 |
INTRODUCTION |
The cyclic nucleotides, cAMP and cGMP, are intracellular second
messengers mediating the actions of a large number of hormones and
neurotransmitters. These cyclic nucleotides act to allosterically regulate the action of a small number of important proteins. Unlike cAMP, which acts mainly through cAMP-dependent protein
kinases (cAKs),1 cGMP is able
to activate three classes of proteins: ion channels, phosphodiesterases, and cGMP-dependent protein kinases
(cGKs). The cAKs and the cGKs are highly homologous protein kinase
families with similar substrate specificities. Phosphorylation of
cellular proteins by both families of kinases leads to alterations in
calcium mobilization, protein phosphatase activity, ion channel
function, gene transcription, smooth muscle contractility, and platelet aggregation (1-4).
The cGKs are classified into two types based on their historical order
of characterization. The type I enzymes (cGKIs) are highly expressed in
lung (5), cerebellum (6), platelets (7), and smooth muscle (8). Two
type I isoforms, I
and I
, arise from the alternative splicing of
a single gene (9-12). These two forms differ in their amino-terminal
autoinhibitory domains but share the same cGMP-binding sites and
catalytic domains (4, 13). In Purkinje cells (6), smooth muscle cells
(14, 15), monocytes (16), and neutrophils (17, 18), the majority of the
cGKI immunoreactivity is soluble and localized to the cytoplasm. A
second type of cGK, termed the type II cGK (cGKII), is highly expressed
in intestinal microvilli (19) and is encoded by a gene distinct from
that encoding cGKI proteins (20, 21). While cGKI isoforms are soluble
proteins, cGKII is particulate and associated with cellular membranes
(19). Both types possess amino-terminal leucine zipper motifs and exist
as homodimers in native tissues (13). In contrast to the cAKs, which
have separate catalytic and regulatory subunits, each monomer of the
cGKs consists of both a regulatory domain and a catalytic domain
contained in the same polypeptide (13).
Many mammalian tissues coexpress isoforms of cAK and cGK, where the cAK
and cGK proteins are thought to play distinct roles in cellular
regulation. The in vitro substrate specificities of the cAKs
and the cGKs are very similar, although a number of proteins have been
identified as specific substrates for either cAKs or cGKs. For example,
the type I regulatory (R) subunit of cAK (22), G-substrate (23),
histone H2B (24), and the bovine lung cGMP-binding cGMP-specific
phosphodiesterase (25, 26) are specific substrates of cGKI in
vitro, while the cAMP response element-binding protein (CREB) has
been shown to be a specific in vitro substrate of cAK (76).
Although in vitro substrate specificity may be an important indicator of in vivo substrate specificity, recent evidence
suggests that colocalization of kinase and substrate in the cell is at least an equally important factor (27).
The cAMP signaling pathway is used to regulate the transcription of
many genes and involves the phosphorylation of specific transcription
factors by the C subunit of cAK (28). In the absence of cAMP, cAK
exists predominantly as an inactive tetrameric holoenzyme composed of
two R subunits and two C subunits. The inactive holoenzyme may be
localized diffusely in the cytoplasm or localized to specific subcellular compartments by interaction of the R subunits with protein
kinase A anchoring proteins (27). Upon cytoplasmic elevation of cAMP,
cAMP binds to each R subunit, causing the holoenzyme complex to
dissociate into a homodimer of R subunits and two catalytically active
C subunits. Once released, the C subunit can phosphorylate cytoplasmic
substrates, and because of its small size (40 kDa), it can also
passively diffuse through the nuclear pores into the nucleus (29). In
the nucleus, the C subunit can phosphorylate nuclear transcription
factors, such as members of the CREB/ATF family, which bind directly to
specific enhancer sequences and alter levels of gene transcription (30,
31). Substrates of cAK therefore include both cytoplasmic and nuclear proteins.
Like the cAKs, the cGKs are capable of activating gene transcription in
a cyclic nucleotide-dependent manner (32-35). For example, in PC-12 cells, stimulation of the NO/cGMP pathway leads to increased expression of the immediate early genes c-fos and
junB. Importantly, this induction can be blocked by the
selective cGK inhibitor KT5823, suggesting that this induction is
dependent on cGK activity (35).
While the mechanisms by which the cAKs activate gene transcription have
been well characterized, little is known about cGK regulation of gene
transcription. In this report, we demonstrate that both transiently
expressed and endogenous cGKs are localized to the cytoplasm in
mammalian cells. Treatment of cells with cyclic nucleotide does not
cause nuclear accumulation of the cGKs, while the C subunit of cAKs
does translocate to the nucleus. As a result of its cytoplasmic
restriction, cGKI
is a weaker activator of CRE-dependent
gene transcription than cAKII. These data suggest that regulation of
gene transcription by cGK involves phosphorylation of cytoplasmic
substrate protein(s) that remain to be characterized.
 |
MATERIALS AND METHODS |
Screening of Mouse Brain cDNA Library for Full-length Murine
cGKI
cDNA--
cDNA library screening was performed
essentially as described previously (20). A 1.0-kilobase pair
EcoRI-SalI restriction fragment from pCGKI.6 was
isolated and labeled by random primer extension in the presence of
[
-32P]dATP. The resulting radiolabeled DNA fragments
were used to screen a mouse brain cDNA library. Clones mcGKI
3D
and mcGKI2.2 were isolated, and their inserts were restriction-mapped.
mcGKI
3D, which contains the entire open reading frame (ORF) of
murine cGKI
, was fully sequenced in both directions using Sequenase
DNA polymerase (U.S. Biochemical Corp.). mcGKI2.2, which lacks the
first 1130 bp of the cGKI
ORF, was partially sequenced to confirm a
2-bp deletion in the ORF of mcGKI
3D. The murine cGKI
cDNA
sequence derived from the sequencing of both clones has been submitted to the GenBankTM data base. Sequence analyses were
performed using DNASTAR software.
Construction of Murine cGKI
Mammalian Expression
Vector--
The pCMV.mcGKI
mammalian expression vector was
constructed by the polymerase chain reaction (PCR) method using the
oligonucleotides 5'-GGA GAT CTC CAC CAT GGG CAC CCT GCG GGA TTT AC-3'
and 5'-GGA TCC AAG CTT ACA TTA GAA GTC TAT GTC-3' with cDNA clone
mcGKI
3D as a template. The resulting 2100-bp PCR fragment containing
the coding region of murine cGKI
flanked by a BglII site
and a BamHI site was digested with BglII and
BamHI and ligated into the BglII site of pCMV.Neo
(36) to create pCMV.mcGKI
. During sequencing of cDNA clone
mcGKI
3D, a two-nucleotide deletion (Fig. 1) was discovered in the
ORF sequence. To correct the deletion in pCMV.mcGKI
, a 600-bp
BsrGI-AflII restriction fragment from cDNA
clone mcGKI2.2, containing the correct sequence, was ligated into
BsrGI- and AflII-digested pCMV.mcGKI
.
pCMV.mcGKI
was sequenced to confirm the entire coding region sequence.
Transient Transfection of Mammalian Cells and in Vitro Kinase
Activity Determination--
10-cm plates of CV-1 or HEK293 cells were
transiently transfected using a calcium phosphate coprecipitation
method (37). 48 h after application of DNA precipitates, plates
were washed twice with ice-cold phosphate-buffered saline (PBS).
Following the addition of 200 µl of homogenization buffer (10 mM sodium phosphate (pH 7.0), 1 mM EDTA, 1 mM dithiothreitol, 250 mM sucrose) containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A,
and 1 µg/ml leupeptin (Sigma), cells were scraped into separate tubes
and sonicated twice for 10 s. For kinase activity determinations, cyclic nucleotide (50 µM) was added to or omitted from
separate tubes containing a phosphotransferase assay mixture consisting of 20 mM Tris (pH 7.5), 10 mM MgAc, 500 µM IBMX, 200 µM ATP, 11 nM
[
-32P]ATP (ICN) (specific activity = 200-300
cpm/pmol), 10 mM NaF, 10 mM dithiothreitol, and
the synthetic phosphate acceptor peptide Kemptide (LRRASLG; 150 µM) or H2Btide (RKRSRAE; 110 µM). When assaying cGKI
activity, protein kinase inhibitor (PKI) peptide (1 µM) was added to all tubes. The assay was initiated by
the addition of cell extracts (0.2 mg/ml), and the phosphotransfer reaction was allowed to proceed for 10 min at 30 °C. The assay was
terminated by spotting aliquots onto P81 phosphocellulose (Whatman).
The P81 phosphocellulose was washed in 10 mM phosphoric acid and counted.
Site-directed Mutagenesis of cGKI
and Construction of cGKI
Deletion Mutant--
The mutations in pCMV.mcGKI
R75A,
pCMV.mcGKI
S79D, pCMV.mcGKI
K404R, pCMV.mcGKI
K407R/R409Q,
pCMV.mcGKI
H419Q, and pCMV.mcGKI
W530R were generated by a
two-step PCR method using mutagenic primers as described previously
(38). The resulting PCR fragments were subcloned into pCMV.mcGKI
using convenient restriction sites, and each construct was verified by
DNA sequencing of the PCR-amplified region.
pCMV.FC/CD, the expression plasmid for the chimeric cGKI deletion
mutant, was constructed by replacing the amino-terminal regulatory
domain (amino acids 1-355) of cGKI
with an amino-terminal Flag
epitope (DYKDDDDK) followed by the amino-terminal (amino acids 1-20)
of murine C
(39). The two-step PCR method was used to create this
chimeric cDNA. Initially, a C
PCR fragment containing a
BamHI site and an amino-terminal Flag epitope was generated using the primers 5'-GGG GGA TCC ACC ATG GAC TAC AAG GAC GAC GAT GAC
AAG GGC AAC GCC GCG GCC GCC AAG AA-3' and 5'-AAG TAC TCC GGA GTC CCA
C-3' with pGEM-4.C
(40) as a template. The resulting PCR fragment
was ligated into pGEM-T (Promega) to create pGEM-T.Flag-C
1. To
generate the cAK/cGK chimera, PCR fragments coding for the Flag-tagged
amino terminus of murine C
and the carboxyl terminus of murine
cGKI
were amplified in separate PCRs. A PCR fragment coding for the
amino terminus of murine C
was generated using primer 5'-CAG GAA ACA
GCT ATG AC-3' and the chimeric primer 5'-GGC TTC ATA TTT TGC TTT TGC
TAG GAA CTC TTT CAC GCT-3' with pGEM-T.Flag-C
1 as a template. A PCR
fragment coding for the carboxyl terminus of murine cGKI
was
generated using primer 5'-AGG CAC GCT TCC ATC AAC-3' and the chimeric
primer 5'-AGC GTG AAA GAG TTC CTA GCA AAA GCA AAA TAT GAA GCC-3' with
pCMV.mcGKI
as a template. The partially overlapping PCR fragments
were isolated, combined with flanking primers 5'-GGG GGA TCC ACC ATG
GAC TAC AAG GAC GAC GAT GAC AAG GGC AAC GCC GCG GCC GCC AAG AA-3' and
5'-AGG CAC GCT TCC ATC AAC-3', and amplified. The resulting fragment
was cut with BamHI and BsrGI, isolated, and
ligated into the BglII- and BsrGI-digested
pCMV.mcGKI
to create pCMV.FC/CD. pCMV.FC/CD was restriction-mapped
and sequenced to confirm the amplified sequence.
Construction of Murine cGKII and cGKIIG2A Mammalian
Expression Vectors and Generation of Polyclonal Antibodies to Murine
cGKII--
pCMV.mcGKII and pCMV.mcGKIIG2A were constructed by PCR. PCR
fragments were generated using the following oligonucleotides as
forward primers: 5'-GAG ATC TGC TAG CCC ACC ATG GGA AAT GGT TCA GTG-3'
(cGKII) and 5'-GAG ATC TGC TAG CCC ACC ATG GCA AAT GGT TCA GTG-3'
(cGKIIG2A). The oligonucleotide 5'-CTC TAT CGA GGG CCC AAG-3' was used
as the reverse primer, and pCMV.His6cGKII (41) was used as
the template in the PCRs to amplify 780-bp DNA fragments coding for the
amino termini of mcGKII and mcGKIIG2A. pCMV.His6cGKII was
digested with BglII. The full-length cGKII insert was
subcloned into the BglII site of pSP73 (Promega) to create
pSP73.His6cGKII. The PCR fragments described above were digested with BglII and NsiI, isolated, and
ligated individually into pSP73.His6cGKII, which had been
fully digested with NsiI and partially digested with
BglII, generating pSP73.mcGKII and pSP73.cGKIIG2A.
pSP73.mcGKII and pSP73.cGKIIG2A were individually cut with
BglII, and 2.4-kilobase pair fragments were isolated and
ligated into BglII-cut pCMV.Neo to generate pCMV.mcGKII and pCMV.mcGKIIG2A, respectively. Both pCMV.mcGKII and pCMV.mcGKIIG2A were
restriction-mapped, and the amplified regions were fully sequenced.
His6cGKII was expressed in Spodoptera
frugiperda (Sf9) cells and purified as described previously
(41). Purified His6cGKII was used to immunize rabbits
for polyclonal antibody production (Research Genetics Inc.).
Luciferase Assays--
CV-1 and HEK293 cells were grown
separately on 10-cm plates to 30 and 50% confluency, respectively, and
transfected using a standard calcium phosphate method (37) with 0.5 µg of the cAMP-responsive reporter construct human chorionic
gonadotropin-luceriferase (HCG-luciferase) (42) as well as the
indicated amounts of pRSV.
gal and expression vectors. The total
amount of plasmid DNA was brought to 30 µg with the parental vector
pCMV.Neo. 24 h after transfection, cells were incubated for an
additional 24 h in Dulbecco's modified Eagle's medium (DMEM)
with or without added cyclic nucleotides. Following treatment, cells
were washed twice with ice-cold PBS, scraped into homogenization
buffer, sonicated, and assayed for both luciferase and
-galactosidase activities as described (43).
Identification of a cDNA Clone Encoding Murine VASP,
Construction of a Flag-tagged VASP Mammalian Expression Vector, and
Western Blot Analysis--
A full-length cDNA sequence coding for
murine VASP (I.M.A.G.E. Consortium clone identification no.
354921/GenBankTM accession no. W45954) (44) was identified
in a search of the expressed sequence tag data base for protein
sequences homologous to human VASP using the BLAST algorithm (45). This
I.M.A.G.E. Consortium (LLNL) cDNA clone was obtained from Genome
Systems Inc. It was sequenced in both directions using Sequenase DNA
polymerase (U.S. Biochemical), and sequence analyses were performed
using DNASTAR software.
The nucleotide sequence of the murine cDNA derived from a mouse
embryo (embryonic day 13.5-14.5) cDNA library differs from that of
the published murine VASP genomic sequence (46), in that it contains a
G instead of an A at nucleotide position 955, a three-nucleotide
deletion from position 1192 to 1194, and an A instead of a G at
nucleotide position 1867. The first difference changes threonine 209 to
an alanine, the second difference eliminates glutamine 292, and the
third difference is located in the 3'-untranslated region. The murine
VASP cDNA sequence has been submitted to the GenBankTM
data base.
A mammalian expression plasmid encoding amino-terminal Flag-tagged
murine VASP protein was constructed using the PCR method. A PCR
fragment containing an amino-terminal Flag epitope (DYKDDDDK) was
generated using the primers 5'-GGA TCC GGT ACC TCC ACC ATG GAC TAC AAG
GAC GAC GAT GAC AAG GGC GGA GGT ATG AGC GAG ACG GTC ATC TGT TCC-3' and
5'-GGA TCC CTC GAG TCA AGG AGA ACC CCG CTT CCT CAG-3' with clone 354921 as a template. The resulting PCR fragment was digested with
BamHI, isolated, and ligated into BglII digested
pCMV.Neo to create pCMV.Flag-VASP. pCMV.Flag-VASP was restriction-mapped, and the inserted DNA was fully sequenced.
For phosphorylation studies, 10-cm plates of CV-1 cells at 30%
confluency were transfected using a calcium phosphate transfection method as described previously (37). Each plate was transfected with 20 µg of pCMV.Flag-VASP. 24 h after transfection, the media was
changed, and the cells were incubated for 24 h in DMEM without serum. Following treatment with cyclic nucleotides, cells were quickly
washed three times with 5 ml of ice-cold PBS, scraped into 400 µl of
ice-cold homogenization buffer, and sonicated for 10 s. Extracts
were quickly diluted with 500 µl of RIPA buffer (20 mM
sodium phosphate (pH 7.0), 300 mM NaCl, 2% sodium
deoxycholate, 2% Triton X-100, 2% SDS, 2 mM EDTA, 2 mM EGTA, 100 mM NaF, 10 mM sodium
pyrophosphate) and vortexed. Fractions were denatured in SDS-PAGE
buffer at 95 °C for 5 min, resolved on 10% SDS-PAGE gels, and
transferred to 0.45-µm nitrocellulose membranes (BA-85; Schleicher
and Schuell). Membranes were blocked for 1 h in TBST (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween
20) supplemented with 5% nonfat dried milk and subsequently incubated
with a 1:1000 dilution of an anti-Flag epitope antibody (M2) (Eastman
Kodak Co.) in TBST supplemented with 5% nonfat dried milk for 1 h. Filters were washed three times for 10 min with TBST and incubated
with an 35S-labeled sheep anti-mouse antibody (0.5 µCi/ml) (Amersham Pharmacia Biotech) in PBS supplemented with 0.5%
bovine serum albumin and 0.1% Triton X-100 as the secondary antibody
for 1 h. Following the final set of three 10-min washes with TBST,
the blots were dried and quantitated. PhosphorImager quantitation
was performed in a PhosphorImager apparatus and analyzed with
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Immunofluorescence Microscopy--
CV-1 cells were grown in DMEM
containing 10% fetal calf serum in eight-well tissue culture chambers
on poly-L-lysine-coated glass slides (Lab-Tek) to 30%
confluency. Cells were transiently transfected using a standard calcium
phosphate method (37) with 0.1 µg of pCMV.mcGKI
; 0.01 µg of
pCMV.Flag-C
3; and 0.04 µg of pCMV.RII
, 0.1 µg of
pCMV.mcGKI
S79D, or 0.1 µg of pCMV.FC/CD. Total plasmid
concentration was maintained at 0.25 µg by the addition of the
parental vector, pCMV.Neo. Following a 12-h incubation with DNA
precipitates, cells were washed once with DMEM containing 10% fetal
calf serum and grown for 24 h. Indicated cells were stimulated
with 8-Br-cAMP (1 mM) or 8-Br-cGMP (1 mM) and
3-isobutyl-1-methylxanthine (500 µM) in DMEM for various
times at 37 °C. Following stimulation, cells were washed twice with
ice-cold PBS and fixed with 4% formaldehyde in PBS for 10 min at room
temperature followed by a 1:1 mixture of methanol and acetone for 5 min. After washing three times with PBS, cells were incubated with a
rabbit polyclonal antibody generated against the carboxyl-terminal 15 amino acids of cGKI (anti-cGMP-PK CT) (Upstate Biotechnology, Inc.) at
a 1:1000 dilution or an anti-Flag epitope antibody (M2) (Eastman Kodak)
at a 1:2000 dilution in blocking buffer (PBS supplemented with 1%
bovine serum albumin, 3% goat serum, and 0.1% saponin (Sigma)) for
1 h at room temperature. After four washes with wash buffer (PBS
supplemented with 0.1% saponin), a 1:1000 dilution of
Cy3-F(ab')2 fragment goat anti-rabbit IgG (Jackson) or a
1:3000 dilution of Cy3-F(ab')2 fragment goat anti-mouse IgG
(Jackson) was incubated with the cells for 1 h in the dark in
blocking buffer. Prior to examination by fluorescence microscopy, cells
were washed four times for 2 min in wash buffer and twice for 2 min in
PBS.
CV-1 cells transiently transfected with 0.2 µg of pCMV.mcGKII or 0.2 µg of pCMV.mcGKIIG2A or A7r5 cells were grown, stimulated, fixed, and
examined as above. Treated cells were incubated with anti-cGKII serum
or anti-cGMP-PK CT at a 1:1000 dilution in blocking buffer for 1 h
at room temperature. After four washes with wash buffer, a 1:1000
dilution of Biotin-SP-F(ab')2 fragment goat anti-rabbit IgG
(Jackson) was incubated with the cells for 1 h in blocking buffer.
After four washes with wash buffer, a 1:1000 dilution of
Alexa-488-conjugated streptavidin (Molecular Probes, Inc., Eugene, OR)
was incubated with the cells for 1 h in blocking buffer in the
dark. Finally, cells were washed four times for 2 min in wash buffer
and twice for 2 min in PBS.
In control experiments, to verify the specificity of the anti-cGMP-PK
CT antibody for endogenous cGKI in A7r5 cells, the anti-cGMP-PK CT
antibody (40 nM) was preincubated for 1 h at room
temperature with a peptide coding for the carboxyl-terminal 18 amino
acids of cGKI (5 µM) (STRESSGEN) prior to incubation with cells.
 |
RESULTS |
Cloning and Sequencing of Murine cGKI
--
A single full-length
cGKI
cDNA clone was isolated from a mouse brain cDNA
library. The cDNA was fully sequenced and shown to contain 2841 bp
(Fig. 1). The murine cGKI
cDNA
contains a short 89-bp 5'-untranslated region, an ORF of 2061 nucleotides, and a 691-bp 3'-untranslated region. The predicted murine
cGKI
protein contains 686 amino acids with a calculated molecular
mass of 77.8 kDa and shows greater than 99% amino acid identity to human cGKI
(9). Despite 139 nucleotide differences in the ORF
sequence, there are only three amino acid differences between murine
cGKI
and human cGKI
: Glu242 to Asp,
Thr280 to Gln, and Asn671 to Ser (Fig. 1).

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Fig. 1.
Nucleotide and predicted amino acid sequence
of murine cGKI . The sequence represents a
composite of the sequences derived from mcGKI 3D and mcGKI2.2. Amino
acid sequence of murine cGKI inferred from the composite nucleotide
sequence is represented below the DNA sequence with the
one-letter amino acid codes. Nucleotide numbers are indicated at the
left of the sequence and amino acid numbers at the
right of the sequence. Amino acid residues from the human
cGKI sequence (9) that are divergent from the predicted murine
cGKI amino acid sequence are shown individually below the
murine cGKI amino acid sequence. The two nucleotides (1672 and 1673)
deleted in mcGKI 3D but present in mcGKI2.2 are
underlined. (GenBank accession no. AF084547)
|
|
Effect of cGKI
Overexpression on CRE-dependent Gene
Transcription--
To determine whether cGKI
was capable of
regulating CRE-dependent gene transcription, we examined
the ability of cGKI
to transactivate the cAMP-responsive HCG
promoter. CV-1 cells (Fig. 2A)
or HEK293 cells (Fig. 2B) were transfected with a constant amount of the cGKI
expression vector along with the HCG-luciferase reporter plasmid (42). Control cells were transfected with the HCG-luciferase reporter plasmid alone. Transfected cells were treated
with or without 8-Br-cGMP (1 mM) for 20 h. CV-1 and
HEK293 cells were chosen for this experiment because regulation of
CRE-dependent gene transcription by cAK has been
characterized by transfection experiments previously in these cell
lines (36, 43, 47). In both CV-1 and HEK293 cells, transfection of wild
type cGKI
only minimally stimulated luciferase gene transcription in
the absence of cyclic nucleotide treatment (Fig. 2, A and
B). CV-1 and HEK293 cells transfected with the
HCG-luciferase reporter plasmid alone showed minimal responses to
8-Br-cGMP treatment (Fig. 2, A and B). When CV-1
cells overexpressing cGKI
were treated with 8-Br-cGMP (1 mM), a small but reproducible increase in luciferase activity was observed (Fig. 2A). In HEK293 cells,
overexpression of cGKI
increased luciferase activity following
8-Br-cGMP treatment 13-fold (Fig. 2B). Thus, the
HCG-luciferase reporter plasmid is more sensitive to overexpression of
cGKI
in HEK293 cells than in CV-1 cells. The greater sensitivity of
the luciferase reporter assay in HEK293 cells may be due to a number of
factors including the levels of accessory transcription factors (48,
49) or cellular phosphatases (50, 51). The data from both CV-1 and HEK293 cells indicate that cGKI
is capable of inducing
CRE-dependent gene transcription in mammalian cell
lines.

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Fig. 2.
Activation of CRE-dependent gene
transcription by cGKI and cAKII.
Transcriptional assay measuring CRE-dependent gene transcription. A and B, CV-1 cells
(A) or HEK293 cells (B) were transiently
transfected with (cGKI) or without (Neo)
pCMV.mcGKI (10 µg). 24 h post-transfection, cells were
treated for 20 h in the absence of serum with (gray
bar) or without (black bar) 8-Br-cGMP (1 mM) and IBMX (500 µM). All plates received a
cAMP-responsive reporter construct (HCG-luciferase) (0.5 µg),
pCMV.Flag-PKI (2 µg), and pRSV. gal (5 µg). C, CV-1
cells were transiently transfected with pCMV.mcGKI (10 µg) and
pCMV.Flag-PKI (2 µg) or pCMV.Flag-C 3 (1 µg) and pCMV.RII (4 µg). 24 h post-transfection, cells expressing cGKI ( ) or
RII 2C 2 ( ) were treated for 20 h
in the absence of serum with varying concentrations of 8-Br-cGMP and
IBMX (500 µM) ( ) or 8-Br-cAMP and IBMX (500 µM) ( ). All plates received a cAMP-responsive reporter
construct (HCG-luciferase) (0.5 µg) and pRSV. gal (5 µg).
Following nucleotide treatment, cells were harvested and assayed for
luciferase and -galactosidase activities. Luciferase activity was
corrected for transfection efficiency by dividing by -galactosidase
activity (RLU/ gal). The error bars depict the
S.D. D, comparison of activation of
CRE-dependent gene transcription by cGKI and cAKII.
Fold Induction represents the increase in luciferase
activity of 8-Br-cGMP (1 mM)-treated cGKI -transfected
cells (black bar) or 8-Br-cAMP (1 mM)-treated
cAK-transfected cells (gray bar) normalized to the increase
in luciferase activity resulting from treatment of cGKI -expressing
cells with 8-Br-cGMP (1 mM). The increase in luciferase
activity resulting from treatment of cGKI expressing cells with
8-Br-cGMP (1 mM) was assigned a value of 1.
|
|
cGKI
Is a Relatively Weak Activator of CRE-dependent
Gene Transcription--
For comparison of cGKI
and cAK activation
of CRE-dependent gene transcription, the same
HCG-luciferase reporter assay was employed. To generate comparable
levels of cGKI
and cAKII in transfected cells, the quantity of each
expression vector transfected was carefully titrated to produce similar
amounts of cGKI
and cAKII protein and kinase activity (data not
shown). For cGK expression, CV-1 cells were transfected with 10 µg of
pCMV.mcGKI
. For cAK expression, cells were transfected with both 1 µg of pCMV.Flag-C
3 and 4 µg of pCMV.RII
(52). Control cells
were transfected with the parental pCMV.Neo vector alone. Extracts from
cGKI
- and cAKII-transfected cells possess similar levels of cGKI
and cAKII protein by quantitative Western blotting using purified
proteins as standards (data not shown). The extracts from cGKI
- and
cAKII-transfected cells also showed similar specific activities with
in vitro kinase assays in which the nonspecific peptide
Kemptide (150 µM) was used as the phosphoacceptor
(extracts from cGKI
transfected cells = 0.73 nmol/min/mg and
extracts from cAKII transfected cells = 0.84 nmol/min/mg). The
maximal cGMP- or cAMP-stimulated protein kinase activities of extracts
from cGKI
-transfected cells and extracts from cAKII-transfected cells were at least 10-fold higher than endogenous kinase activity in
extracts from control CV-1 cells (data not shown).
Once similar levels of kinase activity were obtained, transcriptional
regulation was examined (Fig. 2C). In cells expressing cGKI
or cAKII, stimulated luciferase activity was half-maximal at
100 µM 8-Br-cGMP and 30 µM 8-Br-cAMP,
respectively. Cotransfection with cGKI
gave a maximal 2-fold
stimulation of reporter gene expression over basal levels, whereas
cotransfection with cAK gave a maximal 14-fold stimulation (Fig.
2C). cAKII-transfected cells showed significantly higher
luciferase activity at all cyclic nucleotide concentrations examined
(Fig. 2C). Although CV-1 cells express both endogenous cGKI
and cAK, cells transfected with the reporter gene alone showed no
response to 8-Br-cGMP treatment but a significant response to 8-Br-cAMP
treatment (4-fold) (data not shown). A reduced potency of cGKI
relative to cAKII was also observed in HEK293 cells, where
cotransfection with cGKI
gave a maximal 13-fold induction in
comparison with the 250-fold induction seen with the C subunit of cAK
(data not shown). cAK's maximal induction of luciferase activity was
52-fold higher than cGKI
's in CV-1 cells and 25-fold higher than
cGKI
's in HEK293 cells (Fig. 2D). Our results in both
CV-1 and HEK293 cells indicate that when compared with cAK, cGKI
is
only a weak activator of CRE-dependent gene transcription.
VASP Is Phosphorylated Efficiently by Both cGKI
and cAK in
Vivo--
Since cGKI
and cAKII were expressed at equal amounts, but
showed a 10-fold difference in their ability to regulate transcription, experiments were designed to compare the ability of cGKI
and cAK to
phosphorylate a mutually recognized substrate in vivo.
A cytoplasmic substrate for cGK and cAK has been identified in human
platelets (53). This 46-kDa protein, termed VASP
(vasodilator- and A
kinase-stimulated phosphoprotein), has been
shown to be widely expressed and localized to cytoplasmic focal
adhesions and stress fibers (54). The human VASP protein was
demonstrated to be phosphorylated at three sites to varying extents by
both cGK and cAK. Phosphorylation of one of these sites,
Ser157, caused a shift in mobility from 46 to 50 kDa in a
SDS-PAGE gel. VASP was chosen as a good substrate for comparing cGK and
cAK activity, since Ser157 is phosphorylated comparably by
both cGK and cAK in vivo (55).
A full-length murine VASP cDNA was sequenced (see "Materials and
Methods"), and the murine VASP protein predicted by the ORF was 87%
identical to the human VASP protein (56). All three human VASP cyclic
nucleotide-dependent phosphorylation sites are conserved in
murine VASP (46). To differentiate transfected VASP from endogenous
VASP, we constructed an amino-terminal Flag-tagged VASP mammalian
expression vector, pCMV.Flag-VASP (see "Materials and
Methods").
To verify that Flag-tagged murine VASP was expressed and properly
localized, cells were transiently transfected with pCMV.Flag-VASP. Indirect immunofluorescence analysis with the M2 anti-Flag antibody showed that Flag-tagged VASP localizes to the cytoplasm in the presence
or absence of 8-Br-cAMP (data not shown). Western blot analysis of cell
extracts with the M2 anti-Flag antibody detected a single band with an
apparent molecular mass of 46 kDa (Fig. 3A), the size expected for
nonphosphorylated VASP. No band was seen in nontransfected CV-1 cell
extracts (data not shown).

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Fig. 3.
Time course of Flag-tagged VASP mobility
shift induced by membrane soluble cyclic nucleotides. CV-1 cells
were transiently transfected with pCMV.Flag-VASP (20 µg)
(A) or a combination of pCMV.Flag-VASP (20 µg),
pCMV.mcGKI (2 µg), and pCMV.Flag-PKI (2 µg) (B).
24 h after transfection, cells were serum-starved for an
additional 24 h and then treated for the indicated number of
minutes with 8-Br-cAMP (1 mM) and IBMX (500 µM) (A) or 8-Br-cGMP (1 mM) and
IBMX (500 µM) (B). Extracts were generated
(see "Materials and Methods"), resolved by 10% SDS-PAGE and
immunoblotted with the anti-Flag antibody (M2) as the primary antibody
and an 35S-labeled sheep anti-mouse antibody as the
secondary antibody.
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When Flag-tagged VASP-transfected cells were treated with 8-Br-cAMP,
two immunoreactive species were detected on Western blots. The most
rapidly migrating species corresponded to the previously observed
46-kDa VASP band, and an additional 50-kDa VASP band corresponded to a
phosphorylated form of the VASP protein (Fig. 3A).
Phospho-VASP was observed following 5 min of cyclic nucleotide treatment and remained at the maximal level for at least 1 h (Fig. 3A). Formation of the 50-kDa band was specifically due to
phosphorylation of Ser153 (the murine VASP residue
equivalent to Ser157 of human VASP) (56) by endogenous cAK
because its detection was completely blocked by cotransfection of an
expression vector for mouse PKI protein (data not shown), which
specifically inhibits cAKs but not cGKs (57). Formation of the 50-kDa
band was also prevented by site-directed mutagenesis of
Ser153 to an alanine (data not shown).
To demonstrate activation of transiently expressed cGKI
by
8-Br-cGMP, cells were cotransfected with expression vectors for Flag-tagged VASP, cGKI
, and PKI. cGKI
and endogenous cAK are expressed at similar levels in extracts from CV-1 cells transfected with 2 µg of pCMV.mcGKI
, as determined by in vitro
kinase assays (data not shown). Western blot analysis of extracts from
untreated cells using an anti-Flag antibody detected two bands, a major 46-kDa band and a minor 50-kDa band (Fig. 3B), indicating
that cGKI
has significant basal activity toward VASP in
vivo. Within 5 min of 8-Br-cGMP treatment, nearly all of the
remaining 46-kDa band was converted to the 50-kDa form and remained in
that form for at least 1 h (Fig. 3B). To exclude the
possibility that 8-Br-cGMP was cross-activating endogenous cAK, we
cotransfected sufficient PKI expression vector to completely block cAK
activation (data not shown). Coexpression of PKI had no effect on
8-Br-cGMP-dependent increases in VASP phosphorylation (data
not shown). These findings suggest that both cGKI
and cAK are
equally capable of phosphorylating protein substrates in cells treated
with membrane soluble cyclic nucleotides. Thus, the lack of significant
regulation of CRE-dependent gene transcription by cGKI
is not due to a deficit in cGKI
activation.
Effect of Cyclic Nucleotides on the Subcellular Distribution of
cGKI
and the C Subunit of cAKII--
Nuclear pore complexes mediate
the transport of proteins between the cytoplasm and the nucleus of
mammalian cells. These pore complexes allow passive diffusion of small
proteins (<40-65 kDa) in and out of the nucleus, while larger
proteins must be actively transported (58). cAKs consist of separate R
subunits and C subunits, while cGKs possess a linker sequence, which
connects the regulatory domain and catalytic domain into a single
polypeptide chain. Therefore, unlike the cAKs, activation of the cGKs
by cGMP does not result in the dissociation of the regulatory and the catalytic components of the enzyme and does not cause the release of a
small catalytic subunit (13). The actual size of active cGKs is
increased further by dimerization of cGK subunits (13). Hence, while
the free C subunit of cAK (40 kDa) is able to passively diffuse into
the nucleus to phosphorylate specific transcription factors (29),
cGKI
(150 kDa) would be expected either to be restricted to the
cytoplasm or to require a nuclear localization sequence (NLS) for
transport to the nucleus.
Because CREB is primarily a nuclear protein (28), a restricted
cytoplasmic localization could explain cGKI
's minimal ability to
activate CRE-dependent gene transcription. To examine the
subcellular localization of cGKI
, we transiently transfected CV-1
cells with pCMV.mcGKI
. CV-1 cells express low levels of endogenous
cGKI and possess a flat morphology. Anti-cGKI
serum, which
recognizes the carboxyl-terminal 15 amino acids of murine cGKI
(anti-cGMP-PK CT), detected a single major band with an apparent
molecular mass of 75 kDa in extracts of CV-1 cells transfected with
cGKI
. The amount of cGKI
was more than 10-fold higher than the
small amount of endogenous cGKI found in control CV-1 cell extracts
(data not shown). Staining of transfected CV-1 cells with the
anti-cGMP-PK CT antibody revealed diffuse, cytoplasmic
immunofluorescence (Fig. 4A).
The diffuse staining showed no specific association with major cellular
structures. The addition of 8-Br-cGMP to the medium did not lead to a
change in the cytoplasmic staining pattern or an increase in nuclear
staining (Fig. 4B). Furthermore, no change in the staining
pattern was observed at other times following 8-Br-cGMP treatment (30 min, 2 h, and 4 h; data not shown). Treatment with 8-Br-cGMP
had no apparent effect on the intensity of the cGKI
staining and
suggested that the ability of the antibody to recognize activated
cGKI
was not altered by cyclic nucleotide treatment. Nontransfected
cells showed a low but detectable level of cytoplasmic staining
probably due to endogenous cGKI. Treatment with 8-Br-cGMP had no effect
on this staining pattern either (data not shown). A similar restriction
of cGKI
to the cytoplasm was observed in transfected COS-1, HEK293,
and baby hamster kidney mammalian cell lines (data not shown).

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Fig. 4.
Effect of cyclic nucleotides on the
subcellular distribution of cGKI and the C
subunit of cAKII. Indirect immunofluorescence microscopy analysis
of CV-1 cells transiently transfected with either pCMV.mcGKI
(cGKI ; a and b) or pCMV.Flag-C 3
and pCMV.RII (cAKII; c and d).
24 h post-transfection, 8-Br-cGMP (1 mM) and IBMX (500 µM) (b) or 8-Br-cAMP (1 mM) and
IBMX (500 µM) (d) were added to
(8-Br-cNMP; b and d) or omitted
(Cont.; a and c) for 1 h. Cells
were fixed with a 4% paraformaldehyde solution and a 1:1 solution of
acetone and methanol and then labeled with the anti-cGMP-PK CT antibody
(a and b) or the anti-Flag antibody (M2)
(c and d) as the primary antibody and
Cy3-F(ab')2 goat anti-rabbit (a and
b) or Cy3-F(ab')2 goat anti-mouse (c
and d) as the secondary antibody.
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In contrast, as shown previously (30), the C subunit of cAK was found
to quickly diffuse into the nucleus upon treatment of cells with
8-Br-cAMP (Fig. 4, C and D). Thus, unlike the C subunit of cAK, which can translocate into the nucleus upon activation, cGKI
is restricted to the cytoplasm independent of its activation state.
In CV-1 cells, immunofluorescence microscopy revealed that both
endogenous CREB and a GFP-hCREB chimera protein were targeted to the
nucleus. No significant immunofluorescence was discernible in the
cytoplasm (data not shown). Hence, the restricted cytoplasmic localization of cGKI
is entirely consistent with its being a weak
activator of CRE-dependent gene transcription.
cGKII Does Not Translocate to the Nucleus upon Activation by
cGMP--
To determine the subcellular localization of the cGKII
isoform, rabbit antiserum was prepared using full-length His-tagged cGKII purified from SF9 cells as the antigen (41). Anti-cGKII serum
recognized a single band with an apparent molecular mass of 86 kDa in
extracts from cGKII-transfected HEK293 cells. No bands were detected in
extracts from control or cGKI
-transfected HEK293 cells. Preimmune
serum from the rabbit did not detect bands in any extracts (data not shown).
Immunostaining for transfected wild type murine cGKII in CV-1 cells
using cGKII antiserum, revealed a crescent-shaped, perinuclear staining
pattern (Fig. 5A). This
staining was specific for cGKII, since no significant anti-cGKII
immunofluorescence was observed in cells transfected with parental
vector alone (data not shown). Treatment with 8-Br-cGMP for 1 h
did not affect the staining pattern and did not cause an increase in
nuclear staining (Fig. 5B).

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Fig. 5.
Effect of 8-Br-cGMP on the subcellular
distribution of cGKII and the nonmyristoylated cGKIIG2A
mutant. Indirect immunofluorescence microscopy analysis of CV-1
cells transiently transfected with either pCMV.mcGKII
(cGKII; a and b) or
pCMV.mcGKIIG2A (G2A; c and
d). 24 h post-transfection 8-Br-cGMP (1 mM)
and IBMX (500 µM) were added to (8-Br-cGMP;
b and d) or omitted (Cont.;
a and c) for 1 h. Cells were fixed as in
Fig. 4 and then labeled with the anti-cGKII polyclonal antibody as the
primary antibody, Biotin-SP-F(ab')2 fragment goat
anti-rabbit IgG as the secondary antibody, and Alexa-488 conjugated
streptavidin as the fluorescent marker.
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To rule out the possibility that cGKII was not translocating to the
nucleus because it was strongly membrane-bound, we mutated the
penultimate glycine of murine cGKII to an alanine. The cGKIIG2A mutant
is a nonmyristoylated and soluble protein that is enzymatically similar
to wild type cGKII (59). In contrast to wild type cGKII, the cGKIIG2A
mutant was localized diffusely in the cytoplasm (Fig. 5C).
As described for wild type cGKII, treatment with 8-Br-cGMP had no
effect on the subcellular distribution of the cGKIIG2A protein (Fig.
5D). These data indicate that restriction to the cytoplasm
is not cGKI
-specific but a general property of both cGKI
and cGKII.
Localization of Endogenous cGKI in A7r5 Smooth Muscle
Cells--
Since overexpression of proteins can result in aberrant
subcellular localization, several cell lines were screened for
detectable expression of cGKI and cGKII. The anti-cGMP-PK CT antibody
detected a single cGKI band with an apparent molecular mass of 75 kDa
in cell extracts from N1E-115 neuroblastomas, N2A neuroblastomas, NG-108 neuroblastomas, CV-1 cells, HEK293 cells, A10 smooth muscle cells, and A7r5 smooth muscle cells. The highest levels were detected in extracts from NG-108 neuroblastomas and A7r5 smooth muscle cells. No
band was detected in extracts from COS-1 cells or Y1 adrenal tumor
cells or when identical blots were probed with anti-cGKII serum (data
not shown).
Localization of endogenous cGKI in A7r5 smooth muscle cells by indirect
immunofluorescence revealed diffuse cytoplasmic staining (Fig.
6A), and treatment with 1 mM 8-Br-cGMP had no effect on this staining pattern (Fig.
6B). The cytoplasmic staining was specific for cGKI, since
it was abolished by preabsorption of the anti-cGMP-PK CT antibody with
a peptide coding for the carboxyl-terminal 18 amino acids of cGKI (Fig.
6C).

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Fig. 6.
Immunofluorescence localization of endogenous
cGKI in A7r5 smooth muscle cells. Indirect immunofluorescence
microscopy analysis of cGKI in A7r5 cells stimulated with 8-Br-cGMP (1 mM) and IBMX (500 µM) for 0 h
(Cont.; a) or 1 h (8-Br-cGMP;
b). Cells were fixed as in Fig. 4 and then labeled with
anti-cGMP-PK CT antibody as the primary antibody,
Biotin-SP-F(ab')2 fragment goat anti-rabbit IgG as the
secondary antibody, and Alexa-488-conjugated streptavidin as the
fluorescent marker. Cytoplasmic immunofluorescence was abolished
by preabsorption of the anti-cGMP-PK CT antibody with a peptide coding
for the carboxyl-terminal 18 amino acids of cGKI (Blocked;
c).
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Generation of Full-length Constitutively Active cGKI
Mutants--
The difference in the ability of cGKs and cAKs to
activate CRE-dependent gene transcription could be due to
differences in their substrate specificities, their subcellular
localizations, or both. To define the mechanism(s) by which cGKs and
cAKs differentially regulate gene transcription, a full-length and a
small sized constitutively active cGKI
mutant were generated.
In efforts to produce a full-length constitutively active kinase, three
different classes of cGKI
point mutants were generated. For the
first class of mutant, mutations were made in cGKI
analogous to
those that have been identified in the C subunit of cAK and result in
constitutive activity of the C subunit even in the presence of excess R
subunit (60-64). These residues, His68 and
Trp197, are conserved between the C subunit of cAK and the
catalytic domain of cGKI. His419 and Trp530 of
cGKI
were mutated to glutamine and arginine, respectively, in
accordance with the mutations found for the R subunit-insensitive C
subunit mutants (60-64). A second class of constitutively active mutant was designed based on the observation that regulatory domain arginine residues in both cAK and cGK may mimic substrate site arginines. These pseudosubstrate arginine residues have been shown to
be important in the high affinity of several kinases' regulatory domains for their catalytic domains (65). Specifically, proteolytic cleavage of cGKI
after its pseudosubstrate arginine
(Arg75) generates a kinase with high constitutive activity
(66). Thus, to generate the second class of constitutively active
mutant, we mutated Arg75 to a neutral residue, alanine.
Finally, for the third class of mutation, it was reported that cGKI
undergoes autophosphorylation at two sites in its regulatory domain in
the presence of cyclic nucleotides. Phosphorylation of the second site,
Ser79, leads to a large increase in basal kinase activity
(67). For this third class of constitutively active mutant, we mutated
Ser79 to an acidic residue, aspartic acid, to mimic the
effect of autophosphorylation. One advantage of the second and third
approaches is that mutations in the regulatory domain are less likely
to affect the substrate specificity or specific activity of the enzyme.
The effects of these mutations were tested by measuring the basal and
cGMP-activated kinase activities in extracts made from HEK293 cells
transiently transfected with the mutant expression vectors (Fig.
7). Extracts from HEK293 cells
transfected with pCMV.Neo or a mammalian expression vector encoding a
catalytically inactive mutant (cGKI
K404R) showed no significant
cGMP-dependent kinase activity (Fig. 7). Except for the
cGKI
H419Q mutant, which shows a 5-fold reduction in specific
activity, the cGMP-dependent kinase activities (Fig. 7) and
protein expression levels (data not shown) of all of the constitutively
active mutants were similar to wild type cGKI
. Both the cGKI
R75A
and cGKI
S79D mutants exhibited high basal activity when compared
with wild type cGKI
. The increases in basal kinase activity of the
cGKI
R75A and cGKI
S79D mutants were 12- and 14-fold, respectively.
Specifically, mutation of Ser79 to an aspartic acid
increased the basal kinase activity from 5 to 72% of the total
cGMP-dependent kinase activity (Fig. 7). These results
support the hypotheses that Arg75 is important in the high
affinity of cGKI
's regulatory domain for its catalytic domain (65)
and that autophosphorylation of Ser79 is capable of
activating cGKI
in the absence of cGMP (67). Of interest, the
cGKI
H419Q and cGKI
W530R mutants showed only small increases in
basal kinase activity (Fig. 7), suggesting that the amino acid residues
in the cAK catalytic domain responsible for the tight interaction
between cAK's R subunit and C subunit are less important to inhibition
of cGKI's catalytic domain by its regulatory domain. The cGKI
S79D
mutant showed the highest basal activity and was therefore employed in
further studies of cGKI regulation of transcription.

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Fig. 7.
In vitro activity of full-length
constitutively active cGKI mutants.
In vitro kinase activity. HEK293 cells were transiently
transfected with 30 µg of pCMV.Neo (Neo), pCMV.mcGKI
(WT), pCMV.mcGKI K404R (K404R),
pCMV.mcGKI H419Q (H419Q), pCMV.mcGKI W530R
(W530R), pCMV.mcGKI R75A (R75A), or
pCMV.mcGKI S79D (S79D). 48 h after transfection,
protein extracts were generated (see "Materials and Methods") and
assayed for kinase activity in the presence (gray bar) or
absence (black bar) of cGMP (50 µM) using the
heptapeptide substrate H2Btide (110 µM). Protein kinase
inhibitor peptide (1 µM) was included in all assay tubes
to inhibit endogenous cAK activity. Specific kinase activity is
expressed as nmol/min/mg.
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Generation of a Constitutively Active cGKI Deletion Mutant--
An
additional mutant was sought to determine the effect of cGKI's protein
size on its ability to regulate transcription. Previous studies have
shown that truncation of cGKI's autoinhibitory regulatory domain
results in a fully active, cGMP-independent catalytic domain that
possesses a similar substrate specificity as the cGKI holoenzyme (68).
Because of its small size (40 kDa), it was predicted that the catalytic
domain of cGKI would be able to passively diffuse into the nucleus and
directly phosphorylate Ser133 of CREB in a manner similar
to the C subunit of cAK (29).
The entire amino-terminal regulatory domain of cGKI
was deleted, and
only the catalytic domain was expressed in HEK293 cells. To increase
the stability of the cGKI catalytic domain, we appended the amino
terminus of the C subunit of cAK to the cGKI catalytic domain.
Previously, the first 23 amino acids of cAK's C subunit were reported
to stabilize PKC's catalytic domain and allow for a high level of
expression in mammalian cells (69). To assess its stability and the
extent of its activation in transfected cells, the basal and
cGMP-dependent kinase activities of this chimera were
examined in extracts made from transiently transfected HEK293 cells
(Fig. 8A). Wild type cGKI
,
the cGKI
S79D mutant, and the catalytic domain of cGKI were found to
be expressed at equivalent levels by Western blot analysis (data not
shown) and in vitro kinase assays (Fig. 8A). As
expected, the truncated catalytic domain of cGKI
was found to be
fully active in the absence of cyclic nucleotides (Fig.
8A).

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Fig. 8.
In vitro and in vivo
activity of constitutively active cGKI
mutants. A, in vitro kinase activity.
HEK293 cells were transiently transfected with 30 µg of pCMV.Neo
(Neo), pCMV.mcGKI (WT), pCMV.mcGKI S79D
(S79D), or pCMV.FC/CD (CD). 48 h after
transfection, protein extracts were generated (see "Materials and
Methods") and assayed for kinase activity in the presence (gray
bar) or absence (black bar) of cGMP (50 µM) using the heptapeptide substrate H2Btide. Protein
kinase inhibitor peptide (1 µM) was included in all assay
tubes to inhibit endogenous cAK activity. Specific kinase activity is
expressed as nmol/min/mg. B and C, in
vivo kinase activity. B, Western blot analysis of
Flag-tagged VASP phosphorylation. CV-1 cells were transiently
transfected with pCMV.Flag-VASP (20 µg) and either no cGKI
expression vector (Neo; lane 1), 1 µg of
pCMV.mcGKI (WT; lane 2), 1 µg of
pCMV.mcGKI S79D (S79D; lane 3), or 1 µg of
pCMV.FC/CD (CD; lane 4). 24 h after
transfection, cells were serum starved for an additional 24 h.
Extracts were generated and blotted as in Fig. 3. C,
PhosphorImager quantitation of level of Flag-tagged VASP
phosphorylation. Statistical analysis of experiment was as described
for B. The percentage of VASP phosphorylation was calculated
by dividing the quantity of the 50-kDa band by the sum of the 46-kDa
band and the 50-kDa band.
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In Vivo Activity of Constitutively Active cGKI
Mutants--
To
determine if the cGKI
S79D mutant and the catalytic domain of cGKI
have increased basal kinase activity in vivo, Flag-tagged VASP was transfected alone or cotransfected with cGKI
, cGKI
S79D, or the catalytic domain. The level of VASP phosphorylation on Ser153 was low in serum-starved CV-1 cells expressing
Flag-tagged VASP alone with 3% of the total VASP in the 50-kDa form
(Fig. 8, B and C). Expression of the cGKI
S79D
mutant converted 30% of VASP to the 50-kDa form, while the catalytic
domain converted 70% of VASP to the 50-kDa form (Fig. 8, B
and C). Wild type cGKI
stimulated VASP phosphorylation
significantly less, with only 6% conversion of VASP to the 50-kDa form
(Fig. 8, B and C). Thus, both the cGKI
S79D mutant and the catalytic domain of cGKI showed constitutive activity in vivo, with the catalytic domain being slightly more
active toward VASP than the cGKI
S79D mutant.
Subcellular Localization of Constitutively Active cGKI
Mutants--
In agreement with the earlier localization studies, which
used 8-Br-cGMP to activate wild type cGKI
, the subcellular
localization of the cGKI
S79D mutant was cytoplasmic in the absence
or presence of 8-Br-cGMP (Fig. 9,
A and B). Unlike full-length cGKI
, the Flag-tagged catalytic domain of cGKI was found both in the nucleus and
in the cytoplasm (Fig. 9C). The catalytic domain was evenly distributed between the nucleus and the cytoplasm, consistent with the
passive diffusion previously reported for the C subunit of cAK (29).
The catalytic domain did not concentrate preferentially in the nucleus
(Fig. 9C). As expected, the addition of 8-Br-cGMP to the
medium had no discernible effect on its localization (data not shown).
The catalytic domain of cGKI was detected both in the cytoplasm and in
the nucleus with the M2 anti-Flag antibody (data not shown) and the
Anti-cGMP-PK CT antibody (Fig. 9C), demonstrating that the
anti-cGMP-PK CT antibody was capable of detecting cGKI in the nucleus.
These data corroborate the cytoplasmic restriction of active
full-length cGKI
and strongly suggest that cGKI
is restricted to
the cytoplasm because of its large size.

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Fig. 9.
Localization of constitutively active
cGKI mutants. Indirect immunofluorescence
microscopy analysis of CV-1 cells transiently transfected with either
pCMV.mcGKI S79D (S79D; a and b) or
pCMV.FC/CD (CD; c). 24 h post-transfection,
8-Br-cGMP (1 mM) and IBMX (500 µM) were added
(+; b) or omitted ( ; a and c) for
1 h. Cells were fixed as in Fig. 4 and then labeled with the
anti-cGMP-PK CT antibody as the primary antibody and
Cy3-F(ab')2 goat anti-rabbit as the secondary
antibody.
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Activation of CRE-dependent Gene Transcription in Cells
Overexpressing Constitutively Active cGKI
Mutants--
To correlate
the localization of the various cGKI
mutants with their ability to
increase CRE-dependent gene transcription, the abilities of
wild type cGKI
, the cGKI
S79D mutant, and the catalytic domain of
cGKI to regulate CRE-dependent gene transcription were
measured using the HCG-luciferase reporter. Expression of cGKI
or
the cGKI
S79D mutant did not stimulate CRE-dependent transcription in CV-1 cells (Fig.
10A) under conditions where
a substantial increase in the phosphorylation level of the cytoplasmic substrate VASP was observed (Fig. 8, B and C). In
contrast, the catalytic domain, which could enter the nucleus,
activated transcription from the HCG-luciferase reporter construct
4-fold (Fig. 10A). Unlike full-length cGKI
and the
cGKI
S79D mutant, the catalytic domain was effective at both
elevating the phosphorylation state of VASP (Fig. 8, B and
C) and inducing CRE-dependent gene transcription in CV-1 cells (Fig. 10A), suggesting that the ability of the
catalytic domain to passively enter the nucleus allows it to be a more
efficient activator of CRE-dependent gene
transcription.

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Fig. 10.
Activation of CRE-dependent
reporter by cotransfection with constitutively active cGKI
mutants. Transcriptional assay measuring CRE-dependent
gene transcription. A, CV-1 cells were transiently
transfected with no cGKI expression vector (Neo), 1 µg
of pCMV.mcGKI (WT), 1 µg of pCMV.mcGKI S79D
(S79D), or 1 µg of pCMV.FC/CD (CD).
B, HEK293 cells were transiently transfected with no cGKI
expression vector (Neo), 10 µg of pCMV.mcGKI
(WT), 10 µg of pCMV.mcGKI S79D (S79D), or 10 µg of pCMV.FC/CD (CD). C, HEK293 cells were
transiently transfected with 1 µg of pCMV.FC/CD (cGK/CD)
or 1 µg of pCMV.Flag-C 3 (cAK/C). All plates received
HCG-luciferase (0.5 µg) and pRSV. gal (5 µg). 24 h
post-transfection, cells were serum-starved for 24 h, harvested,
and assayed for luciferase and -galactosidase activities.
Luciferase activity was corrected for transfection efficiency by
dividing by -galactosidase activity (RLU/ gal). The
error bars depict the S.D.
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Regulation of HCG-luciferase activity was also examined in HEK293
cells, because the reporter assay was shown to be more sensitive in
this cell line. When assayed for induction of luciferase gene expression upon transient transfection into HEK293 cells, wild type
cGKI
showed essentially no stimulation of luciferase activity in the
absence of nucleotide treatment (Fig. 10B). In contrast, transfection of the cGKI
S79D mutant elevated the basal luciferase activity 66-fold (Fig. 10B). This induction is consistent
with the induction observed in HEK293 cells expressing wild type
cGKI
and treated with 8-Br-cGMP (13-fold). In HEK293 cells,
cGKI
S79D was significantly less potent as a regulator of gene
transcription than the catalytic domain of cGKI (Fig. 10B).
Whereas the cGKI
S79D mutant stimulated CRE-dependent
gene transcription 66-fold, the catalytic domain of cGKI induced
luciferase activity 1071-fold (Fig. 10B). The C subunit of
cAK and the catalytic domain of cGKI activated
CRE-dependent gene transcription to a similar extent (Fig.
10C), with the 3-fold difference in activation being
primarily due to the low stability of the cGKI catalytic domain
compared with the C subunit of cAK (data not shown). As observed in the CV-1 cells, full-length cGKI
was cytoplasmic in the presence or
absence of 8-Br-cGMP in HEK293 cells (data not shown). Therefore, both
the CV-1 and HEK293 cell data strongly support the conclusion that
active full-length cGKI
is a relatively weak activator of CRE-dependent gene transcription primarily because it is
restricted to the cytoplasm.
Characterization of cGKI
ATP-binding Domain Mutant--
Unlike
CV-1 cells, transfection of full-length cGKI
into HEK293 cells
confers readily measurable CRE-dependent transcriptional responses to 8-Br-cGMP. When cotransfected with HCG-luciferase into
HEK293 cells, wild type cGKI
can mediate a 13-fold increase in
luciferase activity in response to 8-Br-cGMP (Fig. 2B). The magnitude of induction is dependent on the amount of cGKI
expression vector transfected (data not shown) and the 8-Br-cGMP concentration in
the media (Fig. 11). Although
immunofluorescence microscopy localized a majority of the cGKI
immunoreactivity to the cytoplasm in cGKI
-transfected HEK293 cells,
it was possible a small fraction of the total cGKI
was present in
the nucleus and responsible for the small increase in transcription
(data not shown). Typically, proteins larger than 40-65 kDa require a
basic NLS to be actively transported to the nucleus. Inspection of the
cGKI
protein sequence by other investigators for a potential NLS
revealed a single cluster of basic amino acids in the ATP-binding
domain (KILKKRHI; residues 404-411) (33, 34).

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Fig. 11.
8-Br-cGMP activates
CRE-dependent gene transcription in a
concentration-dependent manner in HEK293 cells
overexpressing cGKI . A transcriptional
assay measuring CRE-dependent gene transcription is shown.
HEK293 cells were transiently transfected with ( ) or without ( ) 4 µg of pCMV.mcGKI . All plates received HCG-luciferase (0.5 µg),
pRSV. gal (4 µg), and pCMV.PKI (2 µg). 24 h
post-transfection, cells were treated for 20 h in the absence of
serum with varying concentrations of 8-Br-cGMP and IBMX (500 µM), harvested, and assayed for luciferase and
-galactosidase activities. Luciferase activity was corrected for
transfection efficiency by dividing by -galactosidase activity
(RLU/ gal). Luciferase activity is expressed as the
percentage of maximal activity attained. The error bars
depict the S.D.
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To determine whether the induction of CRE-dependent gene
transcription by full-length cGKI
in HEK293 cells was dependent on
the putative NLS sequence, this sequence was altered to the corresponding amino acids from the C subunit of cAK. The amino acids
encoding this basic sequence are not highly conserved in the C subunit
of cAK (KILDKQKV; residues 72-79) (39). Using site-directed
mutagenesis, we changed two of the basic amino acids in cGKI
's
putative NLS to the equivalent residues in the C subunit of
cAK(Lys407 to Asp and Arg409 to Gln). These
specific amino acid substitutions were chosen because the C subunit of
cAK is not actively transported to the nucleus (29), and these
mutations were unlikely to perturb the structure of the kinase since
they are found in the C subunit of cAK.
To determine the effect of these mutations on the ability of
cGKI
to activate CRE-dependent gene transcription,
we cotransfected wild type cGKI
or the cGKI
K407D/R409Q
mutant along with a CRE-dependent reporter construct
into HEK293 cells. While cGKI
increased luciferase activity
10-fold following 8-Br-cGMP treatment, the activated cGKI
K407D/R409Q mutant only induced a 4-fold increase in
gene transcription (Fig.
12A). Therefore,
substitution of two basic amino acids in the putative NLS sequence
reduced but did not eliminate cGKI
's ability to activate
CRE-dependent gene transcription.

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Fig. 12.
The cGKI
ATP-binding domain mutant is a weak inducer of both
CRE-dependent gene transcription and Flag-tagged VASP
phosphorylation. A, transcriptional assay measuring
CRE-dependent gene transcription. HEK293 cells were
transiently transfected with 20 µg of pCMV.Neo (Neo),
pCMV.mcGKI (WT), or pCMV.mcGKI K407R/R409Q
(MUT). All plates received HCG-luciferase (0.5 µg),
pRSV. gal (5 µg), and pCMV.PKI (2 µg). 24 h
post-transfection, cells were treated for 20 h in the absence of
serum with (gray bar) or without (black bar)
8-Br-cGMP (1 mM) and IBMX (500 µM).
B, in vitro kinase assay. Protein extracts from
untreated cells (Fig. 12A) were assayed for kinase activity
in the presence (gray bar) or absence (black bar)
of cGMP (50 µM) using the heptapeptide substrate H2Btide
(110 µM). Protein kinase inhibitor peptide (1 µM) was included in all assay tubes to inhibit endogenous
cAK activity. C, Western blot analysis of Flag-tagged VASP
phosphorylation. CV-1 cells were transiently transfected with
pCMV.Flag-VASP (20 µg), pCMV.Flag-PKI (2 µg) and no cGKI
expression vector (Neo; lanes 1 and
2), 1 µg of pCMV.mcGKI (WT; lanes
3 and 4), or 1 µg of pCMV.cGKI K407D/R409Q
(Mut; lanes 5 and 6). 24 h after
transfection, cells were serum-starved for an additional 24 h and
then treated for 1 h with (+) or without ( ) 8-Br-cGMP (1 mM). Extracts were generated and blotted as in Fig. 3.
D, PhosphorImager quantitation of level of Flag-tagged VASP
phosphorylation. Statistical analysis of experiment described for
C. Black bar, without 8-Br-cGMP; gray
bar, plus 8-Br-cGMP.
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Because this basic sequence is located within the ATP-binding region of
the catalytic domain, it was possible that the mutations negatively
affected cGKI
's catalytic activity. To determine the relative
effect of the two amino acid substitutions on the catalytic activity of
the cGKI
K407D/R409Q mutant, the basal and cGMP-dependent kinase activities of this mutant were compared with those of wild type
cGKI
in extracts made from the transiently transfected HEK293 cells.
Although expression levels of wild type cGKI
and the
cGKI
K407D/K409Q mutant were similar as determined by Western blot
analysis (data not shown), cGMP-dependent kinase activity
was 60% less in the extract from the cells overexpressing the
cGKI
K407D/R409Q mutant as compared with the extract from the
cells overexpressing wild type cGKI
(Fig. 12B).
The cGKI
K407D/R409Q mutant showed a reduction in the phosphorylation
of substrates in vivo, as determined by VASP transfection experiments (Fig. 12, C and D). Western blot
analysis of extracts from cells transfected with VASP and PKI alone
detected a single 46-kDa band (Fig. 12C). When identically
transfected cells were treated with 8-Br-cGMP (1 mM) for
1 h, both a 46-kDa band and a 50-kDa band were detected (Fig.
12C). Formation of the 50-kDa phosphorylated VASP band with
8-Br-cGMP treatment was due to phosphorylation of Ser153 by
endogenous cGKI, since its formation was not blocked by coexpression of
PKI, although PKI did completely block 8-Br-cAMP induction of the
50-kDa band (data not shown). While 94% of Flag-tagged VASP was
converted to the 50-kDa form in cells expressing wild type cGKI
treated with 8-Br-cGMP, only 56% was converted in similarly treated
cells expressing cGKI
K407D/R409Q (Fig. 12, C and
D). Hence, the cGKI
K407D/R409Q mutant has significantly
reduced basal and cGMP-stimulated kinase activity in vivo.
These data suggest that mutation of basic residues in the putative NLS
decreases the ability of cGKI
to activate gene transcription, not
because it prevents localization of cGKI
in the nucleus but because
it decreases the catalytic activity of the kinase. Thus, mutagenesis of
the putative NLS of cGKI
did not specifically alter either
cGKI
's localization or its ability to regulate transcription.
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DISCUSSION |
cAK and cGK regulation of CRE-dependent gene
transcription was investigated in this study to determine if these two
kinases differentially regulate this process. Because of the large size of active cGKs, we sought to determine whether, like the C subunits of
cAKs, cGKs would translocate to the nucleus following activation by
cyclic nucleotides. Analyses of the subcellular localization of
endogenous cGKI in A7r5 cells as well as transfected cGKI
and cGKII
indicate that the cGKs localize to the cytoplasm regardless of their
activation state. The inability of cGKI
to translocate to the
nucleus and directly phosphorylate CREB renders it a relatively weak
activator of CRE-dependent gene transcription and suggests one mechanism by which cAK and cGK differentially regulate gene transcription. These findings strongly imply that the restricted cytoplasmic localization of the cGKs is an important mechanism for
selective regulation of nuclear functions by the two families of cyclic
nucleotide-dependent protein kinases.
In this study, constitutively active cGKI mutants were generated by two
approaches to determine if cAK's greater ability to activate
CRE-dependent gene transcription was due to differences in
cGK's and cAK's substrate specificities or differences in their subcellular localizations. First, mutation of an autophosphorylation site serine to an aspartic acid to mimic phosphorylation resulted in a
constitutively active cGKI without significantly changing the size of
the protein. Autophosphorylation of cGKI
at Ser79 has
been shown to activate the enzyme and to produce an elongation of the
enzyme, suggesting that both cGMP binding and autophosphorylation activate the enzyme by a similar mechanisms (70). When this activated
form of full-length cGKI
, cGKI
S79D, was expressed and its ability
to transactivate a CRE-responsive promoter was measured, only a minimal
increase in luciferase expression was found when compared with
catalytic domain of cGKI. Like activated wild type cGKI
, the
cGKI
S79D mutant was also restricted to the cytoplasm, suggesting a
mechanism for its poor transactivating ability.
Each subunit of a cGK dimer consists of an amino-terminal regulatory
domain and a carboxyl-terminal catalytic domain. Deletion of cGKI's
regulatory domain generates a monomeric, constitutively active kinase.
In contrast to the cGKI
S79D mutant, the smaller, constitutively
active catalytic domain of cGKI was found in both the cytoplasm and the
nucleus. The catalytic domain strongly activated CRE-dependent gene transcription, demonstrating that the
phosphotransferase activity of cGKI is capable of recognizing members
of the nuclear CREB-like transcription factor family. Likewise, the
catalytic domain of cGKI activated gene transcription to a similar
extent as the free C subunit of cAK, implying that cGKI's restricted cytoplasmic localization and not its unique substrate specificity is
the major reason for cGKI's weak transactivating ability. These data
are entirely consistent with the conclusion that cGKI
is a weak
activator of CRE-dependent gene transcription because the large size of the active kinase prevents nuclear translocation.
During the course of these experiments, a report appeared describing
the identification of an NLS in the ATP-binding domain of human cGKI
that is only functional upon activation of the kinase by cGMP (34). The
identified NLS (KILKKRHI) does not closely resemble the well
characterized monopartite nuclear targeting sequence of SV40 large T
antigen (PKKKRKV) or the bipartite motif of nucleoplasmin (71). In an
attempt to compare these findings with our own, the murine cGKI
expression vector was transiently transfected into baby hamster kidney
cells. Localization of murine cGKI
in baby hamster kidney cells by
indirect immunofluorescence revealed diffuse cytoplasmic staining
either in the presence or absence of 8-Br-cGMP. Similar results were
obtained in HEK293 cells, COS-1 cells, and CV-1 cells, suggesting that
the restricted cytoplasmic localization of cGKI
was common in
mammalian cell lines. The discrepancy between our results and those
published previously is not due to the overexpression of cGKI
, since
similar results were also obtained with endogenous cGKI in A7r5 cells. Finally, mutagenesis of the putative NLS reported by Gudi et
al. (34) in our murine cGKI
shows no specific effect on its
ability to transactivate a CRE-dependent reporter construct
(Fig. 12). Instead, the decreased ability of this ATP-binding site
mutant to transactivate a CRE-dependent reporter construct
correlated with its decreased catalytic activity in vitro
and in vivo.
In this study, we demonstrate that cGKI
is a weak inducer of
CRE-dependent gene transcription. When HEK293 cells were
transiently transfected with cGKI
, 8-Br-cGMP treatment elevated
CRE-dependent gene transcription 13-fold (Fig.
2B). 8-Br-cGMP treatment did not significantly activate
CRE-dependent transcription in the absence of transfected
cGKI
(Fig. 2B). Transcriptional activation was also
mediated by the cGKI
S79D constitutively active mutant alone,
implying that cGKI
kinase activity by itself was capable of
elevating CRE-dependent transcription and that the
8-Br-cGMP was not affecting other signaling systems. Transient
coexpression of PKI did not block cGKI
-dependent
increases in gene transcription, suggesting that this transactivation
is independent of cAK activity. Experiments using a GAL4-CREB fusion
construct suggest that transcriptional regulation by cGKI is at least
partially mediated through CREB, since the cGKI
S79D mutant is
capable of transactivating pGAL4-luc when coexpressed with the
GAL4-CREB fusion protein (data not shown). These findings suggest that
the pathway(s) by which cGKI
activates CRE-dependent
gene transcription may involve specific cytoplasmic cGK substrates that
can signal through CREB.
The exact mechanism(s) by which cGKs weakly regulate CRE-containing
promoters is unknown. It is possible that cGK activation results in the
activation of other protein kinases that are able to translocate to the
nucleus and phosphorylate CREB. A number of growth factor-stimulated
kinases including the RSKs (72) and MAPKAP kinase 2 (73) as well as CaM
kinases (74) have recently been shown to translocate to the nucleus and
phosphorylate CREB. Alternatively, although it is difficult to
determine, it is possible that a small fraction of CREB is cytoplasmic
and in equilibrium with the majority of nuclear CREB bound to DNA.
Finally, novel transcription factors that can be phosphorylated in the cytoplasm and translocate to the nucleus may be the substrate of cGKs.
In this regard, it is interesting to note that C/EBP
can be
phosphorylated in the cytoplasm of PC12 cells by cAK, and following
phosphorylation it translocates to the nucleus and increases gene
transcription (75).
Since the cGKs and the cAKs are highly homologous protein kinase
families that possess similar substrate specificities, many questions
remain regarding the unique roles these kinases may play in cells that
coexpress cAK and cGK isoforms. The results of this study suggest that
the physiological role(s) of these cyclic
nucleotide-dependent protein kinases are more distinct than
currently appreciated and that, in comparison with cAKs, cGKs play only
a minor role in the regulation of CRE-dependent transcription. The findings as well as the reagents generated within
this study should be useful in characterization of the specific
in vivo role(s) of the cyclic
nucleotide-dependent protein kinase families.