From the
Casein kinase II (CKII) is a protein serine/threonine kinase
known to control the activity of a variety of regulatory nuclear
proteins. This enzyme has a tetrameric structure composed of two
catalytic (
Casein kinase II (CKII)
CKII is a tetrameric
enzyme composed of two
In the present study, we have
demonstrated by immunoprecipitation using
It is a general feature, although not necessarily a universal
feature (i.e. no
At
present, distinct functions for CKII
We thank Sharon Rennie and Elzbieta Slominski for
helpful assistance during the course of this work and Dr. Michael Mowat
for synthesis of oligonucleotides and helpful discussions. We also
thank Dr. Stephen Elledge (Baylor College of Medicine) for providing
plasmids used in the two-hybrid studies.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and/or
`) subunits and two
subunits. We
have examined the subunit composition of tetrameric complexes of
purified bovine CKII by immunoprecipitation using
,
`, or
subunit-specific antibodies. These experiments indicate that the
enzyme can exist as homotetramers (i.e.
or
`
) as well as heterotetramers
(i.e.
`
). To further examine
subunit interactions between the
,
`, or
subunits of
CKII, we have utilized the yeast two-hybrid system (Fields, S. and
Song, O.(1989) Nature 340: 245-246). For these studies,
each subunit of human CKII was expressed in yeast as a fusion with the
DNA binding domain or with the transcriptional activation domain of the
yeast GAL4 transcriptional activator. These studies demonstrate that
the
or
` subunits of CKII can interact with the
subunits of CKII, but not with other
or
` subunits. By
comparison, the
subunits of CKII can interact with
,
`,
or
subunits. These results indicate that the CKII holoenzyme
forms because of the ability of
subunits to dimerize, bringing
two heterodimers (
or
`
) into a tetrameric complex.
(
)
is a protein
serine/threonine kinase that is ubiquitously distributed in mammalian
tissues and is likely to play an important role in the control of
proliferative events (for reviews, see Refs. 1-3). Although its
exact biological functions remain poorly understood, genetic studies in
Saccharomyces cerevisiae(4, 5) , in
Schizosaccharomyces pombe (6, 7), and in Dictyostelium
discoideum(8) all suggest that CKII is essential for
viability. Immunofluorscence localization studies have demonstrated
that the enzyme is localized within the nucleus and the cytoplasm of
cells
(9, 10) , observations that are consistent with the
demonstration that CKII phosphorylates a variety of proteins in each of
these compartments. Of particular relevance to a role in the regulation
of proliferation is the demonstration that CKII phosphorylates a host
of regulatory nuclear proteins including transcription factors such as
the serum response factor, c-Myc, N-Myc, c-Myb, c-Jun, and the tumor
suppressor p53 (reviewed in Refs. 3 and 11).
(and/or
`) subunits and two
subunits. The
and
` subunits contain all of the conserved
consensus motifs of protein kinase family members
(12) and have
kinase
activity
(13, 14, 15, 16, 17) .
The precise functions of the
subunit are not known, but
does appear to have a role in regulating the kinase activity of the
subunits
(18, 19, 20) . Although
enhances protein kinase activity exhibited by
toward most protein
substrates, it actually inhibits the phosphorylation of calmodulin
(21). Complementary DNAs encoding the
,
`, and
subunits
of CKII have been isolated from human and chicken cDNA
libraries
(13, 14, 22, 23, 24) .
These studies indicate that the
and
` subunits are closely
related proteins that are encoded by distinct genes. Between mammals
and birds, the
and
` subunits are very highly conserved with
the deduced amino acid sequences of human and chicken
and
`
exhibiting 98 and 97% identity, respectively. The
subunit of CKII
exhibits an even greater degree of conservation. Remarkably, the
deduced amino acid sequences of human and chicken
are identical
and differ from the deduced sequence of frog
by only a single
amino acid
(14, 22, 24, 25) .
Collectively, these studies suggest that the functional properties of
CKII are highly conserved throughout evolution. Furthermore, these
results obviously indicate that the tetrameric structure of CKII is
conserved between species.
- and
`-specific
antibodies that mammalian CKII exists as homotetramers
(
or
`
) and as heterotetramers
(
`
). Furthermore, using a yeast two-hybrid
system originally described by Fields and Song
(26) , we have
demonstrated that the
or
` subunits of CKII can interact
with the
subunits of CKII, but not with other
or
`
subunits. By comparison, the
subunits of CKII can interact with
,
`, or
subunits. These results indicate that the
formation of the CKII holoenzyme involves dimerization of
subunits to bring two
or
`
heterodimers into a
tetrameric complex.
Materials
CKII was purified from bovine
testis
(27) . Antisera specific to the ,
`, or
subunits of CKII were previously described
(28, 29) and
were affinity-purified on peptide affinity columns as described
elsewhere
(30) . The cDNAs encoding the human CKII
and CKII
` subunits were isolated from a human T-cell library
(13) .
The cDNA encoding the
subunit of human CKII was similarly
isolated from a human T-cell library and was a generous gift of Dr. F.
Lozeman and Dr. E. Krebs (University of Washington, Seattle). Each of
these cDNAs had been cloned into the BamHI site of pBluescript
(sk
) to make the following constructs
sk
/CKII
, sk
/CKII
`, and
sk
/CKII
. Plasmids encoding the GAL4 DNA binding
domain (pAS1-CYH2) or the GAL4 transcriptional activation domain
(pACTII) were obtained from Dr. Stephen Elledge (Baylor College of
Medicine, Houston, TX)
(31, 32) . Oligonucleotides were
synthesized on an Applied Biosystems model 380B DNA synthesizer by Dr.
Mike Mowat (Manitoba Institute of Cell Biology). Heat-killed,
formalin-fixed Staphylococcus aureus cells (Pansorbin) was
obtained from Calbiochem. The Bst sequencing kit for DNA sequencing was
purchased from Bio-Rad.
Immunoprecipitations
Purified CKII (0.25 µg)
was immunoprecipitated using CKII subunit-specific antibodies by
incubating the enzyme for 1 h on ice in phosphate-buffered saline with
affinity-purified antibodies (5 µg). Immune complexes were isolated
by the addition of 40 µl of Pansorbin (10% v/v) and further
incubation on ice for 30 min. Immunoprecipitates were collected by
centrifugation and were washed twice with radioimmune precipitation
buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate) and
once with 50 mM Tris-Cl, pH 7.5, prior to solubilization by
the addition of Laemmli
(33) sample buffer and incubation in a
boiling water bath for 3 min. Samples were subjected to
SDS-polyacrylamide gel electrophoresis
(33) followed by transfer
to nitrocellulose
(34) using transfer buffer (25 mM
Trizma (Tris base), 192 mM glycine, 20% methanol) containing
0.005% SDS. CKII subunits were detected using CKII subunit-specific
antiserum (each added at a concentration of 1:1000) followed by the
addition of I-protein A (1 µCi/ml, final
concentration) as described previously
(28) . After extensive
washing of the filter and drying, filters were subjected to
autoradiography. For quantitation, individual
I-containing bands were excised and analyzed by
counting.
Plasmid Constructs
To facilitate cloning of the
cDNAs encoding CKII ,
`, or
into the pAS1-CYH2 and
pACTII vectors, a BamHI site was introduced at the 5` end of
the coding region for each cDNA using the polymerase chain
reaction
(35) . A 718-base pair fragment of CKII
was
amplified from an sk
/CKII
template using the
following primers: 5` CGC GGG ATC CTG TCG GGA CCC GTG CCA 3` (sense
primer) and 5` ATC ATA ATT GTC ATG TCC ATG GAA 3` (antisense primer).
The fragment was digested with BamHI and NcoI and
used to replace a corresponding BamHI/NcoI fragment
of the sk
/CKII
construct. A 383-base pair
fragment of CKII
` was similarly amplified from an
sk
/CKII
` template using the following primers:
5` GCG GGG ATC CTG AGC AGC TCA GAG GAG 3` (sense primer) and 5` CAG GAT
CTG GTA GAG TTG C 3` (antisense primer). The fragment was then digested
with BamHI and PpuM1 and used to replace a corresponding
BamHI/PpuM1 fragment of sk
/CKII
`. A 673-base pair fragment containing the entire coding region of
CKII
was amplified from an sk
/CKII
template using the following primers to introduce BamHI sites
at each end of the cDNA: 5` GCG GGG ATC CTG AGC AGC TCA GAG GAG 3`
(sense primer) and 5` CGG GGG ATC CAG ACT GCA GGA CAG GTG 3` (antisense
primer). The fragment was digested with BamHI and cloned into
the BamHI site of sk
. The sequence of all
amplified fragments was confirmed by sequencing of double-stranded
Bluescript templates using the method of Sanger et
al.(36) , with a Bst sequencing kit. Each of the
cDNAs was subsequently cloned into the BamHI site of pAS1-CYH2
and of pACTII. The pAS1-CYH2/CKII
and pACTII/CKII
constructs each encode fusion proteins containing the entire CKII
with the exception of Met
which is lost through fusion to
the respective domains of GAL4. Similarly, the pAS1-CYH2/CKII
`
and pACTII/CKII
` constructs each encode fusion proteins
containing the complete sequence of CKII
` beginning at
Pro
. The entire sequence of CKII
starting with
Ser
is contained within the fusion proteins that are
encoded by the pAS1-CYH2/CKII
and pACTII/CKII
plasmids. All
pAS1-CYH2 and pACTII plasmids were routinely maintained in
Escherichia coli DH5
.
Transformation and Maintenance of Yeast
Yeast
strains were grown and manipulated according to standard
methods
(37) . For two-hybrid experiments, the yeast strain
DGY63::171 (MAT
a, ade2 trp1-901 leu2-3, 112
his3-
200 gal4
gal80
::pRY171[GAL1
lacZ](38) was
transformed with equal quantities of various pAS1-CYH2 and pACTII
plasmids using the highly efficient method of Gietz et al. (39). Transformants were selected on synthetic complete minus
Trp-Leu plates at 30 °C and grown for 3-5 days. Colonies were
then replicated onto synthetic complete minus Trp-Leu plates containing
50 mM KHPO
, 2% sucrose as a carbon source, and 1
mg/ml 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside
(X-gal) to visualize colonies expressing
-galactosidase.
Measurement of
Assays
for -Galactosidase Activity
-galactosidase activity were performed essentially as
described previously
(40) . Briefly, yeast were grown on
synthetic complete minus Trp-Leu plates for 3-5 days at 30
°C. Several colonies from each plate were scraped from the plate
with an inoculating loop, transferred to 1 ml of distilled water and
thoroughly mixed. OD
measurements were taken so that
-galactosidase activities could be normalized according to the
density of yeast cells. An aliquot (200 µl) of each suspension of
yeast was transferred to a microcentrifuge tube and centrifuged briefly
to pellet the yeast cells. After removing the supernatant, the cells
were resuspended in 500 µl of Z buffer (100 mM
NaPO
, pH 7.0, 10 mM KCl, 1 mM
MgSO
, 38 mM
-mercaptoethanol). A 50-µl
aliquot of 0.1% SDS was added, and the cells were vortexed vigorously
for 15 s. Chloroform (50 µl) was then added, and the cells were
vortexed for an additional 15 s. Assays for
-galactosidase were
immediately initiated by the addition of 100 µl of 4 mg/ml
o-nitrophenyl
-D-galactopyranoside. Following
incubation for 6 min at 37 °C, reactions were terminated by the
addition of 500 µl of 1 M Na
CO
.
Reaction mixtures were centrifuged for 1 min, and the OD
was measured.
-galactosidase activities in Miller units were
calculated according to the following formula: units =
OD
1000/(volume)(time)(OD
)
(41) .
Immunoprecipitation of CKII with Subunit-specific
Antibodies
CKII is a tetrameric enzyme composed of two catalytic
( and/or
`) subunits and two
subunits
(13, 14) . As we had previously
noted
(27) , purified bovine testis CKII consists of comparable
amounts of
and
` subunits (Fig. 1A). Direct
sequence analysis and molecular cloning studies have demonstrated that
CKII
and CKII
` are very closely related proteins that have
distinct carboxyl-terminal domains (13, 14, 27). Antipeptide antibodies
raised against carboxyl-terminal peptides of CKII
or CKII
`
do not exhibit any cross-reactivity on immunoblots
(Fig. 1B, lanes 1-4). Similarly,
antibodies directed against
do not react with
or
`
subunits (Fig. 1B, lanes 5 and 6). On
immunoblots the anti-
antisera recognizes CKII
more
efficiently than anti-
` antisera recognizes its respective antigen
(compare lanes 1 and 2 with lanes 3 and
4).
Figure 1:
Electrophoretic and immunoblot analysis
of purified CKII. A, purified bovine testis CKII (2 µg)
was subjected to electrophoresis on a 12% SDS-polyacrylamide gel and
visualized by staining with Coomassie Blue. The (M
45,000),
` (M
40,000), and
subunits (M
26,000) of CKII are marked on the
right side of the lane (from top to bottom).
B, purified CKII (0.05 µg, lanes 1, 3,
and 5; 0.25 µg, lanes 2, 4, and
6) was subjected to SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose. Individual lanes were incubated with
antipeptide antiserum specific for the
(lanes 1 and
2),
` (lanes 3 and 4), or
(lanes 5 and 6) subunits of CKII. Each antiserum was
utilized at a dilution of 1:1000. Immune complexes were detected with
I-protein A and visualized by autoradiography as
described under ``Experimental Procedures.'' The positions of
CKII
, CKII
`, and CKII
are indicated as in Panel
A.
To determine whether CKII and CKII
` exist
in the same tetrameric complex, we subjected purified bovine testis
CKII to immunoprecipitation using these subunit-specific antibodies
(Fig. 2). In an initial round of immunoprecipitation
(Fig. 2A), purified CKII was immunoprecipitated with
antibodies directed against CKII
, CKII
`, or CKII
,
respectively. Qualitatively, all immunoprecipitations are similar since
,
`, and
are present in each immunoprecipitate. This
result indicates that some of the CKII exists in a heterotetrameric
complex composed of one
subunit, one
` subunit, and two
subunits. However, there are significant quantitative differences
between the immunoprecipitates obtained with the different antibodies.
All subunits of the purified enzyme are quantitatively
immunoprecipitated with anti-
antibodies (Fig. 2A,
lane 9) since none of any of the subunits can be detected in
the immunoprecipitation supernatant (Fig. 2C, lane
10). The quantitative immunoprecipitation of CKII with anti-
antibodies confirms that the tetrameric complex was intact under our
immunoprecipitation conditions. Quantitation of the
I
content resulting from the presence of
I-protein A
attached to immune complexes for the
and
` bands indicates
that the relative ratio of
:
` is 2.4:1 for anti-
immunoprecipitates. Since comparable amounts of
and
` are
present in the testis enzyme (Fig. 1), it is apparent from this
experiment that the antipeptide antibodies raised against
`
recognize
` less efficiently than antibodies directed against
recognize
. By comparison with anti-
immunoprecipitations, all subunits are not quantitatively isolated when
immunoprecipitates are performed with anti-
or with anti-
`
antibodies (Fig. 2A, lanes 1-8).
Furthermore, alterations in the relative intensity of CKII
to
CKII
` are observed when the different antibodies are used for
immunoprecipitation. While the amount of
that is isolated by
immunoprecipitation using
or
antibodies is very similar
(Fig. 2A, lanes 1-4 compared with
lane 9), significantly less
is isolated using
anti-
` antibodies. Also, the signals observed for
and
`
in immunoprecipitates using anti-
` antibodies are very similar
(Fig. 2A, lanes 5-8). However, when it is
considered that
` is detected less efficiently than
, this
result indicates that
` is more abundant than
in
immunoprecipitates performed with anti-
` antibodies. The
:
` ratio is 5.3:1 for anti-
immunoprecipitates and is
only 0.67:1 for anti-
` immunoprecipitates. These data reflect an
enrichment of
relative to
` in anti-
immunoprecipitates
and a corresponding enrichment of
` relative to
in
anti-
` immunoprecipitates. Collectively, these results indicate
that some of the purified CKII is composed of homotetramers (i.e.
and
`
).
Analysis of material isolated in a second round of immunoprecipitation
supports this conclusion (Fig. 2B) as does the analysis
of supernatants obtained following one or two rounds of
immunoprecipitation (Fig. 2C).
Figure 2:
Immunoprecipitation of purified CKII with
subunit-specific antipeptide antibodies. A, pPurified CKII
(0.25 µg) was immunoprecipitated using anti- antibodies
(lanes 1-4), anti-
` antibodies (lanes
5-8), or anti-
antibodies (lane 9).
Immunoprecipitates were electrophoresed on SDS-polyacrylamide gels and
analyzed by immunoblot analysis using a mixture of anti-
,
anti-
`, and anti-
antiserum each at a dilution of 1:1000.
Immune complexes were detected with
I-protein A and
visualized by autoradiography. B, supernatants obtained
following immunoprecipitation with either anti-
antibodies
(lanes 1-3) or anti-
` antibodies (lanes
4-6) were subjected to a second round of immunoprecipitation
with anti-
antibodies (lanes 1 and 4),
anti-
` antibodies (lanes 2 and 5), or anti-
antibodies (lanes 3 and 6). Immunoprecipitates were
analyzed by immunoblotting as in Panel A. Purified CKII was
also analyzed (lane 7, 0.25 µg; lane 8, 0.125
µg). C, supernatants obtained following one or two rounds
of immunoprecipitation were subjected to electrophoresis and analyzed
by immunoblotting as in Panels A and B. As a
standard, purified CKII (0.25 µg) is also analyzed (lane
1). Supernatants are as follows: lane 2, one round of
immunoprecipitation with anti-
; lane 3, two rounds of
immunoprecipitation with anti-
; lane 4,
immunoprecipitation with anti-
followed by anti-
`; lane
5, immunoprecipitation with anti-
followed by anti-
;
lane 6, one round of immunoprecipitation with anti-
`;
lane 7, immunoprecipitation with anti-
` followed by
anti-
; lane 8, two rounds of immunoprecipitation with
anti-
`; lane 9, immunoprecipitation with anti-
`
followed by anti-
; lane 10; one round of
immunoprecipitation with anti-
. The positions of the
,
`, and
subunits of CKII are marked as in Fig.
1.
Supernatants obtained
following the first round of immunoprecipitation were subjected to a
second round of immunoprecipitation using anti-, anti-
`, or
anti-
antibodies (Fig. 2B) or were analyzed
directly on immunoblots together with supernatants obtained following
two rounds of immunoprecipitation (Fig. 2C). When the
supernatant from an anti-
` immunoprecipitate is subjected to
immunoprecipitation with anti-
(Fig. 2B, lane
4), the immunoprecipitate contains primarily
and
subunits. The
:
` ratio of this immunoprecipitate based on
I content is 5.9:1. Similarly, the supernatant obtained
following two rounds of immunoprecipitation with anti-
`
(Fig. 2C, lane 8) demonstrates that
is
much more abundant than
` (
:
` ratio of 7.5:1). A
corresponding enrichment of
` relative to
is observed in the
supernatant obtained following one or two rounds of anti-
immunoprecipitation (Fig. 2C, lane 3;
:
` ratio of 0.3:1) and when the supernatant from an
anti-
immunoprecipitation is subjected to immunoprecipitation with
anti-
` in the second round (Fig. 2B, lane
2;
:
` ratio of 0.1:1.0).
Analysis of Subunit Interactions with the Two-hybrid
System
To examine subunit interactions between the various
subunits of CKII, we utilized the yeast two-hybrid system developed by
Fields and Song
(26) . For these experiments, cDNAs encoding the
,
`, or
subunits of human CKII were each ligated into
the pAS1-CYH2 and pACTII vectors described by Durfee et al.(31) in order to express each subunit as a fusion with the DNA
binding domain (pAS1-CYH2) or with the transcriptional activation
domain (pACTII) of the yeast GAL4 transcriptional activator. The yeast
strain DGY63::171, which expresses
-galactosidase under the
control of the GAL1 promoter that is regulated by the GAL4
transcription factor was then cotransformed with different combinations
of pAS1-CYH2 and pACTII constructs. Initial experiments were performed
to verify that interactions between fusion proteins containing CKII
(or CKII
`) and CKII
could be detected in yeast and to
determine whether any of the DNA-binding domain fusions encoded by
pAS-CYH2/CKII
, pAS1-CYH2/CKII
`, pAS1-CYH2/CKII
nonspecifically stimulated
-galactosidase expression. Interactions
between hybrid proteins are indicated when yeast that are grown on
media containing X-gal turn blue because they express
-galactosidase. Yeast that had been cotransformed with
combinations of
(or
`) and
hybrids turned blue when
grown in the presence of X-gal, indicating that interactions between
the CKII subunits can be detected with the two-hybrid system (data not
shown). Importantly,
-galactosidase expression was not evident in
those yeast expressing only one of the CKII subunit hybrids
demonstrating that none of the CKII subunit hybrids can stimulate
transcription in the absence of a binding partner (data not shown). To
assess interactions between all of the subunits of CKII, yeast were
cotransformed with all combinations of pAS1-CYH2 and pACTII constructs
encoding each of the CKII subunits (). An indication of
interaction between the various fusion proteins was obtained by
measuring
-galactosidase activity in extracts prepared from
various transformants. All transformants obtained using various
combinations of
subunits together with either
or
`
subunits express significant amounts of
-galactosidase activity.
The highest
-galactosidase expression is observed when GAL4 DNA
binding hybrids of the
subunit are cotransformed with GAL4
transcriptional activation hybrids of either
or
`. Less
significant
-galactosidase expression is observed when these
hybrid combinations are reversed such that the GAL4 DNA binding hybrids
of
or
` are cotransformed with GAL4 transcriptional
activation hybrids of
. Although we do not know why different
levels of
-galactosidase expression are achieved when the hybrid
combinations are reversed, these results provide as expected, a clear
indication that the
or
` subunits of CKII interact with the
subunit of CKII in the two hybrid system. Interestingly, when
yeast are cotransformed with the
subunit hybrid of the GAL4 DNA
binding domain together with the
subunit hybrid of the GAL4
transcriptional activation domain, measurements of
-galactosidase
expression are comparable to those observed when the GAL4
transcriptional activation hybrid of
is cotransformed into yeast
with the GAL4 DNA binding domain hybrids of
or
`. By
comparison, minimal
-galactosidase activity was expressed in yeast
transformed with various combinations of hybrids encoding
and/or
`. Of these combinations, only yeast cotransformed with GAL4 DNA
binding domain and GAL4 transcriptional activation domain hybrids of
expressed measureable activity. However, the activity of this
combination was more than 20-fold less than observed for any of the
combinations involving hybrids of
.
subunit has been identified in
Dictyostelium)
(8) , that CKII is a tetrameric enzyme
composed of two
subunits and two
and/or
` subunits.
Data obtained in this study using the yeast two-hybrid system indicate
that the
subunit can interact with another
subunit,
suggesting that the tetrameric CKII complex forms through association
between
subunits which brings two
and/or
`
heterodimers into a tetrameric complex
(Fig. 3). A low level of
-galactosidase activity is observed
in yeast cells cotransformed with GAL4 DNA binding domain fusions of
CKII
and with GAL4 transcriptional activation domain fusions of
CKII
. However, it is unlikely that
interactions
are responsible for tetramer formation since no activity is observed
when any other combinations of CKII
or CKII
` are analyzed.
If one
subunit were unable to interact with another
subunit, we would predict that CKII would exist as a heterodimer rather
than a tetramer.
Figure 3:
Model of
tetrameric CKII complexes. Immunoprecipitations indicate that CKII
exists in homotetrameric complexes ( and
`
) and in heterotetrameric
complexes (
`
). Results obtained with the
yeast two-hybrid system indicate that tetrameric CKII complexes form
through interactions of
subunits.
We have previously demonstrated that the bulk of
newly synthesized subunit is rapidly incorporated into complexes
with
subunits
(42) . By comparison, the
subunit is
synthesized in excess of
and incorporates slowly with
.
These observations imply that CKII complex formation would be governed
by the availability of
subunit. From data presented here, it
appears that formation of the tetrameric CKII complex is mediated
through the
subunit and that there are only modest differences
(i.e. approximately 2-fold) in the interactions of the
or
` subunits of CKII with
. Therefore, the incorporation of
or
` subunits into the tetramer would be determined by the
availability of the respective proteins. We do not know whether the
assembly of tetrameric CKII is an ordered process. However, since the
two hybrid data indicates that
subunits can interact directly,
presumably in the absence of
subunits, then formation of
dimers could precede incorporation of
or
` subunits.
and CKII
` remain
poorly defined. The intracellular localization of the two isozymic
forms has been examined by immunofluorescence
techniques
(9, 10) . In chicken DU249 cells, Krek et
al.(10) suggest that there is a great deal of overlap
between the localization of
and
` and that both isozymes are
predominantly nuclear. By comparison, Yu et al. (9) suggest
that there are dramatic differences between the localization of CKII
and CKII
`. Discrepancies between the two studies are
difficult to resolve, but may result from the different experimental
systems under examination. The results presented in this study
demonstrate that bovine testis CKII exists in homotetrameric complexes
(
,
`
) and in heterotetrameric complexes
(
`
) (see Fig. 3). We have previously
noted that CKII
` is much less prominent in other cell lines than
it is in testis
(28, 42) . Therefore, it is possible
that, in systems in which
` is less abundant than
,
` is
primarily in heterotetrameric (
`
) complexes.
By comparison, in systems such as testis, where
` is as abundant
as
, then
` also exists in homotetrameric
`
complexes. Although we do not know
the tetrameric composition of the CKII under examination in the studies
or Krek et al.(10) or Yu et al.(9) ,
differences in the tetrameric composition of the CKII could determine
whether or not
and
` are actually colocalized. Clearly, it
will be of interest to examine the localization of
and
` in
a cell system in which CKII
` is abundant and a significant
proportion of the enzyme is in homotetrameric
`
complexes. A systematic analysis of
the unique properties of CKII
and CKII
` will undoubtedly
yield a more complete understanding of the functions of the different
tetrameric CKII complexes.
Table:
Transcriptional activation by hybrid
proteins
-D-galactoside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.