From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Regulation of protein kinase A by subcellular
localization may be critical to target catalytic subunits to specific
substrates. We employed epitope-tagged catalytic subunit to correlate
subcellular localization and gene-inducing activity in the presence of
regulatory subunit or protein kinase inhibitor (PKI). Transiently
expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking
the nuclear export signal blocked gene induction but not nuclear
accumulation, demonstrating that nuclear export is not essential to
inhibit gene induction. When the catalytic subunit was targeted to the
nucleus with a nuclear localization signal, it was not sequestered in
the cytoplasm by regulatory subunit, although its activity was
completely inhibited. PKI redistributed the nuclear catalytic subunit
to the cytoplasm and blocked gene induction, demonstrating that the
nuclear export signal of PKI can override a strong nuclear localization
signal. With increasing PKI, the export process appeared to saturate,
resulting in the return of catalytic subunit to the nucleus. These
results demonstrate that both the regulatory subunit and PKI are able
to completely inhibit the gene-inducing activity of the catalytic
subunit even when the catalytic subunit is forced to concentrate in the
nuclear compartment.
The subcellular localization of multifunctional protein kinases is
emerging as a critical regulatory mechanism to target activity to sites
of second messenger generation or to a specific subset of intracellular
substrates (1, 2). One of the important actions of
PKA1 involves the
phosphorylation of transcription factors and the induction of specific
genes. However, the PKA holoenzyme is normally found to be cytoplasmic,
and only the catalytic subunit is thought to be able to enter the
nucleus after its release from the holoenzyme. The translocation of the
C subunit into the nucleus is the key event associated with the
initiation of PKA-mediated induction of transcription. After
translocation to the nucleus, the C subunit phosphorylates
transcription factors such as CREB, specifically stimulating
transcription (3-5).
The C subunit of PKA has been shown to translocate into the nucleus
after the microinjection of purified protein into the cytoplasm of
cultured cells (6, 7). However, microinjection of preformed holoenzyme
injected into the cytoplasm fails to traverse the nuclear membrane and
remains cytoplasmically sequestered (8). The nuclear pore complex (NPC)
is the primary route for proteins to diffuse back and forth across the
nuclear membrane. Studies measuring pore permeability suggest that
proteins larger than 23-26 nm in diameter, which corresponds
approximately to a 40-60-kDa globular protein, are excluded from
passage through the aqueous pore of the NPC (9, 10). Because the C
subunit is approximately 40 kDa, this places it at the upper size limit
of proteins that can diffuse passively into the nucleus. Nevertheless,
investigation into the mechanism of C subunit translocation into the
nucleus has suggested that passive diffusion is sufficient to explain the kinetics and apparent nuclear accumulation of C (11).
The partitioning of C between cytoplasm and nucleus is further
complicated by the recent discovery of the nuclear export properties of
PKI. Residues 37-46 of PKI contain a leucine-rich region that has been
shown to function as a nuclear export signal (NES) (12). PKI appears to
transport the C subunit out of the nucleus in a C/PKI complex utilizing
an ATP and temperature-dependent mechanism (6, 7). PKI is
capable of freely entering the nucleus (6, 8, 13) and actively
shuttling the C subunit back into the cytoplasm where the C subunit can
recombine with R subunits to form inactive holoenzyme and restore cAMP
regulation to the cell.
In this study, we utilize epitope-tagged C subunit to evaluate the
ability of either the RII subunit or PKI to prevent nuclear accumulation and inhibit gene induction of a CRE-luciferase reporter. By adding an artificial nuclear localization signal (NLS) to the tagged
C subunit we are able to compare the ability of RII and PKI to affect a
strong nuclear localization signal and inhibit C subunit activity. Our
results are consistent with the current model developed from
microinjection experiments and further demonstrate that the entire
RII/C holoenzyme complex would be transported to the nucleus if the C
subunit contained an exposed NLS. In contrast to RII, PKI is capable of
exporting a C subunit with a strong NLS, although export is not a
prerequisite for inhibition of gene expression. The export process that
is engaged by PKI appears to be saturable, suggesting that free PKI may
interact with the export machinery.
Cell Culture and Transient Transfection--
JEG3 cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum (Life Technologies, Inc.) in a 10% CO2
incubator at 37 °C. Before transfection, the cells were passaged
onto 24-well plates at 2 × 104 cells/well. The cells
were allowed to grow to approximately 50% confluence. The medium was
reduced to 250 µl/well 3-6 h before transfection. The cells were
transfected by standard calcium phosphate techniques (14), adding 25 µl of the calcium phosphate/DNA precipitate to each well. The
Luciferase activity was corrected for transfection efficiency by
normalizing to relative Construction of Expression Vectors--
The CTAG
construct was made by PCR using a 5' primer containing the
5'-untranslated region and the initial three codons of the coding
sequence adding the NcoI site to the 5' end
(5'-ACGCCGCCGCCATGGGCAACG-3'). The 3' primer is complimentary to the
carboxyl-terminal 9 codons of C
The PKIMUT construct was generated by PCR mutagenesis using
primers mutating the leucines (positions 37, 39, 41, and 44) and the
isoleucine (position 46) in the nuclear export signal to alanines. PCR
primers were generated internal to the PKI Immunocytochemistry--
After 12-15 h of zinc treatment, the
cells were fixed with 4% paraformaldehyde in phosphate-buffered saline
and permeabilized with 0.3% Triton X-100 in phosphate-buffered saline.
The cells were subsequently incubated with the c-Myc antibody (Oncogene Sciences) at a dilution of 1:100 for 1-4 h. The fixed cells were then
probed with the biotin-conjugated horse anti-mouse antibody (Vector) at
1:100, followed by incubation with avidin-FITC (Vector) at 1:1000 in
1 × Hepes-buffered saline. After fluorescein isothiocyanate labeling, the cells were incubated in 5 µM propidium
iodide to label the nuclei. Slides were prepared as described
previously (14). The stained monolayer was examined using a CRM-600
Bio-Rad confocal microscope.
Regulation and Localization of the C Subunit by Association with
R--
The localization of either free C subunit or assembled R/C
holoenzyme was examined by transfecting JEG3 cells with an
epitope-tagged C subunit (CTAG) with and without RII. When
the CTAG construct was transfected alone, expressed C
subunit was localized ubiquitously throughout the cell. Because the
carboxyl-terminal region of the C subunit has been shown to
dramatically affect enzymatic activity (18, 19), it was critical to
determine whether adding the c-Myc epitope to the carboxyl terminus of
the C subunit altered activity of the expressed protein.
Co-transfection of the CRE-luciferase reporter into JEG-3 cells
demonstrated that expression of C was associated with robust
stimulation of transcription, suggesting that the recombinant C was
enzymatically active (Fig.
1B). However, it was possible
that the observed activity was because of CTAG displacing
endogenous C subunit from its association with R in holoenzyme. To
address this issue, co-transfections were performed using a tagged
mutant C subunit (CQRTAG) that does not associate with R
(17). In these experiments, CQRTAG demonstrated comparable levels of transcriptional transactivation of the CRE-luciferase reporter as untagged C subunit and CTAG, suggesting that
the addition of the c-Myc tag to the carboxyl-terminal region of the
protein did not significantly diminish enzymatic activity (data not
shown).
When CTAG and RII were co-transfected to form holoenzyme,
the CTAG localized exclusively to the cytoplasm (Fig.
1C, lower panels), and this cytoplasmic
sequestration correlated with a complete block in transactivation of
the CRE-luciferase reporter construct. This suggests that the expressed
C and R subunits appropriately assemble to form the holoenzyme and that
this precludes entry into the nucleus, consistent with previous
observations that the holoenzyme is too large to diffuse between
cytoplasmic and nuclear compartments (7, 8).
In many of the cells transfected with CTAG alone, there
appeared to be nuclear accumulation of the protein. This observation was also previously reported using fluorescein-conjugated C subunit introduced by microinjection (7, 8). One possible explanation is that
the C subunit contains a weak or perhaps conditional NLS, although
fluorescence bleaching and kinetic observations argue against it (11).
We examined the ability of a well characterized NLS to modify both the
subcellular localization of C and its activity in a gene induction
assay. The SV40 T-antigen NLS was spliced onto the amino terminus of
CTAG to yield NLS-CTAG. After transfection into
JEG3 cells, NLS-CTAG was localized entirely to the nucleus (Fig. 2C, upper
panels). NLS-CTAG expression also resulted in high
levels of transactivation of the CRE-luciferase reporter, demonstrating
that the expressed protein was catalytically active (Fig.
2B).
Nuclear Translocation and Activation of the
NLS-CTAG Holoenzyme--
In co-transfection
experiments with the RII regulatory subunit, it was observed that
NLS-CTAG still localized entirely in the nucleus (Fig.
2C, lower panels). However, although the R
subunit did not prevent nuclear translocation, it did prevent
transactivation of the CRE-luciferase construct (Fig. 2B).
That the C subunit was still localized to the nucleus but was not
catalytically active suggested that the NLS on the C subunit caused
translocation of the entire holoenzyme into the nucleus. To test this
hypothesis, an RIITAG expression vector was made.
RIITAG expressed alone was only found in the cytoplasmic
compartment (Fig. 3B), and
co-transfection with a C Regulation of C Subunit Subcellular Localization and
Transactivation by PKI--
PKI has been shown to actively export the
C subunit from the nucleus via the nuclear export signal resident
within PKI between residues 37 and 46 (12). To directly correlate
PKI-mediated export of the C subunit with the ability of PKI to inhibit
C subunit activation of CRE-luciferase expression, co-transfection
studies were done with CTAG and PKI. Initial titration
studies were performed to determine the level of RSV-PKI necessary to
effectively inhibit the majority of C subunit activity (data not
shown). Co-transfection with sufficient PKI to inhibit C subunit
activity by greater than 70% (Fig.
4B) resulted in near complete
cytoplasmic sequestration of the CTAG protein (Fig.
4A).
Competition Between Import and Export with NLS-CTAG and
PKI--
We examined the relative strength of active import and export
processes by co-transfecting NLS-CTAG in the presence or
absence of PKI. Transfection with NLS-CTAG alone resulted
in complete nuclear localization as shown in Fig. 2. Cotransfection of
NLS-CTAG with PKI yielded three different staining patterns
ranging from completely nuclear to completely cytoplasmic (Fig.
5B). Because endogenous PKI
has been shown to be expressed in a cell cycle-dependent fashion, we speculated that there might be cell
cycle-dependent expression of export factors leading to the
variability in localization observed. However, when cells were
synchronized at various points in the cell cycle, the variability in
localization persisted, demonstrating it was not a cell cycle effect
(data not shown).
One plausible explanation for variable export is that co-transfection
of PKI and NLS-CTAG expression vectors results in a variable ratio of PKI to C protein in individual cells. This hypothesis was tested by incrementally increasing PKI and then observing both the
CRE-luciferase transactivation and the subcellular localization of
NLS-CTAG under each transfection condition. Fig.
6A demonstrates that
increasing concentrations of PKI produced a continual decrease in
CRE-luciferase transactivation. However, quantitation of staining patterns demonstrated a biphasic localization such that increasing concentrations of PKI shifted NLS-CTAG localization toward
the cytoplasm until only about 15% of the cells exhibited nuclear-only staining. Increasing the concentration of PKI past this point resulted
in a relative decrease in the cytoplasmic localization and a return to
nuclear localization such that 55% of transfected cells were scored as
nuclear only. This suggests that there is an optimal ratio of PKI to C
for export and that further increases in PKI compete with the C/PKI
complex for binding to the export machinery.
The Role of Nuclear Export in PKI Regulation of C Subunit
Function--
It has been demonstrated that PKI inhibits C subunit
activity and actively exports the C subunit out of the nucleus in a
C/PKI complex. These activities are encoded in discrete regions of the protein. However, the relative functional impact of these two activities on C subunit-directed gene expression has never been quantitated. The localization of CTAG and its ability to
transactivate CRE-luciferase was examined using various concentrations
of either wild-type PKI or mutant PKI in which the critical hydrophobic residues within the nuclear export signal had been mutated to alanines
(Fig. 7A). Titration of the
wild-type PKI showed a concentration-dependent effect both
with respect to export and inhibition of transactivation of the
CRE-luciferase reporter (Fig. 7). Consistent with previous reports
(12), the mutant PKI failed to export the C subunit out of the nucleus
(Fig. 7C). A comparison of mutant and wild-type PKI
demonstrated that mutant PKI was a somewhat less effective inhibitor of
CRE-luciferase transactivation at low concentrations (Fig.
7B). However, with higher levels of PKI, both the mutant and
wild-type proteins were indistinguishable in their inhibitory activity.
This suggests that export of the C subunit may play a particularly
important role in the regulation of C subunit activity when PKI is at
sub-stoichiometric concentrations relative to C subunit but that export
is not essential for complete inhibition of C subunit-induced gene
expression.
The subcellular localization and trafficking of PKA has received a
great deal of attention, and several competing mechanisms have been
described that may modulate this process. The localization of
holoenzyme is most often suggested to be a mechanism to bring PKA
closer to selective substrates, increasing substrate specificity as
well as the rate of phosphorylation. It is equally plausible that
localizing mechanisms could serve to place PKA close to sites of cAMP
generation and allow regional changes in cAMP to promote PKA
activation. Several mechanisms for PKA localization have been elucidated. A collection of anchoring proteins for PKA (AKAPs) have
been described that bind with nM affinities to the type II regulatory subunits, and these AKAPs are themselves associated with
cytoplasmic membranes, cytoskeletal components, or cytoplasmic organelles (20, 21). Recently several novel AKAPs have been isolated
that can also bind to type I regulatory subunits although with much
lower affinities (22, 23). The C Our results demonstrate that unbound C subunit localizes to both
cytoplasmic and nuclear compartments and promotes gene activation. However, when C was incorporated into an RII-containing holoenzyme, no
nuclear C was detected, and transcription from the CRE-luciferase reporter returned to base line. These results are in good agreement with previous studies using microinjection of fluorescently tagged C
and R proteins (11) as well as studies examining the endogenous C
subunit by direct immunofluoresence (26, 27). The R/C holoenzyme exceeds the size that is capable of diffusing at significant rates through the NPC. In addition, cytoplasmic AKAPs are potentially capable
of binding holoenzyme and preventing its redistribution to the nucleus.
When cAMP dissociates the holoenzyme and releases active C subunit,
this 40-kDa monomer is now presumed to be small enough to diffuse into
the nucleus where it can phosphorylate nuclear targets such as CREB and
other transcription factors, modulating their activity.
The C subunits do not appear to contain an endogenous NLS, although a
consensus sequence (KRVK) resembling the human c-Myc NLS is located at
amino acid 189. Mutations of this sequence have been shown to have no
effects on nuclear accumulation or transcriptional activity of
C.2 We introduced an
exogenous SV40 T-antigen NLS onto the amino terminus of C to test the
ability of either RII or PKI to maintain the cytoplasmic localization
and compete with a well characterized signal for nuclear import. The
presence of an exogenous NLS on the C subunit transported nearly 100%
of the protein into the nucleus, and NLS-C was an effective inducer of
reporter gene activity. This experiment demonstrates that the C subunit
is localized completely to the nucleus when an exposed NLS is
fused to the protein and argues that no endogenous NLS is recognized in
the free C subunit.
When an RII expression vector was added to the transfections along with
the NLS-CTAG, the ability of C to induce gene activity was
completely inhibited, but surprisingly, the C subunit remained nuclear.
This implies that the NLS-CTAG is forming holoenzyme effectively and that the NLS is capable of targeting this entire holoenzyme to the nucleus where it remains inactive. We tested this
idea by constructing a tagged RII expression vector and by following
the localization of RII in the presence or absence of NLS-C. As
expected, RII remained cytoplasmic when transfected on its own or when
transfected with wild-type C The egress of C out of the nucleus has been shown to be facilitated by
PKI, which not only binds and inhibits C subunit activity but also acts
as an adapter to target C to the nuclear export machinery (12). There
are three isoforms of PKI ( When CTAG was transfected together with PKI, the
CTAG subunit did not appear in the nucleus to a significant
extent, and gene expression was severely inhibited. The complex of C
and PKI may be too large to diffuse effectively through the NPC, and
the C/PKI that does enter the nucleus might be rapidly exported.
However, when the NLS-CTAG was transfected with various
concentrations of PKI, a more complicated pattern emerged. As the ratio
of PKI to NLS-CTAG expression vector increased, the
induction of CRE-luciferase progressively decreased to basal levels.
The nuclear localization of NLS-CTAG at first diminished
with increasing PKI, but then as more PKI expression vector was added
to the transfections, the tagged C subunit reappeared in the nucleus
and decreased in the cytoplasm. We interpret this to mean that
overexpression of PKI interferes with the nuclear export machinery,
decreasing the capacity to export the C/PKI complex. This contrasts
with previous reports suggesting that the NES on PKI is masked and only
becomes available when C binds to PKI (12, 25). One possibility might be that free PKI interacts with components of the export machinery and
prevents their normal function without actually being a target for
export itself in the absence of C.
Because PKI acts as both an inhibitor and exporter of C subunit, we
examined the relative importance of the export process in the control
of gene expression. A PKI expression vector was constructed with
mutations in the NES (12) that rendered it incapable of interacting
with the export machinery but did not affect C subunit binding. As
expected, the mutant PKI did not alter the cellular distribution of
co-transfected CTAG, which remained distributed in both the
cytoplasm and nucleus. However, when the ability of this mutant PKI to
inhibit gene induction by C subunit was compared with wild-type PKI, we
found only a slight reduction in inhibitory activity, which was most
evident at low levels of PKI transfection. Our results suggest that the export function of PKI is not essential for terminating gene induction by PKA and that the role of PKI export is more likely related to
resetting the system for subsequent gene induction responses.
The ability of the C subunit of PKA to regulate gene expression is well
established and thought to be particularly important in the hormonal
regulation of genes and the activity-dependent activation
of gene expression in neurons. In at least some cases, the C subunit
must gain access to the nucleus to phosphorylate nuclear substrates
such as CREB and members of the ATF family of transcription factors.
However, because C must also be able to phosphorylate cytoplasmic
enzymes and membrane-bound receptors and ion channels, it is perhaps
not surprising that the endogenous C subunit does not have a strong
nuclear localization signal. To terminate gene expression and reset the
system, the C subunit must be rapidly returned to a holoenzyme form.
The observation that PKI can act to transport C out of the nucleus
suggests that it may play an essential role in the temporal regulation
of gene expression by cAMP. Further studies to alter the function of
PKI in vivo may uncover the physiological significance of
PKI as both an inhibitor and transporter of C subunit in the cell.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
168-luciferase reporter, containing 168 base pairs of the human
gonadotropin
promoter (3, 15), was used at a concentration of 2.5 ng/well in all transfections as a cAMP responsive reporter.
CTAG, NLS-CTAG, and RIITAG were
used at a concentration of 2.5 ng/well unless otherwise stated. The
RSV-PKI
construct was generated by synthesis of the nucleotide
sequence inferred from the PKI
amino acid sequence (16). RSV-PKI
was used at 37.5 ng/well unless otherwise designated. To control for promoter competition between MT-1 driven constructs, ZEM3 (an empty
vector containing the metallothionein promoter) was added to each well
to keep levels constant across conditions in each experiment.
Bluescript KS+ was added to each reaction to bring the total DNA to 250 ng/well. Transfection efficiency was normalized by the addition of 50 ng/well of the constitutive reporter RSV-
-galactosidase to correct
for variability between wells and transfection conditions. After the
addition of DNA, the cells were placed in a 3% CO2
incubator for 24 h. To induce expression of the MT-1-driven
constructs, the cells were grown in 2.5% fetal bovine serum
supplemented Dulbecco's modified Eagle's medium with 100 µM ZnCl2 for 12-15 h at 10%
CO2. After zinc treatment, cells used in the reporter
assays were washed once with cold phosphate-buffered saline, lysed, and
collected. The assays were performed as described previously (14).
-galactosidase activity in each sample of
lysed cells. Transfections were performed in triplicate, and the S.D.
for each data point was determined.
and the 12amino acid c-Myc
epitope
(5'-CTTAAGCCCGGGGATCAAGTCTAGGAGGAGTCTTTATTCGAAGACGAGGTATTTGAGTCATTTGAGGAACGGTGTGAA-3') with an ApaI site added for subcloning. 10 ng of the C
expression vector was used as a template to PCR amplify the
1099-nucleotide NcoI-ApaI fragment that was
subsequently cut with NcoI and ApaI and subcloned
into CEV (17), a MT-1-driven C expression vector with the 3' poly(A)
site from the human growth hormone gene. The NLS-CTAG
construct was made using a 5' primer containing a NcoI site
followed by the coding sequence for the first 26 amino acids of the
SV40 large T antigen nuclear localization signal and the initial eight
codons of C
(5'-GAATTCCCATGGCCTCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCGGCAACGCCGCCGCCGCCAAG-3'), and the same 3' primer as was used to generate the
CTAG construct. The PCR product was NcoI- and
ApaI-digested, and the resulting 1192-base pair fragment was
subcloned into ZEM3. The same PCR-based strategy was used to
incorporate the SV40 NLS to the amino-terminal end of NLS-Cwt, with a
3' primer complimentary to the carboxyl-terminal coding region of C
followed by an ApaI site proximal to the termination codon.
RIITAG was made using a 3' primer complimentary to the carboxyl-terminal region of RII
fused to the c-Myc epitope used in
the construction of NLS-CTAG and CTAG with a 5'
primer complimentary to the RII
amino-terminal coding region that
introduced a NcoI site at the initiator methionine.
coding sequence, amplifying a 350-nucleotide fragment and a 150-nucleotide fragment with
25 nucleotides of overlap between the 3' end of the 350-nucleotide fragment and the 5' end of the 150-nucleotide fragment. The two purified fragments were annealed and then underwent two cycles of PCR
without primers to complete the synthesis of the mutagenized PKI
before the addition of primers. Primers against the 5' and 3' ends of
PKI
, containing NcoI and ApaI restriction
sites, respectively, were added to the reaction, and 13 more cycles
were completed. The resulting band purified product was then subcloned
into the MT vector already described. The generation of MT-PKI was done using the same 5' and 3' primers with NcoI and
ApaI sites. The amplified PKI
-coding region was
subsequently subcloned into the MT vector. Multiple clones of both
MT-PKI and MT-PKIMUT were selected and tested.
RESULTS
View larger version (38K):
[in a new window]
Fig. 1.
Subcellular localization and transcriptional
activity of free and RII-bound C subunit. JEG-3 cells were
transiently transfected with either CTAG alone or
CTAG in the presence of the regulatory subunit, RII .
Panel A, expression of the Myc-tagged C
subunit
CTAG and RII
are regulated by the mouse metallothionein
promoter, which is stimulated by the addition of 100 µM
ZnCl2. Panel B, induction of CRE-luciferase
activity normalized against the constitutive reporter
RSV-
-galactosidase. Panel C, subcellular localization of
CTAG protein (green) as determined by
immunocytochemistry using the c-Myc antibody and laser-scanning
confocal microscopy. The cells were counter-stained with propidium
iodide (red) to visualize the nucleus.
View larger version (31K):
[in a new window]
Fig. 2.
Nuclear localization of the C subunit and
concomitant CRE transcriptional activity in the presence or absence of
the RII regulatory subunit. Panel
A, the SV40 NLS was added to the amino-terminal end of the C
construct. Panel B, transcriptional activity of the
NLS-CTAG protein in the presence and absence of coexpressed
RII
. Panel C, confocal imaging of NLS-CTAG
(green) and propidium iodide staining of the nucleus
(red). The yellow regions represent
colocalization of NLS-CTAG in the nucleus.
expression vector had no effect on the
localization of the RIITAG (Fig. 3C). However,
when RIITAG was co-transfected with NLS-C
(diagrammed in
Fig. 3A), there was complete nuclear translocation of the
RIITAG protein (Fig. 3D). This confirmed that an
active and exposed NLS on the C subunit was sufficient to translocate
the entire holoenzyme to the nucleus.
View larger version (39K):
[in a new window]
Fig. 3.
Translocation of the regulatory subunit into
the nucleus by association with the nuclear-targeted
NLS-C subunit. Panel A,
localization of the regulatory subunit was determined by placing the
c-Myc epitope on the carboxyl terminus of RII
, which was
subsequently subcloned into the MT-1/hGH vector. The C
and NLS-C
constructs were both subcloned into the same vector to equalize
expression levels. Panel B, expression of RIITAG
alone resulted in cytoplasmic sequestration. Thin sections taken with
the confocal microscope demonstrated that in the plane of the nucleus,
there was no detectable RIITAG protein (green)
colocalized with the propidium iodide counter-stain (red).
Panel C, co-transfection of RIITAG with
wild-type C
. Panel D, co-transfection of
RIITAG with NLS-C
.
View larger version (35K):
[in a new window]
Fig. 4.
Subcellular localization and functional
activity of CTAG in the presence of PKI. Panel
A, JEG-3 cells were transfected with either CTAG alone
or co-transfected with CTAG and CMV-PKI . Panel
A, overexpression of CTAG alone gave ubiquitous
distribution throughout the cytoplasm and nucleus, consistent with
previous experiments. The addition of CMV-PKI
restricted the
localization of CTAG to the cytoplasm. Panel B,
overexpression of CTAG resulted in transactivation of the
CRE-luciferase reporter. Co-transfection of CTAG with
CMV-PKI
resulted in inhibition of CRE-luciferase activity.
View larger version (53K):
[in a new window]
Fig. 5.
Localization patterns observed when the
NLS-CTAG subunit has been cotransfected with
PKI . Panel A, cells transfected
with NLS-CTAG alone. Panel B, co-transfection of
NLS-CTAG with PKI
resulted in the emergence of three
different localization patterns. In the top row, three
examples of predominantly nuclear localization are presented. The
second row in panel B (Cyto + Nucleus)
depicts examples where strong cytoplasmic and nuclear staining are both
evident. The bottom row (Cyto) shows cells with
predominantly cytoplasmic sequestration of NLS-CTAG with
minimal to no protein present in the nucleus.
View larger version (28K):
[in a new window]
Fig. 6.
NLS-CTAG transcriptional activity
and subcellular localization with increasing PKI
concentrations. JEG-3 cells were transfected with
NLS-CTAG alone or co-transfected with various
concentrations of PKI
. Panel A, the transcriptional
activity of NLS-CTAG is plotted as -fold induction
calculated relative to the normalized levels of CRE-luciferase activity
in cells transfected with just the reporter constructs. The amount of
NLS-CTAG (2.5 ng/well) is constant in all conditions, and
the level of RSV-PKI
is increased as shown in the figure.
Panel B, the subcellular localization was characterized by
visual examination of cells under each transfection condition. The
three localization patterns (Nucleus (
), Cyto + Nucleus (
), and Cyto only (
)) observed correspond
to those depicted in Fig. 5.
View larger version (21K):
[in a new window]
Fig. 7.
Effects of mutagenesis of the NES of
PKI on subcellular localization and
transcriptional regulation by CTAG. Site-directed
mutagenesis was performed to mutate the leucines and isoleucine within
the hydrophobic NES of PKI
. The wild-type and mutant PKI were then
subcloned into the MT/hGH expression vector used to express
CTAG. Panel A, a diagram of the PKI constructs
with the amino acid sequence of the NES shown. Panel B, the
ability of wild-type or mutant PKI
to inhibit C subunit-mediated
transactivation of the CRE-luciferase construct is shown. Panel
C, the subcellular localization patterns observed for
CTAG in the presence of various concentrations of wild-type
(MT-PKI) or mutant (MT-PKImut) PKI
is shown.
DISCUSSION
and C
1 isoforms of C have also
been shown to be amino-terminal myristylated (24), and this lipid
modification might hypothetically play a role in membrane association,
although no evidence for this exists to date. The heat-stable inhibitor
of PKA, PKI, has also been shown to facilitate nuclear export of C
subunits back to the cytoplasm (7, 25), and this may help to terminate
the transcriptional response. We have used an epitope-tagging strategy
to localize C subunits that were free or bound to either RII or PKI.
The nuclear distribution of C subunit was correlated with the
transcriptional activation of a cAMP-responsive reporter gene.
. However, in the presence of NLS-C, the
RII was transported into the nucleus, leaving no visible cytoplasmic
staining. Despite the known array of cytoplasmic AKAPs, which bind RII
with high affinity, a strong NLS was capable of transporting all of the
holoenzyme to the nucleus. Although all cells appear to have AKAPs, we
have not investigated which AKAPs are present in JEG-3 cells, and it is
possible that if higher affinity AKAPs had been present, some of the
holoenzyme might have remained in the cytoplasmic compartment.
,
, and
) (28, 29), and in addition
to the C subunit interacting domain, each has a highly conserved
leucine-rich NES. These leucine-rich nuclear export sequences have been
shown to interact with CRM1 (exportin 1) and together with Ran, form a
complex that can be exported through the NPC (30, 31). Once in the
cytoplasm, GTPase-activating proteins bind and stimulate the hydrolysis
of Ran-bound GTP, causing the complex to dissociate. This process returns the C subunit to the cytoplasm where it is presumed to bind to
R subunits when cAMP has returned to basal levels. PKI may therefore
function to terminate the nuclear actions of C subunit and rapidly
reset the PKA system to respond again.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM32875 (to G. S. M.) and HD33057 (to R. L. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Training Grant
2T32-GM07270 in molecular and cellular biology.
§ To whom correspondence should be addressed: Dept. of Pharmacology, Box 357750, University of Washington, Seattle, WA 98195. Tel.: 206-616-4237; Fax: 206-616-4230; E-mail: mcknight{at}u.washington.edu.
2 J. C. Wiley, L. A. Wailes, R. L. Idzerda, and G. S. McKnight, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, protein kinase A; R, regulatory; C, catalytic; PKI, protein kinase inhibitor; NLS, nuclear localization signal; NES, nuclear export signal; PCR, polymerase chain reaction; NPC, nuclear pore complex; CRE, cAMP response element; RSV, Rous sarcoma virus; AKAP, A kinase-anchoring protein; CMV, cytomegalovirus; CREB, cAMP responsive element binding protein; hGH, human growth hormone.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|