(Received for publication, October 25, 1994; and in revised form, January 17, 1995)
From the
Protein kinase CKII (formerly casein kinase II) can be isolated
as a heterotetramer, containing two catalytic ( or
`) and two
regulatory (
) subunits. We have characterized the forms of CKII in
HeLa cells using antibodies specific for the
or
` subunits.
Following metabolic labeling with [
S]methionine,
whole cell soluble extracts were analyzed by immunoprecipitation and
gel electrophoresis. Both
and
` coprecipitate with
and
with each other. However, when extracts are depleted of
, a pool
of CKII containing only
` and
is identified. Similarly,
depletion of
` revealed a pool exclusively of
and
.
Therefore, we propose that there are three distinct isoforms of CKII
within HeLa cells with different catalytic subunit stoichiometries
(
,
`
, and
`
). With our immunodepletion
procedure we have characterized the isoforms by activity analysis,
turnover of pulse-labeled subunits, and by localization in subcellular
fractions obtained from labeled cells. We have also analyzed complex
formation between the catalytic and regulatory subunits by examining
the differences in the rate of signal incorporation into subunits in
immunoprecipitates obtained from continuously labeled and pulse-labeled
cells. We have found that the
and
`
isoforms assemble relatively slowly
(12-16 h), whereas complex formation of the
`
isoform occurs more rapidly
(2-4 h). Analysis of isoform complex formation in subcellular
fractions from pulse-labeled cells revealed that the majority of
nuclear CKII is assembled in the nucleus from free catalytic and
regulatory subunit polypeptides.
Protein kinase CKII ()(casein kinase II) is a
ubiquitous protein serine/threonine kinase present in all eukaryotic
cells (for reviews, see Hathaway and Traugh, 1982; Edelman et
al., 1987; Tuazon and Traugh, 1991). In mammalian cells, CKII has
been implicated in signal transduction pathways, and increased kinase
activity has been observed in a variety of cell types after serum
(Carroll and Marshak, 1989) or growth factor stimulation (Sommercorn
and Krebs, 1987; Klarlund and Czech, 1988; Ackerman and Osheroff, 1989;
DeBenedette and Snow, 1991; for review, see Issinger, 1993). Also,
mitogenic stimulation of quiescent fibroblasts can be reduced or
eliminated by antisense oligonucleotides complementary to CKII mRNAs or
by microinjection of CKII antibodies, indicating that CKII is necessary
for cell cycle progression (Pepperkok et al., 1991, 1994;
Lorenz et al., 1993). The observations that p34
kinase and CKII are targets for one another and that
phosphorylations are cell cycle-dependent further imply a regulatory
role for CKII in the cell cycle (Russo et al., 1992;
Litchfield et al., 1992). Consistent with this has been the
identification of a number of nuclear substrates, including oncogene
products such as c-myc and c-myb; transcription
factors including SRF, c-erbA, c-jun, Max, CREB; and
the tumor suppressor p53 (Lüscher et al. 1989, 1990; Manak et al., 1990; Glineur et al.,
1989; Baker et al., 1992; Lin et al., 1992; Berberich
and Cole, 1992; Lee et al., 1990; Meek et al., 1990).
However, in most cases, the exact function of the phosphorylation is
not known.
In most organisms and tissue types, it has been proposed
that there are two forms of the enzyme of heterotetrameric subunit
stoichiometry: and
`
(for review, see Pinna, 1990). Subunits
and
` are thought to be catalytic based on homology to
conserved subdomains in other kinases (Lozeman et al., 1990;
for review, see Hanks et al., 1988). It has been shown by in vitro reconstitution studies that the
and
`
subunits have phosphotransferase activity in the absence of the
subunit (Cochet and Chambez, 1983; Hu and Rubin, 1990; Jakobi and
Traugh, 1992) and in vivo, by overexpression of subunits in
mammalian and insect cells, that the
subunit enhances activity of
the catalytic subunit 5-10-fold (Grankowski et al.,
1991; Filhol et al., 1991; Heller-Harrison and Czech, 1991).
In addition to activity characterization, the subcellular distribution of CKII in mammalian cells has also been investigated. CKII has been shown to be present both in the nucleus and cytosol in all tissue types examined to date (Inoue et al., 1984; Meggio and Pinna, 1984; Filhol et al., 1990a) and cultured cell lines (Yu et al., 1991; Pepperkok et al., 1991; Serrano et al., 1987). It has also been found in mitochondrial and microsomal fractions (Edelman et al., 1987; Tuazon and Traugh, 1991) and has been identified as an extracellular matrix-associated kinase or ecto kinase (Kübler et al., 1983). It is described as having mainly nuclear localization, although the cytosolic to nuclear protein ratio may depend on the cellular growth state (Filhol et al., 1990a; Lorenz et al., 1993). The widespread dissemination of CKII in a cell and its association with different cellular proteins such as HSP90 (Miyata and Yahara, 1992) and p53 (Filhol et al., 1992) and heterochromatin (Filhol et al., 1990b), suggest that the enzyme exists in a number of pools or states in a cell and that different enzyme pools may have different activity or function. However, it is important to distinguish between identification of different enzyme pools based on criteria such as localization and cellular protein association, and identification of different forms of the enzyme based on subunit stoichiometry.
In both yeast and higher
organisms, the and
` subunits are encoded by separate genes
and are highly homologous to each other, sharing 85% amino acid
identity in human (Lozeman et al., 1990; Padmanabha et
al., 1990). However, it is not known if there are biochemical
differences between the catalytic subunits. Furthermore, to date it has
not been possible to determine isoform composition for a preparation of
CKII; SDS-PAGE and sedimentation velocity analysis indicate only the
presence of subunits (typically, major
and
bands with a
minor abundance
` band are seen), and polypeptide association
results in the formation of tetrameric complex(es) (for review, see
Pinna, 1990). There is evidence that different tissue types may express
the catalytic subunits at varying levels (for reviews, see Tuazon and
Traugh, 1991; Issinger, 1993); however, if all three subunits are
present simultaneously, it is not possible to tell which forms of CKII
are present.
In this report we describe an immunodepletion assay using catalytic subunit specific antibodies that allowed us to identify CKII isoforms in HeLa cells. Using this immunoprecipitation technique enabled us to engage in isoform characterization, in which we set out to determine if there were any biochemical differences between isoforms based on turnover, subcellular localization, and complex formation. Our results indicate that marked isoform-specific differences exist in the rate of complex formation between newly synthesized polypeptides.
Antibody to CKII was made as described (Yu et al., 1991). To immunoprecipitate the
subunit with
antiserum, denaturation of extracts was required. Labeled
extracts were heated to 90 °C for 15 min in lysis buffer with SDS
added to a final concentration of 1%. After heating, SDS-containing
extracts were diluted 10-fold in 50 mM Tris-Cl, pH 7.5, 50
mM NaCl, and either
antiserum alone was added, or a
combination of
,
`, and
antisera was added to the
immunoprecipitation. All subsequent steps in the immunoprecipitation
prior to gel electrophoresis were carried out as described above.
For immunoblot analysis, proteins were
transferred electrophoretically to nitrocellulose filters (Schleicher
& Schuell) and blocked in 1% polyvinylpyrrolidone. A combination of
subunit antiserum and rabbit antiserum anti-CSH124 representing a
common internal epitope between
and
` (Yu et al.,
1991), diluted at 1:500, was used as primary antibody. Donkey
anti-rabbit immunoglobulin, F(ab`)
horseradish
peroxidase-conjugated and diluted at 1:2,000 was used as secondary
antibody. Signal detection was by enhanced chemiluminescence (ECL;
Amersham) with x-ray film (Kodak) exposure.
Figure 1:
Immunoprecipitation of CKII from HeLa
cells using catalytic subunit-specific antibodies. CKII was
immunoprecipitated from unlabeled cells or cells labeled with
[S]methionine for 6 h. Whole cell extracts were
prepared and divided so that equal protein was used for
immunoprecipitation reactions with either nonimmune sera (NRS, lane 1),
subunit antiserum (lane 2), or
`
subunit antiserum (lane 3). Panel A, autoradiogram of
SDS-PAGE of immunoprecipitates from labeled extract. Panel B,
immunoblot of immunoprecipitates from unlabeled extract. Cross-linked
antibodies were used for three immunoprecipitation reactions (lanes
1-3). After electrophoresis and transfer to nitrocellulose,
the blot was probed simultaneously with a combination of anti-
and
`, and anti-
antibodies. Molecular mass standards are
indicated (kDa). The positions of CKII
,
`, and
subunits are shown in panels A and B. Panel B also contains immunoglobulin heavy chain (arrows).
In addition to
including a negative control for the immunoprecipitation in each of the
two types of immunoprecipitation procedures, we also performed two
additional experiments to assay for specificity of the two catalytic
subunit antibodies. In one experiment, dicistronic bacterial expression
strains that expressed and
, or
` and
subunits
together (dicistronic recombinant plasmid was a gift of Joan Brooks at
New England Biolabs) were used to test for catalytic subunit antibody
cross-reactivity. When extracts from strains that express
simultaneously either
and
subunits, or
` and
together, were used for immunoprecipitation, we determined that it was
only possible to precipitate
and
with
antiserum, and
to precipitate
` and
with
` antiserum (data not shown).
In an additional experiment, it was determined that the two peptides
used to raise
and
` antisera blocked corresponding HeLa cell
extract immunoprecipitation reactions, and peptide to
did not
block precipitation of
` with
` antibody, nor did peptide to
` block precipitation of
with
antibody (data not
shown).
Figure 2:
Immunoprecipitation of CKII isoforms by
immunodepletion assay. Whole cell extracts were prepared from unlabeled
cells or cells labeled with [S]methionine for 6
h, and equal protein was used in each of seven two-step
immunoprecipitation reactions. The first immunoprecipitation reactions (1st IP) represent the immunodepletion step carried out with
either nonimmune (NRS, lanes 1, 2, and 5), anti-
(lanes 3 and 4), or
anti-
` (lanes 6 and 7) antibodies. The second
immunoprecipitation reactions (2nd IP) were performed with
supernatants from the first immunoprecipitation reactions (cleared
extracts) and were carried out with either nonimmune (lane 1),
anti-
(lanes 2, 3, and 7), or
` (lanes 4, 5, and 6) antibodies and subjected
to electrophoresis. Panel A, autoradiogram of second
immunoprecipitation reactions from labeled immunodepleted extracts (2nd IP). Panel B, immunoblot of second
immunoprecipitation reactions from unlabeled immunodepleted extracts
probed with anti-
and
`, and anti-
antibodies. Both
immunoprecipitation steps were performed with cross-linked antibodies.
Molecular mass standards are indicated (kDa). The positions of CKII
,
`, and
subunits are indicated in panels A and B. Panel B also contains immunoglobulin
heavy chain (arrows).
The
[S]methionine label result was verified by
performing a similar immunodepletion experiment with unlabeled extract
and cross-linked antibodies. Equal amounts of extract were divided into
seven immunoprecipitation reactions, and three consecutive clearing
steps were performed using cross-linked antibodies. Products from
immunoprecipitations with alternate catalytic subunit antibodies were
subjected to electrophoresis, transferred to nitrocellulose, and probed
with antisera in a manner identical to the experiment in Fig. 1B. The immunoblot in Fig. 2B shows a result similar to that obtained with
[
S]methionine-labeled lysates. The
and
subunits coimmunoprecipitated in the absence of the
`
subunit (lane 7), and free
` subunit was detected,
independent of the
subunit (lane 4). In this reaction,
the presence of small amounts of contaminating IgG light chain that
eluted from the protein A beads and migrated in the same region of the
gel rendered detection of
subunit difficult.
Figure 3:
Activity assay of isoforms associated with
immunodepleted immunoprecipitation reactions. Whole cell extracts were
prepared from unlabeled cells, and 0.5 mg of protein in the extract was
used in each of seven two-step immunoprecipitation reactions. The first
immunoprecipitation reactions were carried out with either nonimmune,
anti-, or anti-
` antibodies (1st IP). Cleared
extracts from the first immunoprecipitation reactions were used in the
second immunoprecipitation reactions with either nonimmune (NRS), anti-
, or anti-
` antibodies (2nd
IP). Protein A bead immune complexes from the second
immunoprecipitation reactions were the source of enzyme in the activity
assay using two synthetic peptide substrates (see ``Materials and
Methods''). Panel A, activity assay performed with
RRRDDDSDDD peptide. Panel B, assay performed with RRREEETEEE
peptide using a second aliquot of protein A beads. Activity was
calculated as pmol of phosphate incorporated/min/mg of whole cell
extract. Activities associated with immunoprecipitates arise from
single isoforms or isoforms in combination and are indicated by symbols in A and B.
To compare isoform specific
activities, the relative abundance of each isoform was calculated from
the subunit signal associated with isoforms immunoprecipitated
from HeLa cells radiolabeled for 12 h with
[
S]methionine. The
subunit signal (PSL
units) for each isoform was calculated as a percentage of the total
signal obtained with the three isoforms and compared with the
isoform relative activity values shown in Table 1. Comparison of
columns 2 and 3 in Table 1indicates that the isoform activity
contributions closely reflected isoform abundance and suggests that
under our activity assay conditions there were no significant
differences in the specific activities of isoforms.
Figure 4:
Immunodepletion analysis of CKII from
subcellular fractions. Subcellular fractions were prepared from
unlabeled and [S]methionine-labeled cells and
analyzed by immunodepletion assay. For labeled extracts, 0.2 mg of
protein was used in each of four two-step immunoprecipitation reactions
for both cytosolic and nuclear fractions. Antibodies used to clear
extracts are indicated (1st IP), and products from second
immunoprecipitation reactions with cleared extracts (2nd IP)
were subjected to electrophoresis and autoradiography (panels C and D). For unlabeled extracts, 0.1 mg of protein was
used in each of seven two-step immunoprecipitation reactions for both
cytosolic and nuclear fractions. Antibodies used for first
immunoprecipitation reactions are indicated (1st IP). Activity
assay was performed with synthetic peptide substrate RRRDDDSDDD using
protein A bead immune complexes from the second immunoprecipitation
reaction as the source of enzyme (2nd IP). Relative activities
associated with immunoprecipitates are plotted (cpm) and arise
from single isoforms or isoforms in combination; they are indicated by symbols, for cytosolic (panel A), and nuclear (panel B) fractions. Autoradiograms of SDS-PAGE for
S-labeled immunoprecipitates that correspond (connecting lines) to activity assay immunoprecipitates are
indicated for cytosolic (panel C, lanes 1-4)
and nuclear (panel D, lanes 1-4) fractions.
Molecular mass standards are indicated (kDa). The positions of
,
`, and
subunits are shown (arrows) for cytosolic
and nuclear fractions (panels C and D). For direct
comparison, autoradiograms in panels C and D were
processed in parallel.
Figure 5:
Determination of turnover for CKII ,
`, and
subunits by pulse-chase analysis. Cells were labeled
for 12 h with [
S]methionine, washed, and chased
with complete medium for intervals up to 36 h. Whole cell extracts were
prepared from plates harvested at 0, 8, 16, 24, and 36 h. Protein in
the extracts was quantitated, and an equal amount of protein was used
in each immunoprecipitation reaction using anti-
and anti-
`
antibodies in combination. Panel A, autoradiogram of
immunoprecipitates after electrophoresis. Molecular mass markers are
indicated (kDa); and
,
`, and
subunit polypeptides are
indicated by arrows.Panel B, PhosphorImager analysis
of gel from panel A. Signals (PSL units) were plotted as a
function of time and best fit lines drawn. Times that correspond to
half-maximum signal (0 h) represent turnover numbers and are 24, 22,
and 27 h for
,
`, and
subunits,
respectively.
Figure 6:
Determination of turnover for CKII
isoforms by pulse-chase analysis. Cells were labeled with
[S]methionine for 12 h, washed, and chased with
complete media for intervals up to 36 h. Whole cell extracts were
prepared from plates harvested at 0, 8, 16, 24, and 36 h. Samples were
quantitated by protein assay, and equal amounts of protein in the
extracts were used in two-step immunoprecipitation reactions. Extracts
were divided into two sets of immunoprecipitation reactions, and each
set was cleared with either
or
` antibody.
Immunoprecipitation of cleared sets of extracts with alternate
catalytic subunit antibodies generated the
and
`
isoforms, which were
subjected to SDS-PAGE. Panel A, autoradiogram of
immunoprecipitates. Subunits
and
are indicated by arrows. Panel B,
autoradiogram of
`
immunoprecipitates. Subunits
` and
are indicated by arrows. PhosphorImager analysis was performed on gels from panels A and B, and signals for each isoform subunit
were plotted as a function of time. Panel C, best fit plot of
subunit signals associated with
isoform. Turnover numbers were calculated as times that
correspond to half-maximum signal (0 h) and are 27 and 29 h for
and
subunits, respectively. Panel D, best fit plot of
subunit signals associated with the
`
isoform. Turnover numbers are 29 and 24 h for
` and
subunits, respectively.
Figure 7:
Analysis of complex formation between
catalytic and regulatory subunits. Lag in subunit signal
appearance relative to catalytic subunit signal was evaluated by
continuous labeling analysis. Cells were labeled for 16 h with time
points harvested at 2-h intervals. Whole cell extracts were prepared,
samples quantitated by protein assay, and equal amounts of protein in
the extracts immunoprecipitated with
and
` antibodies in
combination. Panel A, autoradiogram of SDS-PAGE of
immunoprecipitates containing
,
`, and
subunits (arrows) for all isoforms. Panel C, PhosphorImager
analysis of gel in panel A. The signal (PSL units) was plotted
as a function of time for each subunit immunoprecipitated with
anticatalytic subunit antibodies. Panel B, autoradiogram of
SDS-PAGE of free
subunit immunoprecipitated from denatured
supernatants of immunoprecipitation reactions in panel A (see
``Materials and Methods''). The signal associated with the
subunit is plotted in panel C. For direct comparison,
autoradiograms in panels A and B were processed in
parallel. Panel D, signals from panel C are replotted
as a ratio of
/catalytic subunit and normalized for each of four
ratios: free
/
or
` and complex-associated
/
or
`.
Figure 8:
Determination of rate of subunit
synthesis. The rate of
subunit synthesis was evaluated by direct
immunoprecipitation from extracts prepared from a 16-h continuous
labeling time course with dishes harvested at 2-h intervals. Denaturing
immunoprecipitation was performed as described under ``Materials
and Methods'' on equal amounts of protein in the extracts.
Reactions included, in addition to
subunit antibody,
and
` subunit antibodies as controls. Panel A, autoradiogram
of SDS-PAGE of immunoprecipitated
,
`. and
subunits.
Positions are indicated (arrows), and signals associated with
each subunit are plotted in panel B. Panel C, signals
from panel B are replotted as a ratio of subunit/subunit and
normalized for each of three ratios:
/
,
/
`, and
/
`.
Figure 9:
Analysis of isoform complex formation.
Cells were labeled continuously for 16 h, dishes were harvested at 2-h
intervals, cell extracts prepared, and protein quantitated and
normalized as standard. The and
`
isoforms were immunoprecipitated in
two-step immunoprecipitation reactions and subjected to
electrophoresis. Analysis of
subunit appearance was determined
for isoforms by autoradiography and imaging analysis. Panel A,
autoradiogram of
immunoprecipitates.
and
subunits are indicated by arrows. Panel
B, autoradiogram of
`
immunoprecipitates.
` and
subunits are indicated by arrows. Panel C contains signal plots of four
subunits for the two isoforms from panels A and B. Panel D, signals from panel C are replotted as a
ratio of
/catalytic subunit and normalized for two ratios:
/
and
/
`.
To determine the rate of complex formation for the
`
isoform, anti-
and anti-
`
immunoprecipitates generated from the first immunoprecipitation step in
this experiment were evaluated, and
subunit signal incorporation
was found to be slow for both mixed isoform immunoprecipitates (data
not shown). From this, we conclude that both the
and
`
isoforms exhibited similar
slow complex formation times. We believe that this result does not
depend on the label incorporation protocol because substitution of the
continuous label procedure by a pulse-chase method that used short
pulses of 2 h followed by 14-h chases yielded similar results for the
three CKII isoforms. Complex formation for the major abundance
isoforms,
and
`
, took 10-14 h of chase to reach
completion (data not shown).
Figure 10:
Analysis of minor isoform complex
formation. Cells were pulsed labeled with
[S]methionine for 30 min, washed, and chased
with complete medium for intervals up to 4 h. Extracts were prepared
from plates harvested at 0 h, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h.
Protein in the extracts was quantitated and equal amounts of protein in
the extracts subjected to two-step immunoprecipitation and
electrophoresis. In addition to imaging analysis of subunits associated
with the
`
isoform, included as
controls are
isoform, and anti-
and anti-
` mixed isoform immunoprecipitates. Subunit signals
associated with each type of immunoprecipitation were quantitated (PSL
units), plotted as a ratio of
/catalytic subunit signal, and
normalized. Closed circles indicate the plot of the minor
abundance
`
isoform; open circles indicate the
isoform plot.
/
and
` plots for anti-
immunoprecipitations are
shown by open squares and closed triangles,
respectively, and for anti-
` immunoprecipitations, by closed
squares and open triangles,
respectively.
Figure 11:
Analysis of nuclear isoform complex
formation. Complex formation of nuclear isoforms was determined by
pulse-chase analysis of nuclear fractions. Cells were labeled for 2 h,
washed, chased with complete medium up to 14 h, and dishes were
harvested every 2 h. Subcellular fractionation was coupled to two-step
immunoprecipitation as described under ``Materials and
Methods,'' and isoform immunoprecipitates from nuclear lysates
were evaluated by autoradiography and imaging analysis by SDS-PAGE. Panel A, autoradiogram of anti- immunoprecipitates that
contain
and
`
isoforms. Signals for subunits are plotted in panel E.
Complex-associated
subunit is shown by closed circles,
and
and
` subunits are indicated by open circles and crosses, respectively. Panel C,
autoradiogram of
`
isoform
immunoprecipitated from supernatants of anti-
immunoprecipitates (Panel A). Subunit signals are plotted in panel F.
The
subunit signal is shown by closed circles and the
` subunit by crosses. Panel D, autoradiogram of
free
subunit immunoprecipitated from supernatants of
`
immunoprecipitates, and E (closed squares),
signal plot. For direct
comparison, autoradiograms in panels A and D were
processed in parallel. Panel B, autoradiogram of anti-
`
immunoprecipitations that contain
`
and
`
isoforms. Signal plots are in F. Open circles indicate complex associated
subunit, and
and
` subunits are shown by closed
symbols. Panel G, signals from panels E and F are replotted as a ratio of
/catalytic subunit and
normalized for each of five ratios:
/
(crosses) or
` (closed circles) for anti-
mixed isoform
immunoprecipitates;
/
(open squares) or
` (closed triangles) for anti-
` mixed isoform
immunoprecipitates; and
/
` for
`
immunoprecipitates (open
circles).
Since this result was similar to
that obtained with immunoprecipitates from whole cell lysates, we
tested if free subunit could be immunoprecipitated from the
nuclear fraction. Supernatants from
`
isoform immunoprecipitates that had been cleared three times with
antibody and once with
` antibody were used for
immunoprecipitation with
subunit antibody. The experiment shown
in Fig. 11D indicates the presence of free
subunit in the nuclear fraction. The loss of signal during the time
course mirrored that seen with continuously labeled whole cell lysate (Fig. 7B), suggesting incorporation of
into
complexes and also suggesting that the major abundance nuclear isoforms
are assembled in the nucleus. Our choice of a pulse-chase as opposed to
a continuous label protocol allowed us to follow incorporation of only
previously synthesized free
subunit into complex. The presence of
free nuclear
subunit was unexpected. In contrast to the catalytic
subunits,
subunit lacked a putative nuclear localization motif
(Jakobi et al., 1989) and therefore would not be expected to
translocate as free polypeptide.
Cytosolic fractions obtained from
the pulse-chase experiment were analyzed in a manner identical to that
for the nuclear fraction. Immunoprecipitation of
and
`
isoforms with
antibody is shown in Fig. 12A,
isoform
immunoprecipitates are shown in Fig. 12B. Quantitative
analysis of change in
subunit signal for the major abundance
isoforms did not reveal the same magnitude or duration of increase as
exhibited for the nuclear fraction (Fig. 12, E and F). However,
/
and
/
` signal ratio plots
shown in Fig. 12G indicate some increase in
subunit association. In contrast, evaluation of
`
immunoprecipitates does not reveal
an increase in
subunit signal (Fig. 12, C and E) or in complex formation (Fig. 12G).
Immunoprecipitation of the
subunit from supernatants of
`
immunoprecipitates shows the
presence of free subunit in this fraction, which also exhibited a
signal decrease similar to that of free nuclear
subunit (Fig. 12D). Consistent with the higher abundance of
nuclear holoenzyme isoforms, twice as much free
subunit was
immunoprecipitated per mg of protein in the extract from the nuclear
fraction than from the cytosolic fraction at 0 h (Fig. 11E and 12E). The observation that free subunit can be
immunoprecipitated from subcellular fractions does not depend on the
labeling procedure. We have immunoprecipitated free catalytic and
regulatory subunit from nuclear and cytosolic fractions using a
continuous label protocol (data not shown). Our ability to
immunoprecipitate free subunit is not limited to the two fractions
reported here. We have observed complex formation for the major
isoforms from free regulatory and catalytic subunit in extracts
obtained from the 100,000
g pellet which resulted
after centrifugation of the soluble cytosolic fraction. (
)This fraction may contain CKII from organelles and/or the
particulate membrane fraction.
Figure 12:
Analysis of cytosolic isoform complex
formation. Complex formation of isoforms in the cytosolic fraction was
analyzed by pulse-chase analysis. Cytosolic extracts from the
subcellular fractionation experiment that yielded the nuclear fractions
in Fig. 11were used in two-step immunoprecipitation reactions.
Immunoprecipitates were evaluated by autoradiography and imaging
analysis of SDS-PAGE gels. Panel A, autoradiogram of
anti- immunoprecipitations that contain
and
`
isoforms. Signals for subunits are plotted in panel E. Closed circles indicate complex-associated
subunit;
and
` are shown by open circles and crosses, respectively. Panel C, autoradiogram of
`
isoform immunoprecipitated from
supernatants of anti-
immunoprecipitates (panel A).
Subunit signals are plotted in panel F. The
subunit
signal is shown by closed circles and the
` subunit by crosses. Panel D, autoradiogram of free
subunit
immunoprecipitated from supernatants of
`
immunoprecipitates. Panel E, closed squares indicate the
signal plot. For direct comparison,
autoradiograms from panels A and D were processed in
parallel. Panel B, autoradiogram of
isoform immunoprecipitated from
supernatants of anti-
` immunoprecipitates (not shown). Subunit
signals are plotted in panel F. Open circles indicate
complex-associated
subunit, and the
subunit signal is
indicated by closed squares. Panel G, signals from panels E and F are replotted as a ratio of
/catalytic subunit and normalized for each of four ratios:
/
(closed circles) or
` (open circles)
for anti-
mixed isoform immunoprecipitates;
/
for
immunoprecipitates (closed
squares); and
/
` for
`
immunoprecipitates (open
triangles).
In this report we describe an immunodepletion assay that allowed us to determine the forms of CKII holoenzyme present in HeLa cells. Furthermore, this assay facilitated biochemical analysis of isoforms, and our in vivo metabolic labeling investigations revealed differences in the rate of complex formation between regulatory and catalytic subunits for different isoforms.
Based on
the presence of the and
` catalytic subunits in HeLa cells
(Pyerin et al., 1987; Yu et al., 1991), there are
three possible tetrameric forms of the enzyme of subunit stoichiometry:
,
`
, and
`
. In support of this we have
demonstrated the existence of three holoenzyme isoforms in HeLa cells,
and because our assay shows only coimmunoprecipitation of subunits,
tetrameric stoichiometry for isoforms is inferred. Purified CKII from
HeLa cells has been characterized previously by Pyerin et
al.(1987). Plasma membrane protein extract contained CKII
,
`, and
subunits by SDS-PAGE and yielded a molecular mass
estimate of 120 kDa by sedimentation velocity analysis, which is
consistent with the existence of tetrameric complexes. Pyerin et
al.(1987) proposed that their preparation contained two isoforms
of subunit stoichiometry,
`
and
`
. Comparison of these data and the
results presented in this paper is problematic because different types
of extracts were used. The results presented in this report ( Fig. 2and Fig. 4and Table 1) show that the
`
isoform is the least abundant of
the three holoenzyme isoforms.
Analysis of specific activity and
subunit composition of holoenzyme forms of CKII to date has been
limited to the isoform, which by
reconstitution of recombinant
and
subunit polypeptides
produced in Escherichia coli, has been demonstrated to have a
tetrameric subunit composition; kinetic parameters for this isoform
have been determined (Hu and Rubin, 1990; Grankowski et al.,
1991). Reconstitution of recombinant
` and
subunits resulted
in enhancement of catalytic subunit activity
(Bodenbach et al., 1994). However, neither K
or V
values nor subunit stoichiometry has been
reported for this isoform. Determination of subunit stoichiometry and
specific activity may prove to be difficult for the
`
isoform because in vitro reconstitution from purified
,
`, and
polypeptides
is predicted to generate additional isoforms. Filhol et al. (1994) separated bovine adrenocortical
and
`
isoforms by phosphocellulose chromatography, but it was not known
if the
`
preparation contained the
`
isoform.
Our CKII subcellular
localization analysis revealed that the three holoenzyme isoforms are
present in both the cytosol and nucleus. However, activity analysis
showed approximately 3-fold greater activity in the nuclear fraction.
Evaluation of [S]methionine-labeled
immunoprecipitates indicates that this is due to greater polypeptide
abundance (Fig. 4). These data confirm the result of Krek et
al.(1992), obtained by immunolocalization of transfected CKII
and
subunits in HeLa cells. They also agree with the
predominant nuclear localization described for other tissue types (for
review, see Pinna, 1990) but are inconsistent with our earlier
localization study that concluded that CKII is mainly cytosolic (Yu et al., 1991). The data obtained previously were based on
reactivity of antibodies made to the amino terminus of the
subunit and to an internal epitope of the
subunit, both of which
by immunofluorescence, exhibited nearly exclusive cytoplasmic staining.
The behavior of these antibodies in immunoprecipitation reactions
suggests that under native conditions, epitopes may not be accessible:
(i) antibody to
subunit will not immunoprecipitate free or
complex associated
without heat denaturation of extract; and (ii)
antibody to the amino terminus of
does not quantitatively
immunoprecipitate. (
)Although it is not possible to relate
immunoprecipitation and immunofluorescence assays directly, incomplete
epitope recognition by these antibodies may help explain the lack of
nuclear staining obtained by immunofluorescence. In contrast,
evaluation of the carboxyl-terminal anti-
and anti-
`
antibodies by immunofluorescence revealed strong nuclear and weak
cytoplasmic staining (data not shown). This is consistent with
immunoprecipitation of a greater amount of polypeptide from nuclear
extracts by both antibodies.
Our analysis of CKII complex formation
in subcellular fractions indicated that and
`
are assembled in the nucleus
from free catalytic and regulatory subunit polypeptides. In contrast to
the catalytic subunits, which contain putative nuclear localization
motifs, the
subunit lacks a similar signal sequence, and
therefore would not be expected to be present in the nuclear fraction
in uncomplexed form. It is possible that
subunit translocation is
chaperone-mediated, but we cannot currently evaluate this possibility
because our anti-
subunit immunoprecipitation protocol is
predicted to abolish all associations between proteins. The location of
complex formation for the corresponding cytosolic
and
`
isoforms is less certain. These could be assembled in the cytosol
but at a faster rate than their nuclear counterparts, or alternatively,
complex formation could occur in the nucleus followed by export of
holoenzyme to the cytosol. Our ability to immunoprecipitate free
regulatory and catalytic subunit from the cytosolic fraction is
consistent with either of these two mechanisms of assembly. Our
hypothesis for assembly of the
and
`
isoforms is that complex formation from
free polypeptides occurs in situ where these forms of CKII are
found in a cell, and this includes both nuclei and the cytosol. We
propose that the most likely model for the apparent rapid assembly of
the minor abundance
`
isoform is that
in contrast to the major abundance isoforms, the
`
form of CKII is assembled at a
single site (probably the cytosol) and exported as a holoenzyme to
other cellular locations. Our data are also consistent with a more
rapid rate of complex formation occurring in situ where this
isoform is found. Our analysis of holoenzyme complex formation from
free subunit polypeptides raises the question of whether tetramers are
assembled from monomers or whether dimer formation of free catalytic
and regulatory subunits is a prerequisite for assembly of tetrameric
complexes.
Biosynthesis of CKII has been evaluated in lymphoid cell
lines (Lüscher and Litchfield, 1994), and the
presence of excess subunit over that incorporated into complex
was demonstrated in a pulse-chase experiment by comparison of
subunit signals obtained with anti-
and anti-
subunit
immunoprecipitations. In HeLa cells, our data suggest that the
subunit is not present in excess over that required to form complex.
Quantitation of CKII
by continuous labeling analysis indicated
that free subunit is not synthesized in excess of what is required for
complex formation (Fig. 7C). In addition, when a fixed
population of
subunit molecules was labeled by a pulse-chase
labeling procedure, we did not observe in subcellular fractions more
free
subunit than that required for the assembly of complexes
from the cytosolic and nuclear fractions combined (Fig. 11E and Fig. 12E). Also, our comparison of the rates
of synthesis for the
,
`, and
subunits indicated a high
degree of similarity (Fig. 8). We do not know if differences
between the cell lines used or in experimental protocols may account
for differences between our results and those obtained by
Lüscher and Litchfield(1994).
Our findings of three different forms of holoenzyme, coupled with different modes of complex formation between isoforms, support the concept that CKII, as an enzyme, exists in multifunctional forms, and different modes of regulation may exist for different isoforms. This research opens the path to evaluate the behavior of the enzyme in the context of cell cycle regulation.