concentration to
0.02 µg/ml (0.15 nM) induced a striking drop (9-fold) in
its specific activity. Similarly, increasing the enzyme concentration
from 38 nM to 380 nM led to a 2-fold decrease in its
specific activity. Linearity versus time of the protein kinase
reaction could not be obtained at higher enzyme concentrations. A
similar experiment was performed with the isolated subunit. The
catalytic subunit does not form oligomeric structures (data not shown),
and no change in its specific activity could be detected upon dilution (Fig. 8).
) or the () subunit was determined as
described under ``Experimental Procedures'' at different
concentrations. For each condition, a time course study was performed
to check the linearity of the reaction.
and subunits with an
stoichiometry(14, 15) . When the ionic strength
is lowered to 0.2 M NaCl, this protomeric kinase can associate
to form ring-like structures with a shape, size, and Stokes radius
compatible with a circular association of four protomers
(). At 0.1 M NaCl, CK2 is a mixture of: (i) protomers, (ii) ring-like structures, (iii) long thin filaments,
and (iv) thick filaments. Remarkably, raising the ionic strength of the
medium (e.g. to 0.4 M NaCl) resulted in the
disappearance of these polymeric organizations and the total recovery
of the enzyme in its protomeric form. As mentioned previously by others (21, 22) , this obviously suggests that electrostatic
interactions are mostly concerned in the self-organization of CK2 into
polymeric structures. On the other hand, the intramolecular association
between the and the subunits in the
protomer is different in nature and
requires drastic conditions to be ruptured(17) .) subunit of the kinase does not
self-polymerize (data not shown). This observation strongly suggests
that the subunit in the protomer plays a crucial role in the
initiation of the self-association process and the stabilization of the
various characterized polymeric forms of the enzyme. This would be in
line with the suggestion by Glover (21) that the subunit,
which is self-phosphorylatable in the native enzyme, may trigger
inter-protomer association due to an enzyme-substrate interaction. protomers and that thin filaments are likely to be made of
linearly associated protomers, it is not yet possible to clearly
understand how the filamentary structures are inter-related. According
to their average width, the thick filaments could be made of a linear
association of ring-like structures or result from side by side
association of two thin filaments. On the other hand, thin filaments
might result from splitting of thick ones. However, we have regularly
observed that thin filaments are on the average much longer than the
thicker ones. Careful kinetic studies taking into account the ionic
strength of the medium as well as the protein concentration and
temperature remain to be carried out to clarify the relationship
between the filamentary structures. Putative models representing the
different molecular forms of CK2 which would be compatible with our
observation are proposed in Table 2.
) moiety is indeed an
active state of the kinase is supported by the fact that the peptide
substrate present in the enzyme environment was extensively
phosphorylated during the sucrose gradient sedimentation. Our data
clearly establish that the active ring-like structure of CK2 is
stabilized by known activating agents such as MgCl or
polyamines. For instance, spermine at submillimolar concentration binds
to CK2(26) , induces the formation of the catalytically active
ring-like structure, and prevents filament formation. The fact that
MgCl at high concentration had the same effect is in
agreement with data showing that spermine can substitute for high
MgCl concentrations in supporting optimal kinase
activity(28) . From these data it is expected that the
subunit, which is required for the polyamine interaction(26) ,
may play a crucial role in governing the molecular organization and the
activity of the enzyme. promote the dissociation of CK2
thick filaments and stabilize the ring-like structure. 2) Striking
changes in CK2 activity as a function of the ionic conditions have
often been reported(28, 29) . Maximal catalytic
activity was detected around 0.2 M NaCl as the enzyme adopts
mostly a ring-like structure. Increasing the salt concentration to 0.3 M NaCl strongly inhibited the kinase activity, and this
inhibition was correlated with the dissociation of the ring-like
structure into protomers. These observations suggest that CK2 filaments
and protomers are relatively inactive molecular forms of the kinase,
whereas the ring-like structure represents the most active form of the
enzyme.P/min/mg of
CK2) (Fig. 5). Analysis of the enzyme by sucrose gradient
sedimentation showed that in this range of concentration the protein
mostly adopted a ring-like structure. Decreasing the enzyme
concentration promoted the dissociation of this structure into
protomers, and this was concomitant with a striking drop of the
specific activity of the kinase. Increasing the enzyme concentration
favored the association of the ring-like structures into filaments and
could explain the observed decrease of the kinase specific activity. subunit
of the kinase which appears to be required for the self-polymerization
of the enzyme is also required for the CK2-p53
interaction(32) . Altogether these observations suggest that in
the intact cell, CK2 may be present (or targeted) into different
subcellular localizations through interaction with cellular components,
resulting in the control of its oligomeric organization, and
consequently in the targeting of its activity from one substrate to
another in response to intracellular specific signals.
)
We acknowledge the expert technical assistance of
Christian Closse in electron microscopy. We are indebted to Sonia Lidy
for editing the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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