(Received for publication, December 22, 1995; and in revised form, February 14, 1996)
From the
Five mutants of protein kinase CK2 subunit in which
altogether 14 basic residues were singly to quadruply replaced by
alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A;
R228A; and R278A, K279A,R280A) have been purified to near homogeneity
either as such or after addition of the recombinant
subunit. By
this latter procedure five mutated tetrameric holoenzymes were obtained
as judged from their subunit composition, sedimentation coefficient on
sucrose gradient ultracentrifugation, and increased activity toward a
specific peptide substrate as compared with the isolated
subunits. The kinetic constants and the phosphorylation efficiencies (V
/K
) of all the
mutants with the parent peptide RRRADDSDDDDD and a series of
derivatives, in which individual aspartic acids were replaced by
alanines, have been determined. Three mutants, namely
K74A,K75A,K76A,K77A; K79A,R80A, K83A; and R191A,R195A,K198A display
dramatically lower phosphorylation efficiency and 8-50-fold
higher K
values with the parent peptide,
symptomatic of reduced attitude to bind the peptide substrate as
compared with CK2 wild type. Such differences either disappear or are
attenuated if the mutants R191A,R195A, K198A; K79A,R80A,K83A; and
K74A,K75A,K76A,K77A are assayed with the peptides RRRADDSADDDD,
RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the
phosphorylation efficiencies of the other substituted peptides decrease
more markedly with these mutants than with CK2 wild type. These data
show that one or more of the basic residues clustered in the
191-198, 79-83, and 74-77 sequences are implicated in
the recognition of the acidic determinants at positions +1,
+3, and +4/+5, respectively, and that if these residues
are mutated, the relevance of the other acidic residues surrounding
serine is increased. In contrast the other two mutants, namely R228A
and R278A,K279A, R280A, display with all the peptides V
values higher than CK2 wild type,
counterbalanced however by somewhat higher K
values. It can be concluded from these data that all the five
mutations performed are compatible with the reconstitution of
tetrameric holoenzyme, but all of them influence the enzymatic
efficiency of CK2 to different extents. Although the basic residues
mutated in the 74-77, 79-83, and 191-198 sequences
are clearly implicated in substrate recognition by interacting with
acidic determinants at variable positions downstream from serine, the
other basic residues seem to play a more elusive and/or indirect role
in catalysis.
Protein kinase CK2, formerly termed casein kinase-2 (or -II), is
a ubiquitous Ser/Thr protein kinase normally composed by the tight
association of two catalytic ( and
`) and two non catalytic
subunits that appears to play a central albeit still enigmatic
role in cell regulation(1, 2) . The presence among the
myriad of its substrates of many proteins implicated in gene expression
and signal transduction (3) , the increase of CK2 activity in
transformed and proliferating tissues(4) , and development of
leukemias in transgenic mice transfected with CK2
subunits (5) suggest the involvement of CK2 in both normal and
uncontrolled cell proliferation. Though CK2 is endowed with basal
catalytic activity toward most of its substrates and by contrast to
previous reports, it seems not to be subjected to any kind of direct
regulation by growth factors(6) , its activity can be modulated
by polycationic effectors acting through its
subunit, which has
been shown to exert a dual function of positive as well as negative
regulation over the catalytic
subunit(7, 8, 9) . The negative effect of the
subunit, especially evident with some substrates exemplified by
calmodulin, is mediated by an acidic cluster located in the N-terminal
part of the molecule. This would imply the interaction with basic
residue(s) of the catalytic subunit. Other properties of CK2 would
imply the presence of crucially relevant basic residues in the
catalytic subunit, namely inhibition by heparin (10) and other
polyanionic compounds, like poly(Glu, Tyr)4:1 (11) and
substrate specificity. This latter is invariably determined by multiple
acidic residues located at positions between -2 and +5 (and
probably farther) relative to the target amino acid (mostly Ser and
rarely Thr)(12) . Heparin inhibition is reduced but not
abolished by mutations affecting lysyl residues 74/75 (13) and
75/76(14) . On the other hand the substitution of Asp for
His
, homologous to PKA (
)Glu
(interacting with Arg at position -2 in PKA
substrates)(15, 16, 17) , affects the
phosphorylation of peptide substrates whose recognition is partially
dependent on an acidic residue at position -2(18) . In
contrast the most powerful determinants of CK2 specificity normally are
acidic residues located downstream from serine, the ones at positions
+3 and +1 playing an especially crucial role, although they
have also been shown to be effective at more remote
positions(12, 19, 20) .
In order to
identify the basic residues responsible for substrate recognition,
inhibition, and intrinsic down-regulation, we have applied the
``charged-to-alanine'' scanning mutagenesis strategy (21) to a number of basic residues of the human subunit
that are conserved across various species but divergent from the
homologous residues of other protein kinases. Six such mutants in which
collectively 16 residues have been singly to quadruply mutated to
alanines have been obtained, and three of them have been shown to be
seriously defective in catalytic activity(18) . Here we
describe the purification of five of these mutants, either as such or
combined with the
subunit to give heterotetrameric holoenzymes,
and we analyze their kinetic properties with a set of peptide
substrates varying for the replacement of individual aspartyl residues
between positions -2 and +5 within the structure of the
reference peptide RRRADDSDDDDD.
In order to obtain CK2 holoenzymes with mutated
subunits, 1.5 g of bacteria pellets expressing mutated
subunits were resuspended and sonicated together with 1.5 g of bacteria
expressing the wild type
subunit in 30 ml of buffer A. The same
purification procedure was applied as for
subunit alone, but
after the phosphocellulose column a further purification step was
performed by pooling all the fractions containing both the
and
the subunits (as judged by SDS-PAGE) and subjecting them to MonoQ
fast protein liquid chromatography. The column was eluted with a linear
gradient from 0.1 to 1 M NaCl, and the eluent was analyzed by
OD monitor. The fractions in the OD peaks were assayed for CK2
activity, and the presence of the holoenzyme was assessed by 12%
SDS-PAGE showing both the
(mutated) and the
subunits. The
reconstituted enzyme was generally eluted from the column at a salt
concentration of 0.5-0.6 M (corresponding to the
prominent peak of both OD and catalytic activity). The fractions
containing the purified holoenzyme were pooled and dialyzed for
4-5 h against 5 mM Tris-HCl, pH 7.5, 50 µM phenylmethylsulfonyl fluoride, and 50% glycerol and stored at
-20 °C. The specific activities of CK2 holoenzymes were: CK2
wild type, 300 units/mg; K74A,K75A,K76A,K77A, 27 units/mg;
K79A,R80A,K83A, 93 units/mg; R191A,R195A,K198A, 40 units/mg; R228A, 450
units/mg; and R278A,K279A,R280A, 370 units/mg. One unit is defined as
the amount of enzyme transferring 1 nmol of phosphate to the peptide
substrate RRRADDSDDDDD per min under the experimental condition
detailed below.
Five mutants of CK2 subunit (22) have been
purified by submitting to phosphocellulose chromatography the extracts
of bacteria expressing the mutated
subunits (see
``Experimental Procedures''). The Coomassie-stained SDS-PAGE
gels of the final preparations are shown in Fig. 1A.
All mutants display a prominent 44-kDa band with the same mobility as
wild type. In several cases a doublet rather than a single band
is visible, probably indicative of limited proteolysis occurring during
the isolation and purification procedure. This conclusion is
corroborated by the finding that anti-
antibodies (see
``Experimental Procedures'') recognize not only the 44-kDa
band but also the ones with lower molecular masses (not shown).
Figure 1:
SDS-PAGE analysis of CK2 mutants. The
Coomassie-stained gels are shown. A, mutants of CK2
subunit after phosphocellulose purification. The arrow denotes
the position of wild type
subunit. The identification of mutated
subunits was also corroborated by immunoreaction with anti-
subunit antiserum (not shown). Also the minor bands with slightly
higher mobility immunoreacted, consistent with their identification as
proteolytic derivatives of the
subunit (see text). Lane
1, wild type-
subunit; lane 2, R228A; lane
3, R191A, R195A,K198A; lane 4, R278A,K279A,R280A; lane 5, K74A,K75A, K76A,K77A; lane 6, K79A,R80A,K83A. B, CK2 holoenzymes reconstituted with mutants of the
subunit and wild type
subunit and purified by phosphocellulose
chromatography and MonoQ fast protein liquid chromatography (see
``Experimental Procedures''). The arrows denote the
positions of wild type
and
subunits. Lane 1, wild
type CK2; lane 2, R228A; lane 3, R191195K198A; lane 4, K74A,K75A, K76A,K77A; lane 5,
R278A,K279A,R280A; lane 6,
K79A,R80A,K83A.
The
phosphotransferase activity of the purified mutants, either as such or
after addition of equimolar amounts of pure subunit, was
determined using the peptide substrate RRRAADSDDDDD(29) . As
shown in Table 1two mutants, R228A and R278A,K279A,R280A,
display an activity significantly higher to that of
w.t., whereas
the phosphorylation rate by the other mutants is much lower, being too
low for a reliable measurement in the case of mutant K79A,R80A,K83A.
Upon addition of equimolar amounts of
subunit, the catalytic
activity of
w.t. increases, as already observed(30) . A
similar or even higher increment of activity is observable adding
subunit to all the mutants. This also causes the appearance of
significant activity with the mutant whose activity is undetectable in
the absence of the
subunit.
These data would indicate that all
the mutants are still capable of associating with the subunit to
give the heterotetrameric holoenzyme. This conclusion was corroborated
by sucrose gradient ultracentrifugation experiments showing that the
addition of the
subunit causes a change in the sedimentation
coefficient similar to that induced by
w.t., consistent with the
reconstitution of
tetramers (Fig. 2).
Figure 2:
Sucrose density gradient
ultracentrifugation of CK2 holoenzymes reconstituted with variably
mutated subunits. Equimolar amounts of
subunit were
combined with 18 µg of
subunits prior to sucrose gradient
ultracentrifugation. Analysis of CK2 activity was done as described in (20) using casein (1 mg/ml) as substrate. CK2
w.t.
(
), K74A,K75A,K76A,K77A (
), K79A,R80A, K83A (
),
R191A,R195A,K198A (
), R228A (
), and R278A,K279A, R280A
(
). The arrows indicate the positions of wild type
subunit alone and reconstituted CK2 holoenzyme
(
2
2).
Once established that all the mutants are still
capable of associating with the subunit, a strategy was developed
for preparing mutated holoenzymes for sake of comparison with CK2 w.t.,
either recombinant or native. The most successful approach was to mix
together the bacteria expressing the mutated
subunit and those
expressing the wild type
subunit and to apply the normal
purification procedure of CK2 (see ``Experimental
Procedures''). In such a way all the five mutants could be
purified to near homogeneity as heterotetrameric holoenzymes, as judged
from both their SDS-PAGE Coomassie patterns, showing the
and the
subunits in approximately the same ratio as CK2 w.t. (Fig. 1B) and sucrose gradient ultracentrifugation
revealing peaks of activity with the same sedimentation coefficient as
CK2 w.t. (see Fig. 2). Heat denaturation curves, another
criterion for judging the reconstitution of normal CK2 holoenzyme that
is much more heat stable than the isolated
subunit(30) ,
are shown in Fig. 3. Four mutants exhibited heat stability
comparable with that of CK2 w.t. holoenzyme; the mutant K79A,R80A,K83A,
however, exhibited a reduced heat stability, suggesting that
susceptibility to protection by the
subunit is partially
compromised in it. All mutants displayed K
values
for ATP comparable with that of CK2 w.t. (17 µM) ranging
between 10 and 25 µM.
Figure 3:
Thermal stability of CK2 holoenzymes. The
catalytic activities of CK2 w.t. (), K74A,K75A,K76A,K77A
(
), K79A,R80A,K83A (
), R191A,R195A,K198A (
), R228A
(
), and R278A,K279A,R280A (
) were determined after
preincubation of 0.1 µg of each enzyme at 40 °C for the time
indicated. The samples were immediately ice-cooled, and the residual
activity was determined as described under under ``Experimental
Procedures.''
The kinetic constants of all the
CK2 mutants with the optimal peptide substrate RRRADDSDDDDD and with a
series of six peptide derivatives in which individual aspartic acids
have been replaced by alanines were determined and compared with CK2
w.t. The V and K
values as
well as the overall phosphorylation efficiencies expressed by the V
/K
ratios are summarized
in Table 2.
All the Asp Ala substitutions, with only
the exception of the one at position +2, are more or less
detrimental to the phosphorylation efficiency of the peptide substrates
by CK2 w.t.; two substitutions, however, are especially deleterious,
namely the ones at positions +3 and +1, both causing a
10-fold drop in phosphorylation efficiency, accounted for by both a
raise of K
and a decrease of V
.
An overall examination of the data of Table 2allows a rough subdivision of the mutants into two
categories: (i) mutants whose affinity for the parent peptide
(expressed by K) is only slightly decreased
(whereas the V
is actually increased) and whose
phosphorylation efficiency is altered by the structure of the peptide
substrate in a manner similar to that of CK2 w.t. (R228A and
R278A,K279A,R280A) and (ii) mutants whose affinity for the parent
peptide is substantially decreased and whose phosphorylation efficiency
is altered by modifications of the peptide substrate in a sharply
different manner as compared with CK2 w.t. (K74A,K75A,K76A,K77A; K79A,
R80A,K83A; and R191A,R195A,K198A).
In order to facilitate a comparative analysis, the relative efficiencies of CK2 w.t. and the mutants are represented in Fig. 4A as histograms normalized to the phosphorylation efficiencies of the parent peptide conventionally set equal to 1 for each mutant. It can be seen that although the profiles of mutants R228A and R278A,K279A,R280A are roughly superimposable to that of CK2 w.t., the histograms of mutants K74A, K75A,K76A,K77A; K79A,R80A,K83A; and R191A,R195A, K198A are dramatically altered. With the last mutant, e.g. the phosphorylation efficiency of the peptide lacking the acidic residue at position +1 (which is normally negligible as compared with the parent peptide) is actually the highest, surpassing by 3-fold that of the parent peptide. In contrast the relative phosphorylation efficiencies of the peptides with acidic gaps at all positions other than +1 are drastically reduced as compared with CK2 w.t. In the case of mutant K74A,K75A,K76A,K77A, the relative phosphorylation efficiencies of three peptides, with Ala for Asp substitutions at positions +3, +4/+5, and +2 are increased, whereas those of the other three peptide derivatives are decreased. The mutations occurring in K79A,R80A,K83A more specifically affect the phosphorylation of the peptide lacking the crucial acidic residue at position +3, whose phosphorylation efficiency is now comparable with that of the parent peptide, without dramatic alterations in the phosphorylation efficiency of peptides RRRADDSDADDD and RRRADDSDDDAA. In contrast the phosphorylation efficiency of peptides with acidic gaps at positions -1, +1, and -2 is drastically reduced.
Figure 4: Relative phosphorylation efficiencies of synthetic peptides by the wild type and mutated forms of CK2. The histograms have been constructed with data drawn from Table 2. The reference peptide, indicated by c (control), is RRRADDSDDDDD, and the other peptides are indicated by numbers (from -2 to +4/+5) denoting the position(s) relative to serine where aspartic acid has been replaced by alanine (see also Table 2). In A the phosphorylation efficiencies are normalized to that of the reference peptide (c) set equal to 1 for each form of CK2. Upper panel, CK2 wild type. Middle panels, mutants whose selectivity profiles are not markedly different from that of CK2 w.t. Lower panels, mutants whose selectivity profiles are deeply altered (see text). In B the mutants of the lower panels of A are further analyzed by expressing the phosphorylation efficiency of each peptide as a percentage of that of the same peptide phosphorylated by CK2 wild type.
The more specific effect of the mutation occurring in the 79-83 stretch as compared with that in the 74-77 stretch is highlighted if their phosphorylation efficiencies are expressed as percentages of the corresponding phosphorylation efficiency by CK2 w.t. (Fig. 4B). In these histograms the only outstanding efficiency bars with mutants K79A,R80A,K83A and R191A, R195A,K198A are those referring to the peptides RRRADDSDDADD and RRRADDSADDDD, respectively, whereas the outcome with mutant K74A,K75A,K76A,K77A is more promiscuous, with three peptides (having acidic gaps between +2 and +5) being represented by bars that surpass that of the parent peptide.
A summary of the mutations examined in this paper and of their effects on holoenzyme reconstitution, catalytic activity, substrate recognition, and susceptibility to polyanionic inhibitors (31) is reported in Table 3.
This paper describes the reconstitution, purification, and
kinetic characterization of five CK2 mutants in which the subunit
underwent substitution of basic residues with alanines.
Charged-to-alanine mutagenesis has been successfully used to identify
residues implicated in substrate recognition by other protein kinases,
namely PKA(15, 17, 21) , myosin light chain
kinase(32) , and phosphorylase kinase(33) . In all
these cases such residues were found to be acidic in nature consistent
with the knowledge that these kinases recognize basic specificity
determinants. These specificity determinants moreover are located
upstream from the phosphorylatable amino acid, notably at positions
-2 and -3. In contrast, the main specificity determinants
for CK2 are acidic residues located on the C-terminal side of the
target amino acid. Our strategy therefore was to mutate basic residues
that are conserved in CK2 from different species but are replaced by
nonbasic residues in other protein kinases with special reference to
the basophilic ones. Consequently the basic residues mutated by us
(listed in Table 3) are not homologous to residues (either acidic
or basic) mutated in previous studies and in particular in the
pioneering study of Gibbs and Zoller (21) in which all charged
residues of yeast PKA were mutated to alanine. Two of the residues
mutated in (21) , Cys
and Glu
, were
found to be implicated in ATP binding. Both residues are highly
conserved throughout the protein kinase family, CK2 included, and
therefore were not modified in our study. On the other hand none of our
mutations significantly modifies the K
for ATP nor
prevents the association with the
subunit to give tetrameric
holoenzyme. In one case, however, where Lys
,
Arg
, and Lys
were mutated into alanines, the
resulting holoenzyme displays a reduced heat stability as compared with
CK2 w.t.. This suggests that the interactions of this mutated
subunit with the
subunit, which is responsible for
thermostability(24) , are weakened. Also with this mutant,
however, the association with the
subunit promotes a severalfold
increase of basal activity with peptide substrate, apparently even
higher than that observed with CK2 w.t., although a precise evaluation
is hindered by the extremely low activity in the absence of the
subunit (see Table 1).
The kinetic constants of all the mutants with a set of seven peptides including the optimal substrate RRRADDSDDDDD (29) and its derivatives in which the aspartyl residues acting as specificity determinants have been variably replaced by alanine were calculated and analyzed. The main outcome of this study is that one or more of the basic residues replaced in three mutants, namely K74A,K75A,K76A,K77A; K79A,R80A,K83A; and R191A,R195A,K198A, are directly implicated in substrate recognition by interacting with definite acidic determinants of the peptide substrate.
In particular
it is clear that one or more of the basic residues substituted by Ala
in the mutant R191A,R195A,K198A are responsible for the recognition of
the acidic determinant at position +1. The substitution of
Asp(+1) with Ala in the peptide RRRADDSDDDDD in fact, which is one
of the most detrimental substitutions with CK2 w.t., does not decrease
but actually increases the phosphorylation efficiency by mutant
R191A,R195A,K198A. This conclusion is also in agreement with the
knowledge that the basic residues Arg, Arg
,
and Lys
are homologous to the PKA hydrophobic residues
Leu
, Pro
, and Leu
that
interact with the hydrophobic residue found in many PKA substrates at
position +1(34, 35) .
By similar arguments it
can be concluded that one or more of the basic residues
Lys, Arg
, and Lys
are
specifically implicated in the recognition of another crucial
specificity determinant, namely the acidic residue at position +3,
because the replacement of this residue is almost ineffective with
mutant K79A,R80A,K83A, whereas it is dramatically detrimental with CK2
w.t.
The case of the first part of the basic cluster 74-83 is
more complicated because the mutation of the four lysyl residues
74-77 gives rise to a mutant whose low phosphorylation efficiency
as compared with CK2 wild type can be improved in relative terms by the
substitution of all the aspartyl residues downstream from +1 (see Fig. 4B). It seems likely therefore that this basic
quartet contributes to the recognition of the determinant at position
+3 (together with the triplet Lys, Arg
,
and Lys
), but it also interacts with acidic residues
downstream from this that are conversely poorly interacting with
Lys
, Arg
, and Lys
. It should be
remembered in this connection that although our reference peptide stops
at position +5, acidic residues have been shown to act as
specificity determinants for CK2 at even more remote
positions(19, 20) . The different functions of the
first and second part of the 74-83 basic cluster is highlighted
by the finding, summarized in Table 3, that inhibition by heparin
is totally abolished by the 74-77 mutation while being unaffected
by the Lys
, Arg
, and Lys
mutation(31) .
The implication of the 74-83 region in substrate recognition by CK2 discloses a situation where the smaller lobe of protein kinases, committed with ATP binding and catalysis, also contributes to the interactions with the phosphoacceptor substrate. Up to now the residues responsible for these interactions had been identified almost exclusively in the larger lobe of PKA, with special reference to subdomains VIII, VI, and IX(16, 35, 36) . In contrast, the 74-83 basic stretch of CK2, based on the common architecture of protein kinases, would be located at the lower edge of the smaller lobe in the proximity of the cleft between the two lobes at the borderline between subdomains II and III.
Actually the whole inclusion of the
74-83 segment into subdomain III is an arbitrary consequence of
multiple amino acid sequence alignment of the catalytic domains of 75
Ser/Thr protein kinases(37) . In contrast, if CK2 is
manually aligned to PKA alone, the 74-79 sequence would fall into
subdomain II (see Table 4). An interesting outcome of such an
alignment, resting on higher similarity between CK2 and PKA, is the
homology between CK2 Lys
, implicated in the recognition of
the acidic determinant at position +3 (see above) and PKA
Lys
, representing a hinge between helices B and C, whose
side chain actually faces that of the aspartyl residue situated at
position +3 in the inhibitor peptide (protein kinase inhibitor)
bound to the catalytic subunit of PKA(16, 38) . It
seems likely therefore that this residue might play a general role in
the recognition of determinants at position +3. In agreement with
this conclusion is the observation that PKA Lys
is
invariably homologous to an acidic residue in the members of the
protein kinase C family, which are known to recognize basic
determinants at positions +2 and
+3(39, 40) . The members of the Cdc2/Cdk family,
moreover, which are know to select substrates with basic residues on
the C-terminal side of the crucial p+1
proline(40, 41) , display a series of acidic residues
clustered at the very end of subdomain II matching the basic
Lys
-Lys
-Lys
-Lys
cluster of CK2 if the manual alignment of Table 4is
followed.
Our kinetic data invariably show that whenever the
structural elements committed with the recognition of the crucial
acidic determinants at positions +1 and +3 are mutated, then
the relevance of the other acidic determinants that normally play a
subsidiary role is increased. This is especially true of the two acidic
residues located upstream from serine at positions -1 and, less
dramatically, -2; both these positions, which are relatively
unimportant with CK2 w.t., become crucial (especially the former) with
mutants R191A,R195A,K198A; K79A,R80A,K83A; and K74A,K75A,K76A,K77A.
These observations may also provide the structural basis accounting for
the efficient phosphorylation of ``atypical'' CK2 sites
lacking the acidic determinant at position +3 and in which the
presence of acidic residues at position -2 and even more at
position -1 is essential(42) . Using these atypical
peptide substrates, it was possible to show that CK2 His contributes to the recognition of the acidic determinant at
position -2(18) .
It should finally be noted that even
the two mutants that are almost indistinguishable from CK2 w.t. reveal
significant differences by the kinetic scrutiny of this work. Their
almost unchanged phosphorylation efficiency in fact results from
significantly and reproducibly higher V values
counterbalanced by higher K
values. The behavior
of mutant R228A is especially intriguing because this mutation also
dramatically increases CK2 sensitivity to inhibition by heparin (see Table 3). A possible interpretation is that heparin, besides
inhibiting CK2 by competing with some of the substrate binding elements
(namely the
Lys
-Lys
-Lys
-Lys
basic quartet and the p+1 loop) might also stimulate CK2
activity by interacting with Arg
. This would be
consistent with the finding that the mutation of the
Lys
-Lys
-Lys
-Lys
cluster not only suppresses inhibition by heparin but even
induces a stimulation by it(31) , as expected assuming the
existence of an up-regulating heparin binding site in CK2
subunit. Additional mutations are in progress in order to check these
possibilities and to identify the residues responsible for the
recognition of the determinants located upstream from serine and
downstream from position +5.