(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
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.
Casein kinase 2 (CK2) ()is a ubiquitous
serine-threonine protein kinase present in both soluble and nuclear
extracts of eukaryotic
cells(1, 2, 3, 4) . The enzyme is
transiently stimulated in cells following treatment with various growth
factors or serum
stimulation(5, 6, 7, 8) , and it has
been reported to accumulate in the nuclei when quiescent cells are
stimulated to proliferate(9) .
CK2 exhibits several
distinctive properties: it uses either ATP or GTP as the phosphate
donor to phosphorylate serine or threonine residues in protein
substrates. It is selectively inhibited by
heparin(10, 11) , and it can be activated by naturally
occurring polycationic compounds such as
polyamines(12, 13) . Moreover, the kinase needs high
concentrations of MgCl (20 mM) for optimal
catalytic activity(24) .
In most animal species, CK2 has
been isolated as a heterotetramer composed of three dissimilar
subunits, i.e. 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) .
Biochemical studies with the purified
enzyme have shown that the dissociation of the tetrameric structure of
CK2 into its 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.
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 and
subunits(23) .
The present study reports that self-polymerization of recombinant Drosophila CK2 generates in vitro three different and well-defined polymeric forms. A characterization by electron microscopy and sucrose density gradient sedimentation analysis as well as by light scattering and gel filtration revealed that the generation of each form was a fully ordered and reversible process which was mostly dependent upon the ionic strength of the medium. In suboptimal conditions for catalysis, CK2 is heterogeneous and mainly composed of short thick filaments. By contrast, under optimal catalytic conditions, the enzyme exhibits a ring-like structure. These observations provide a correlation between the ring-like structure and high CK2 specific activity and strongly suggest that assembly as a specific quaternary structure is the form in which the kinase expresses its optimal activity.
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
() 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.
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 protomer, which
self-assembles when the ionic strength of the medium is lowered.
Figure 2: Electron microscopy of recombinant CK2 in 0.1 M and 1 M NaCl. Purified recombinant Drosophila CK2 (15 µg) was incubated for 2 h at 4 °C in 100 µl of 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50% glycerol, containing either 1 M or 0.1 M NaCl. Samples were then prepared for electron microscopy as described under ``Experimental Procedures.'' Bars = 100 nm. A, recombinant CK2 in 1 M NaCl. B, recombinant CK2 in 0.1 M NaCl. C, gallery of selected images of different molecular forms of CK2 in 0.1 M NaCl.
The
behavior of the isolated 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.
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
() 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.
Figure 7:
Velocity sedimentation of CK2 in the
presence of spermine or MgCl. 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.
Figure 8:
Changes in CK2 specific activity upon
dilution of the enzyme. Specific activity of recombinant Drosophila CK2 () 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.
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 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) .
The
isolated catalytic () 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.
While the present study indicates that the CK2 ring-like structures
are probably an association of four protomers and that thin filaments are likely to be made of
linearly associated protomers, it is not yet possible to clearly
understand how the filamentary structures are inter-related. According
to their average width, the thick filaments could be made of a linear
association of ring-like structures or result from side by side
association of two thin filaments. On the other hand, thin filaments
might result from splitting of thick ones. However, we have regularly
observed that thin filaments are on the average much longer than the
thicker ones. Careful kinetic studies taking into account the ionic
strength of the medium as well as the protein concentration and
temperature remain to be carried out to clarify the relationship
between the filamentary structures. Putative models representing the
different molecular forms of CK2 which would be compatible with our
observation are proposed in Table 2.
With regard to this
remarkable self-polymerization property, CK2 may be considered an
associating-dissociating enzyme. An important feature of dissociating
enzymes with regard to this regulation is that the
association-dissociation process can be modulated in the presence of
their substrates or in response to appropriate regulatory
ligands(27) . Examination of the molecular organization of CK2
under different catalytic conditions revealed that the ring-like
structure was the only conformation recovered in sucrose gradients
containing saturating concentrations of substrates and cofactors, i.e. under optimal catalytic conditions. The fact that the
()
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.
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 promote the dissociation of CK2
thick filaments and stabilize the ring-like structure. 2) Striking
changes in CK2 activity as a function of the ionic conditions have
often been reported(28, 29) . Maximal catalytic
activity was detected around 0.2 M NaCl as the enzyme adopts
mostly a ring-like structure. Increasing the salt concentration to 0.3 M NaCl strongly inhibited the kinase activity, and this
inhibition was correlated with the dissociation of the ring-like
structure into protomers. These observations suggest that CK2 filaments
and protomers are relatively inactive molecular forms of the kinase,
whereas the ring-like structure represents the most active form of the
enzyme.
Early studies by Glover (21) have shown that
polymerization of CK2 depends on the enzyme concentration. Our data are
in agreement with his observations. Our dilution experiments showed
that in 0.2 M NaCl, CK2 at 38 nM had a maximal
specific activity (i.e. 135 nmol of P/min/mg of
CK2) (Fig. 5). Analysis of the enzyme by sucrose gradient
sedimentation showed that in this range of concentration the protein
mostly adopted a ring-like structure. Decreasing the enzyme
concentration promoted the dissociation of this structure into
protomers, and this was concomitant with a striking drop of the
specific activity of the kinase. Increasing the enzyme concentration
favored the association of the ring-like structures into filaments and
could explain the observed decrease of the kinase specific activity.
One may speculate on the functional advantages of the ring-like structure conformation of the kinase with regard to its activity. Reversible interactions between subunits in the ring-like structure may permit a large flexibility in functional regulations such as the following. 1) The ring-like structure in quadrupling the subunits may enhance the substrate binding surface for maximal catalytic activity. 2) Because the advantage of a polymeric state has long been understood as the basis for cooperativity and allosteric regulation, the ring-like structure of CK2 may generate an enzyme species more responsive to regulation by allosteric effectors.
The data presented in this work
show that the catalytic function of CK2 is strongly influenced by its
quaternary structure. Although these data have been obtained in
vitro, they raise new possibilities with regard to the regulation
of the kinase activity in the living cell. At present, it is difficult
to determine the molecular organization of the kinase in the intact
cell. It has been reported that CK2 might form multimolecular complexes
with specific intracellular components such as HSP90(30) ,
cytoskeleton, double-stranded DNA(31) , or the nuclear protein
p53(32) . It is of interest to mention that the subunit
of the kinase which appears to be required for the self-polymerization
of the enzyme is also required for the CK2-p53
interaction(32) . Altogether these observations suggest that in
the intact cell, CK2 may be present (or targeted) into different
subcellular localizations through interaction with cellular components,
resulting in the control of its oligomeric organization, and
consequently in the targeting of its activity from one substrate to
another in response to intracellular specific signals.
As a new approach in the study of CK2 regulation, we propose that the polymerization behavior of the enzyme could be used to supplement the usual enzyme activity assay as a means for identifying and characterizing possible physiological regulators of this ubiquitous and pleiotropic protein kinase.