(Received for publication, August 29, 1994; and in revised form, November 18, 1994)
From the
The heat-stable protein kinase inhibitor (PKI) is a potent and
specific inhibitor of the catalytic (C) subunit of the cAMP-dependent
protein kinase. We report the isolation of a polyclonal antibody raised
to purified recombinant PKI. Using this antibody, the
intracellular distribution of endogenous PKI
was assessed by
immunostaining. The PKI
expression and intracellular distribution
varied as a function of cell cycle progression. PKI
expression
appeared low in serum-starved cells and in cells in G
and
increased as cells progressed through S phase. Its distribution became
increasingly nuclear as cells entered G
/M. Nuclear levels
of PKI
remained high through cell division and decreased again as
cells reentered G
. The cell cycle regulated expression and
nuclear distribution suggests a specific role for PKI
in the
nucleus during the G
/M phases of the cell cycle. Consistent
with this, microinjection of PKI
antibody into serum-starved cells
prevented their subsequent cell cycle progression. Similarly,
overexpression of C subunit in cells arrested at the G
/S
boundary prevented their subsequent division. Together these results
support the idea that PKI
plays an important role in the
inhibition of nuclear C subunit activity required for cell cycle
progression, although a determination of the relative amounts of
endogenous nuclear PKI and C-subunit will be required to substantiate
this hypothesis.
The heat-stable protein kinase inhibitor (PKI) ()is a
highly specific inhibitor of the catalytic (C) subunit of
cAMP-dependent protein kinase. PKI binds to the active site of C with
high affinity specifically in the presence of cAMP when C is
dissociated from the holoenzyme complex. The existence of an inhibitor
protein was first suggested in 1964(1) , and PKI was
subsequently purified (2) and studied extensively by a variety
of techniques (for review, see Ref 3). Several different isoforms of
PKI have been identified, including PKI
(4) , PKI
1,
and PKI
2 (5, 6) . PKI
1 and PKI
2 result
from alternative splicing of the RNA at the amino terminus(6) .
Analysis of peptide analogs determined that high affinity binding of
PKI to C-subunit could be attributed primarily to 20 residues near the
amino terminus. This region contains a cluster of arginine residues
that mimics the basic subsite present in peptide substrates and a
second hydrophobic site required for high affinity binding of PKI to
the C subunit.
In spite of extensive biochemical studies, the
physiological roles of PKI remain unclear. It was originally proposed
that PKI was simply a mechanism to dampen the kinase response by
inactivating low levels of enzyme (for review, see Ref 3). However, the
association of testicular PKI with microtubules (7) and the
isolation of several PKI isoforms (4, 5, 6) that exhibit tissue- and
developmental stage-specific expression suggest additional roles for
these inhibitors(8) . Several studies have implicated a role
for PKI in cell cycle progression. In amphibians (9) and
mammals(10) , microinjection of PKI into quiescent oocytes
resulted in the induction of mitosis. Similarly, injection of an
amino-terminal PKI peptide (PKI) into mammalian
cells induced chromatin condensation, microtubule reorganization, and
changes in cell shape similar to those that occur just prior to
mitosis(11) . Recent studies demonstrated that PKI entered the
nucleus following its injection into the cytoplasm in REF52
cells(12) , suggesting a specific role for PKI in the
regulation of nuclear stores of C subunit.
To better understand the
physiological roles of PKI, an antibody was raised to purified
recombinant PKI and used to characterize the expression and
distribution of endogenous PKI
.
A polyclonal antibody was raised to purified recombinant
PKI and purified on a PKI
affinity column. Both serum and the
affinity-purified PKI
antibody specifically recognized as little
as 10 ng of purified recombinant PKI
on Western blots (Fig. 1, lane1). In contrast, neither reagent
reacted detectably with purified recombinant PKI
1 (lanes2-4). Preimmune serum (not shown) and antibody
preincubated with excess PKI
(lane5) failed to
react with PKI.
Figure 1:
Specificity of the PKI antibody.
Purified recombinant PKI
(10 ng, lanes1 and 5) and increasing amounts of purified recombinant PKI
1 (lanes2-4) were subjected to
SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidine
difluoride membranes, and probed with the affinity-purified PKI
antibody diluted 1:5000 (lanes1-4), and
affinity-purified PKI
antibody was preincubated with an excess
(0.5 µg/µl) of purified recombinant PKI
(lane5).
We previously reported that PKI entered the
nucleus following its microinjection into the cytoplasm of rat
embryonic fibroblasts(12) . To assess if endogenous PKI
exhibited a similar intracellular distribution, exponentially growing
cells were fixed and stained with the PKI antibody. Fig. 2demonstrates that endogenous PKI
could be detected in
the nucleus of Wistar rat thyrocytes and rat embryonic fibroblasts
(REF52). Similar results were observed in mouse 10T1/2 fibroblasts (not
shown). The specificity of the immunostaining for PKI
was
demonstrated in several ways. First, preincubation of the antibody with
purified PKI
(Fig. 2c) or deletion of the primary
antibody (not shown) completely abolished the staining pattern. Second,
the distribution of microinjected PKI
or of a C:PKI
complex
as detected by immunostaining with the PKI
antibody precisely
mirrored that observed following the injection of fluorescently labeled
proteins. Thus, similar to the results obtained following injection of
FITC-labeled PKI
, microinjected PKI
was detected in the
nucleus following immunostaining with the PKI
antibody (Fig. 3, a and c). We previously reported that
a microinjected C:PKI complex was restricted to the
cytoplasm(13) . Consistent with these results, the PKI
antibody reacted with an injected C:PKI
complex primarily in the
cytoplasm (Fig. 3, b and d). Lastly, the PKI
antibody failed to detect microinjected PKI
1 (not shown).
Together, these results document the specificity of the antibody
staining for PKI
.
Figure 2:
Immunostaining with the PKI antibody.
Exponentially growing Wistar rat thyrocytes (a) and REF52 (b and c) cells were fixed in 3.7% formaldehyde in
phosphate-buffered saline and stained with the affinity-purified PKI
antibody as described under ``Materials and Methods.'' Panelc illustrates REF52 cells stained with
affinity-purified PKI antibody that had been preincubated with an
excess of purified PKI
prior to
staining.
Figure 3:
Immunostaining of microinjected PKI
and C:PKI
complex by PKI
antibody. PKI
(128
µM)(c) and C:PKI
(100 µM)(d) were
injected into the cytoplasm. The distributions of the PKI either as a
free protein or as a complex were analyzed by staining with
affinity-purified PKI
antibody followed by a tetramethylrhodamine
isothiocyanate-conjugated anti-rabbit antibody. In parallel, the
FITC-labeled PKI
(128 µM) (a) and
C:FITC-PKI
(100 µM) (b) were also injected
into the cytoplasm.
In asynchronous cells, both the intensity and
intracellular distribution of PKI staining was heterogeneous,
suggesting that the expression or intracellular distribution of
PKI might vary as a function of cell cycle progression. To
determine if this was the case, REF52 cells were synchronized in
G
by serum deprivation and then stimulated to enter the
cell cycle with serum-supplemented growth medium. Similar to other
reports(17) , serum-starved REF52 cells synthesized DNA between
12 and 20 h (Fig. 4A) after serum stimulation and
maximal numbers of mitotic cells were observed between 24-28 h
after serum stimulation (not shown). The relative expression and
distribution of PKI
in synchronized cells varied as a function of
cell cycle progression. Serum-starved REF52 cells exhibited very low
levels of PKI
staining that were uniformly distributed throughout
the cell (Fig. 5a). The fluorescence was slightly more
intense in the nuclei of a few of the starved cells (not shown). Cells
in S phase (16 h after serum stimulation) exhibited a greater overall
fluorescence intensity compared with starved cells (Fig. 5c). Increased fluorescence was found both in the
cytoplasm and in the nucleus. As the cells proceeded through S phase,
the distribution of PKI
became increasingly nuclear. Nuclear
fluorescence was maximal in cells analyzed at 20-24 h after serum
stimulation (Fig. 5e), a time that corresponds to late
S or G
.
Figure 4:
DNA synthesis in synchronized REF52 cells. A, REF52 cells were synchronized in G by serum
deprivation or (B) at the G
/S boundary by
incubation in isoleucine-deficient medium followed by aphidicolin
treatment. The cells were subsequently washed and stimulated with
Dulbecco's modified Eagle's medium containing 20% fetal
calf serum and pulse labeled with bromodeoxyuridine at 4-h intervals.
Following fixation and staining with a bromodeoxyuridine-specific
antibody as described previously(15) , the number of labeled
nuclei was scored.
Figure 5:
PKI expression in synchronized REF52
cells. Cells were synchronized by serum deprivation (panelsa, c,and e) or by
isoleucine/aphidicolin treatment (panelsb, d, and f). Following fixation, the cells were stained
with the PKI
antibody as described under ``Materials and
Methods.'' PKI
expression was low in serum-starved cells (a) but increased in cells stimulated with serum for 16 h
(corresponding to S phase) (c) or 24 h (corresponding to late
G
or M) (e). PKI
expression appeared greater
in cells arrested at the G
/S boundary (b) than in
serum-starved cells (a). PKI
staining was further
increased at 3 h (corresponding to S phase) (d) and 12 h
(corresponding to late G
or M) (f)
following removal of aphidicolin and stimulation with fresh
medium.
To discriminate between the effects of cell
cycle progression and serum stimulation, the distribution of PKI
was assessed in cells arrested at the G
/S boundary by
isoleucine deprivation followed by treatment with aphidicolin, a
specific inhibitor of DNA polymerase
(18) . Both the
isoleucine-deficient and aphidicolin-supplemented medium contained
serum. Under these conditions, REF52 cells synthesized DNA between 0
and 8 h (Fig. 4B) and entered mitosis between 12 and 15
h following removal of aphidicolin and the addition of fresh growth
medium. Compared with cells in G
(Fig. 5a),
cells arrested at the G
/S boundary exhibited higher levels
of PKI
expression (Fig. 5b). PKI
staining was
further increased, especially in the cytoplasm, as cells entered S
phase (3 h after refeeding growth medium) (Fig. 5d). As
the cells proceeded through S phase, nuclear PKI
staining
increased and reached maximal levels just before cell division (12 h
after refeeding growth medium) (Fig. 5f).
PKI
expression was also analyzed as cells proceeded through mitosis.
Nuclear PKI
staining (Fig. 6a) was always far
greater in cells with condensed chromatin (Fig. 6b)
than in interphase cells. This effect was not due to differences in
cell volume or architecture as confocal microscopy also revealed a
dramatic increase in PKI
staining in cells with condensed
chromatin (not shown). As cells proceeded through mitosis, PKI
staining remained distinct and in close proximity to the chromosomes
even after nuclear envelope breakdown although PKI
did not appear
to be chromosome-associated (Fig. 6, c-f). Similar results were observed using
confocal microscopy (not shown). PKI
expression remained high in
cells following division and reformation of the nuclear envelope (Fig. 6, g and h) but eventually declined to
the levels found in asynchronous cells.
Figure 6:
PKI expression during mitosis.
Staining with the PKI
antibody was performed as described under
``Materials and Methods.'' Following PKI
staining,
chromosomes were stained with Hoechst 33258 (10 µg/ml) for 5 min at
25 °C.
The cell cycle-dependent
alterations in PKI expression and intracellular distribution
suggested a specific role for PKI
in cell cycle progression. To
examine this further, the effects of the PKI
antibody on cell
cycle progression were assessed. The antibody was injected into the
cytoplasm of serum-starved REF52 cells, which express very low levels
of PKI
so that the antibody would bind and sequester newly
synthesized PKI
in the cytoplasm, functionally depleting nuclear
stores of PKI
. Following injection, the cells were stimulated with
growth medium and fixed 36 h later, a time at that most of the cells
should have divided. The number of surviving injected cells was
compared with the total number of cells injected. Injection of the
PKI
antibody, but not of control IgG, significantly reduced the
ability of the injected cells to divide (Fig. 7A). To
determine if the effect of the PKI
antibody was related to
increased levels of free C subunit, C subunit was injected into cells
arrested at the G
/S boundary, and the ability of the
injected cells to divide following stimulation was assessed. Similar to
the results obtained with the PKI antibody, overexpression of C subunit
also inhibited cell division (Fig. 7B).
Figure 7:
Microinjection of the PKI antibody
and C-subunit into synchronized cells. A, affinity-purified
PKI
antibody (15 mg/ml) and control rabbit IgG (15 mg/ml) were
injected into the cytoplasm of serum-starved REF52 cells. Approximately
2 h after injection, the cells were stimulated with fresh growth medium
and fixed 36 h following stimulation. After staining with an
FITC-labeled anti-rabbit antibody, the number of surviving injected
cells was scored and compared with the number of cells injected.
Approximately 80% of REF52 cells injected into the cytoplasm survive
microinjection. If every surviving cell divided, the cell number would
increase by a factor of approximately 1.6. The number of cells injected
with the PKI
antibody remained constant (injected = 301,
FITC
= 236), while the number of cells injected
with control IgG increased by approximately 1.6-fold (injected =
306, FITC
= 481). (B). Purified C
subunit (2 mg/ml) was coinjected with rabbit IgG (5 mg/ml) into the
cytoplasm of REF52 cells arrested at the G
/S boundary.
Cytoplasmic injection was performed since C-subunit rapidly enters the
nucleus following introduction into the cytoplasm (27) and
higher numbers of cells survive cytoplasmic compared with nuclear
injection. Cells were arrested at the G
/S boundary so that
the biological effects of C subunit could be monitored in shorter times
since injected C subunit is unstable (not shown). As a control, rabbit
IgG (5 mg/ml) was injected alone. Following stimulation for 22 h, the
cells were fixed and stained, and the number of surviving injected
cells was scored (for C-subunit, injected = 460, FITC
= 354; for IgG, injected = 427, FITC
= 830). In the assays where the cells were not expected to
divide, the survival of C-subunit injected cells was same as the
survival of IgG-injected cells. Over 300 cells were analyzed for each
sample in two independent experiments.
We report the isolation of a rabbit polyclonal antibody
raised to recombinant PKI. This antibody specifically recognized
purified recombinant PKI
on Western blots and both free PKI
and PKI
in the form of a C:PKI
complex following
microinjection into REF52 cells. The ability of the antibody to react
with a C:PKI
complex suggests that it recognizes determinants on
PKI other than those involved in binding to C subunit. The PKI
antibody failed to recognize PKI
1 either on Western blots or
following its injection into REF52 cells. These results are consistent
with the recognition by the antibody of determinants in the
carboxyl-terminal region of PKI
, which are not well conserved in
PKI
(5, 6, 8) . In this regard, the
PKI
antibody differs significantly from the antibody raised
against the testis-specific form of PKI (PKI
) isolated by Tash et al.(7) .
Immunostaining experiments revealed
that both the apparent level and intracellular distribution of PKI
varied in a cell cycle-dependent manner. PKI
expression appeared
much lower in serum-starved cells than in cycling cells. Whether this
is a consequence of PKI degradation or changes in conformation or
binding to components that render it undetected by the antibody under
these conditions remains to be determined. Compared with both cells
arrested in G
and at the G
/S boundary, PKI
expression increased dramatically as cells proceeded through S phase.
Quantitative measurements made using confocal microscopy revealed that
total cellular fluorescence increased in cells in S phase compared with
cells in G
. As cells entered S phase, PKI
appeared
first in the cytoplasm consistent with the new synthesis of PKI
.
It remains possible, however, that the increased fluorescence is due to
the release of PKI
, which was previously sequestered or not
detected with the PKI
antibody. This seems unlikely since binding
of PKI
to C subunit, its only known physiological binding protein,
did not abolish antibody recognition. As cells progressed through S
phase and into G
and M, the distribution of PKI
became
increasingly nuclear. Quantitative confocal microscopy revealed that
nuclear fluorescence increased without a concomitant change in total
cellular fluorescence. As cells progressed through mitosis, PKI
expression remained high and decreased again only after cell division
as cells reentered G
. These results differ somewhat from
those reported earlier for the testis-specific form of PKI (PKI
),
which was found to be localized to cytoplasmic microtubules in
interphase cells and to the spindle apparatus in mitotic
cells(7) . Whether this is a consequence of cell type-specific
differences or differences in the species of PKI recognized by the
respective antibodies is unclear. In REF52 cells, the fluorescently
labeled brain-specific form of PKI (PKI
1) could also be detected
in the nucleus following microinjection in the cytoplasm. (
)
The up-regulation of PKI expression in S phase and
its subsequent accumulation in the nucleus during G
/M
support a specific role for PKI in the nucleus as cells enter mitosis.
Numerous studies have suggested a role for PKI in the inhibition of C
subunit activity required for mitotic entry. In several species, oocyte
maturation is accompanied by decreased cAMP levels and cAMP-dependent
protein kinase activity(19, 20) . Microinjection of C
subunit inhibits oocyte maturation; conversely, microinjection of PKI
induces maturation(9, 21) . In somatic cells,
microinjection of PKI induced many of the cellular changes that
accompany mitosis, including changes in cell shape and chromosome
condensation(11) . Consistent with these results,
microinjection of the PKI
antibody abolished the ability of
injected cells to divide. As reported previously(11) , similar
effects were observed following microinjection of free C subunit. Taken
together, these results support a specific role for PKI in the
inhibition of C subunit activity required for cell cycle progression,
although at present there is no direct evidence that there is
sufficient PKI
to inhibit nuclear C-subunit activity. Most of the
quantitation studies so far were done with total cell
extracts(3) . No values for PKI concentrations in synchronized
cells or in subcellular compartments is yet available.
There is a
burst of phosphorylation as cells enter mitosis and a corresponding
decrease in phosphorylation as cells exit mitosis. Changes in
phosphorylation are likely to mediate, in part, the multiple structural
alterations that occur during mitosis including chromosome
condensation, nuclear envelope breakdown, spindle formation,
cytoskeletal changes and disassembly of organelles. p34 activity, which is maximal in late G
, is responsible
for a number of mitosis-specific phosphorylation events.
Phosphorylation of histone H1 by p34
results in
chromosome condensation, and p34
-mediated
phosphorylation of the nuclear lamins results in lamin disassembly. In
contrast, cAMP-dependent protein kinase inhibits
p34
mediated lamin release(22) , and addition of
cAMP to mitotic extracts results in the loss of p34
kinase activity(23) . Consistent with these results, PKI
and p34
appear to function cooperatively to effect many
of the cellular changes that occur at mitosis. Microinjection of either
p34
(11, 17) or PKI (11) alone,
induced changes in cell shape, chromatin condensation, and alterations
in actin and tubulin architecture. Coinjection of p34
and PKI together induced additional changes including nuclear
envelope breakdown, suggesting that p34
and
cAMP-dependent protein kinase perform distinct and complementary roles
in regulating entry into mitosis. The roles of cAMP-dependent protein
kinase may include the maintenance of higher order chromatin structure,
nuclear envelope integrity, and microtubule architecture, all of which
must be dismantled prior to mitosis. In addition, a number of cellular
processes including transcription and endocytosis are specifically
repressed during mitosis. The activity of the transcription factor cAMP
response element binding protein is dependent upon phosphorylation by
cAMP-dependent protein kinase (for review, see (24) ). It also
needs to be turned off prior to mitosis.
The nuclear localization of
PKI makes it ideally situated to inhibit nuclear C subunit, unlike
regulatory subunits that appear to be localized in the cytoplasm in
many (25, 26, 27, 28) but not all
cell types(29, 30) . Intracellular cAMP levels have
been reported to fluctuate as a function of cell cycle progression with
a dramatic increase observed in cells in S phase and the lowest levels
observed during mitosis(21) . This would result in holoenzyme
dissociation during S phase and the accumulation of active C subunit in
the nucleus as cells enter G. Up-regulation of nuclear PKI
expression at this time would provide a mechanism for extinguishing the
activity of C in the nucleus without affecting its activity in the
cytoplasm. In an earlier report, we found that microinjection of a
C:PKI complex enhanced the rate of export of C subunit from the
nucleus(12) . These results suggest that there may be multiple
mechanisms through which nuclear stores of C subunit could be
inactivated.
A number of earlier studies provided evidence for PKI
regulation (for review, see (3) ). For example, PKI activity
increased following serum stimulation in starved Chinese hamster ovary
cells (31) and follicle-stimulating hormone treatment in
cultured Sertoli cells(32) . In chick kidney cells, vitamin D
down-regulated both PKI activity (33, 34) and mRNA
levels(35) . Additionally, the expression of the and
PKI mRNAs is both tissue- and developmental
stage-specific(8) . Our results suggest that PKI is subject to
both positive and negative regulation in a cell cycle-dependent
fashion. It will be interesting to determine if PKI is regulated
posttranscriptionally, as is the case for the type I regulatory
subunit(35) , or is subject to regulation at the level of
transcription. Phosphorylation of the single tyrosine residue in PKI
reduced its inhibitory activity on C subunit(36) , providing a
potential mechanism through which PKI activity could be regulated
through the activity of other cellular signaling pathways.
cAMP-dependent protein kinase functions in a diverse array of cellular
processes including metabolism, secretion, differentiation, gene
expression, proliferation, and growth inhibition. PKI may be part of a
coordinated cellular control network that helps to modulate the
activity of this important protein kinase.