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INTRODUCTION |
The cGMP-dependent protein kinases, also known
as protein kinase G (PKG),1
are the main effector enzymes activated by elevated cGMP levels due to
stimulation by nitric oxide and natriuretic peptides (reviewed in Refs.
1-3). These enzymes have many essential regulatory roles in diverse
physiological processes, including platelet activation, kidney
function, smooth muscle relaxation, gene expression, and chemotaxis
(for recent reviews, see Refs. 4-6). In mammals there are three
isoforms of PKG that are transcribed from two distinct genes. The
and
isoforms of type 1 PKG are widely distributed and originate
from alternative splicing of the same gene (7). The expression of type
2 PKG is more restricted to the kidney, the intestinal brush border,
and certain tissues of the brain (8-11).
The primary structure of PKG is highly homologous to
cAMP-dependent protein kinase (PKA), particularly in areas
that encode nucleotide binding domains in the amino-terminal half of
the molecule, and in regions involved in ATP binding and
phosphotransferase activity in the COOH-terminal half of the molecule
(see Refs. 4-6). A major difference between the two cyclic
nucleotide-dependent protein kinases is that the PKA
holoenzyme is composed of regulatory and catalytic subunits encoded by
distinct genes, whereas the corresponding functional units of PKG are
encoded as a contiguous polypeptide. Activation of PKA results in
dissociation of the catalytic subunit, which then accumulates in the
nucleus where some important substrates are located (1, 12). Several of the functional domains of PKG deduced by comparison with PKA (such as
nucleotide binding, active site) have been described in great detail by
extensive biochemical analyses of purified protein (1, 13, 14). The
area of least homology between PKA and PKG lies in the first 100 amino
acid residues and constitutes the principal difference between the two
splice variants of type 1 PKG (15). The first 54 amino acids encode a
leucine zipper structure that is thought to mediate homodimer
formation. Proteolytic removal of this region and part of the adjacent
pseudosubstrate motif from purified enzyme results in monomers with
constitutive activity. More recently it was shown that the length of
PKG greatly increases upon activation by cGMP, suggesting that a PKG
monomer might exist in a closed conformation that is maintained by
interaction of the active site with the pseudosubstrate motif (16-19).
PKG encodes a nuclear localization signal in the in the COOH-terminal
half of the molecule that is hidden within the catalytic region. It has
been reported that activation of PKG by cGMP leads to unmasking of this
motif, which results in translocation of the enzyme into the nucleus
(20). The significance of the nuclear localization signal in PKG has
been disputed, however, as nuclear translocation has not been confirmed
by other laboratories (21). Although recent mutational studies have
provided some useful insight into the importance of individual amino
acid residues, few studies have addressed the properties of specific
domains of PKG in vivo.
In addition to its role in dimerization, the leucine zipper region is
important for interaction of the enzyme with specific proteins known as
G-kinase anchoring proteins or "GKAPs," which are often also a
substrate (22-24). The concept of anchoring proteins is a common theme
for the serine/threonine protein kinase superfamily where they serve
the important role of localizing the kinase in the vicinity of its
specific substrates (25, 26). Supportive evidence for the importance of
the leucine zipper motif in targeting PKG has recently emerged using
the yeast two-hybrid assay, which has identified several
muscle-associated proteins that function as GKAPs by binding to this
region (22-24). Although in the past, type 1 PKG was widely believed
to be a soluble enzyme, immunofluorescence studies, largely with
leukocytes, have indicated that PKG also localizes to discrete cellular
compartments (27, 28). Similar evidence has demonstrated that
stimulation of cells can lead to alterations in PKG localization that
may be important to enzyme function. Other laboratories have not
reproduced similar studies, and whether such compartmentalization
occurs in nonleukocyte cells remains to be determined.
This study used a molecular approach to investigate the functions of
specific domains of PKG1
in the form of GFP fusion proteins. Results
shown here demonstrate that the COOH-terminal catalytic region is
capable of nuclear translocation but is restricted from nuclear entry
by interaction with the amino-terminal regulatory region. Visualization
of GFP fluorescence in different cell types indicates that both active
and inactive forms of PKG colocalize with the known substrate for PKG:
vasodilator-stimulated phosphoprotein (VASP). Both dominant negative
and constitutive fusion proteins described here should provide useful
tools for future studies of physiological roles for PKG.
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EXPERIMENTAL PROCEDURES |
Cell Culture/Transfection and Reagents--
Cyclic nucleotides,
BDPEtide, and H89 were from Biomol Research Laboratories, Inc.
(Plymouth Meeting, PA). The phosphatase inhibitor mixture and protease
inhibitor mixture were from Calbiochem. Nonidet P-40 and Tween
20 were from Fisher. The p81 phosphocellulose was obtained from Upstate
Biotechnology Inc. (Lake Placid, NY), and [32P]ATP was
from Amersham Pharmacia Biotech. Unless specified, all other chemicals
were from Sigma.
Human lung epithelial cells (A549) and human embryonic kidney (HEK-293)
cells were passaged by trypsin treatment prior to confluence and
maintained in RPMI 1640 medium (Life Technologies, Inc.) containing
10% fetal bovine serum (Omega Scientific) and supplemented with
streptomycin, penicillin, and L-glutamine. The day before
transfection, cells were plated at 50% confluence on either 100-mm
dishes or six-well plates. The cells were transfected to express
exogenous DNAs using LipofectAMINE PlusTM (Life
Technologies, Inc.) according to the manufacturer's instructions. A
routine experiment involved addition of DNA/liposome mixture to cells
in RPMI (1.5 ml/5 ml/100-mm dish, 0.3 ml/1 ml/well) for 4 h
followed by addition of an equal volume of normal medium for a further
36 h.
Construction and Enzyme Assay of Green Fluorescent Fusion
Proteins--
The cloning of the cDNA encoding human PKG1
from
lung RNA using reverse transcriptase polymerase chain reaction has been described previously (29). A region encompassing amino acids 1-347
that contains the entire regulatory region homologous to the PKA-R1
subunit was generated using polymerase chain reaction. This
1-kilobase fragment was subcloned in frame with GFP into the
EcoRI/BamHI sites of the pEGFP-N1 vector
(CLONTECH, Palo Alto, CA). The full-length PKG1
coding region and a catalytically inactive mutant (T516A, see Ref. 29)
were similarly subcloned into the pEGFP-N1 vector using the same
forward primer and a reverse primer that removed the stop codon
and produced a fusion protein with EGFP at the COOH terminus. The
catalytic regions of PKG1
encompassing amino acids 348-671 were
amplified by polymerase chain reaction and subcloned into the EYFP-C1
vector (CLONTECH, Palo Alto, CA) such that the PKG
sequence was in frame at the COOH terminus of GFP. Similar fusion
proteins of PKG1
were generated by adding a primer encoded FLAG
epitope (DYKDDDDK) in place of GFP. The FLAG-tagged PKG fusion proteins
were all subcloned into the pCDNA3 expression vector (Invitrogen,
Carlsbad, CA). The murine FLAG epitope-tagged VASP protein was a gift
from Dr. Michael Uhler (University of Michigan) and has been described
elsewhere (21, 29). The kinase activity of the expressed enzymes was
measured in cell homogenates using BPDEtide as substrate as detailed
previously (21, 29).
Immunoprecipitation and Western Blotting--
To prepare crude
extracts from transfected cells, monolayers were washed briefly with
PBS followed by lysis in extraction buffer (50 mM HEPES, pH
7.0, 1% Nonidet P-40, 1× protease inhibitor mixture) by rocking for
20 min at 4 °C. Homogenates were clarified by centrifugation at
10,000 × g for 10 min and then frozen at
80 °C
until needed. For direct electrophoretic analysis 30 µl of
homogenates were mixed with 10 µl of 5× PAGE sample buffer, boiled
for 5 min, then 10 µl was loaded per lane. For immunoprecipitation studies, antibodies against GFP (Molecular Probes, Eugene, OR) were
added to 1 ml of lysate for 1 h at 4 °C, followed by addition of 30 µl of protein A-Sepharose (50% v/v in PBS) for 30 min.
Alternatively, 30 µl of anti-FLAG(M2)-conjugated Sepharose beads
(Sigma) was added directly to 1 ml of lysate for 1 h at 4 °C.
Immunoprecipitates were subsequently washed two times in extraction
buffer and one time in PBS. The pellets were resuspended in 30 µl of
1× PAGE sample buffer, and 25 µl was loaded per lane. In some cases
the proteins were eluted from the beads with 100 µM FLAG
peptide prior to electrophoretic analysis.
Electrophoresis of proteins was performed on 10% mini-gels (Bio-Rad)
followed by electrophoretic transfer to nitrocellulose. The blots were
blocked with 5% bovine serum albumin in PTS buffer (PBS containing
0.05% Tween 20) for 30 min at room temperature, then antibodies were
added (1 µg/ml for monoclonals, 1/1000 for serum) for 16 h at
4 °C. Following addition of 1/3000 peroxidase-conjugated secondary
antibody (Calbiochem) for 30 min, the bands on the blots were
visualized using chemiluminescence according to manufacturer's instructions (Pierce). At least three washes (5 min each) using excess
PTS buffer were performed between each incubation step.
VASP Dephosphorylation--
To demonstrate the effect of the
phosphorylation state of VASP on electrophoretic mobility, HEK-293
cells were transfected to express FLAG-VASP, which was
immunoprecipitated using anti-FLAG-Sepharose as detailed above.
Following precipitation and washing with lysis buffer, the beads were
washed two times with phosphatase buffer (50 mM Tris-HCl,
pH 7.2, 5 mM MgCl2, 1 mM
dithiothreitol). The immunoprecipitates were subsequently
treated with 1.5 µg/ml protein phosphatase 2A (Calbiochem), in the
same buffer for 20 min at 30 °C. Control tubes containing
phosphorylated VASP were incubated in phosphatase buffer without
protein phosphatase 2A. The VASP was subsequently eluted from the beads
with 100 µM FLAG peptide before analysis by Western
blotting with anti-FLAG antibodies.
Immunofluorescence Microscopy--
All epifluorescence
microscopy and digitization of images was done using a Nikon Eclipse
inverted microscope (model TE300) equipped with Hoffman optics and
appropriate filter sets for epifluorescence microscopy. The image
capture system was purchased from C-Imaging Systems (Cranberry
Township, PA) and included a Hamamatsu CCD digital camera (model
C4742-95) and SimplePCI software. For colocalization experiments,
images captured using the 100× oil immersion lens were overlaid using
Photoshop 4 software (Adobe, San Jose, CA).
For live imaging of fluorescence, cells were grown in six-well plates
and transiently transfected to express GFP fusion proteins. After
24 h the plate was sealed with parafilm and placed directly on the
microscope stage. Visualization was not continued beyond 30 min. High
magnification and immunohistochemistry was performed cells grown on
coverslips in six-well plates. Following transfection the coverslips
were briefly washed in PBS followed by fixation in 3.7%
paraformaldehyde (Electron Microscopy Sciences, Washington, PA) for 30 min at room temperature. Coverslips were subsequently washed in PBS,
extracted with 0.1% Triton X-100 for 5 min, and then blocked with 10%
bovine serum albumin in PBS for 1 h at room temperature.
Monoclonal antibodies against FLAG epitope were used at 10 µg/ml in
PBS containing 1% fetal bovine serum for 20 h at 4 °C. After
four washes with PBS containing 1% fetal bovine serum, rhodamine-conjugated anti-mouse IgG antibodies were added for 1 h.
After additional washing, the coverslips were mounted on glass slides
using Vectashieldtm fluorescence mounting medium (Vector
Laboratories, Burlingame, CA). Visualization of F-actin was
accomplished by growing and fixing cells on coverslips as detailed
above, followed by staining with rhodamine-phallicidin according to the
manufacturer's instructions (Molecular Probes, Eugene, OR).
Antibody Production--
Antiserum specifically recognizing the
COOH terminus of type 1 PKG or phosphorylated serine 239 of VASP was
generated by immunizing rabbits with synthetic decapeptides coupled to
KLH using standard methods. The peptide used as antigen had the
sequence CAKLRKVS*KQEE, where a phosphorylated serine was included as
indicated. The peptide used as antigen for PKG had the sequence
CADDNSGWDIDF. In both cases the amino-terminal Cys-Ala served to couple
with carrier. Sera were tested by enzyme-linked immunosorbent assay and
Western blotting after sequential booster inoculations until the
effective titer reached 1/1000.
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RESULTS |
Generation and Expression of Green Fluorescent PKG Fusion
Proteins--
To gain some insight into the functions of different
structural regions of type 1 PKG in vivo, we sought to
create GFP fusion proteins that would permit visualization without
fixation/extraction artifacts. The largest construct incorporated the
full-length coding sequence for PKG1
with GFP fused at the COOH
terminus (PKG-GFP). The design of other PKG constructs was based upon
known biochemical properties of purified PKG and its homology with the closely related PKA enzymes (Fig. 1).
Truncations of PKG1
were generated such that either the
amino-terminal regulatory regions (GFP-G1C) or the COOH-terminal
catalytic regions (G1
R-GFP) were essentially replaced with GFP. With
PKG-GFP as the exception, because of similarity in the size of
GFP compared with the regulatory and catalytic regions of PKG (35-40
kDa), the corresponding fusion proteins were expected to be similar in
size to the endogenous PKG monomer (75, 85, and 110 kDa for GFP-G1C,
G1
R-GFP, and PKG-GFP, respectively). When transiently transfected
into HEK-293 fibroblasts all of the constructs were translated into
proteins of the expected sizes as detected by Western blots probed with
either anti-GFP or anti-COOH-terminal-specific anti-PKG antibodies
(Fig. 2). As expected for the G1
R-GFP
construct in which the COOH-terminal catalytic region was replaced with
GFP, this protein was not detected by the PKG-specific antibodies that
recognize the COOH terminus, but was recognized by anti-GFP antibodies.
The catalytic properties of these proteins were examined in homogenates
from transfected HEK-293 cells using BPDEtide as a substrate for
in vitro kinase assays. These cells have previously been
shown to express modest amounts of type 1 PKG (29), and in
mock-transfected cells the presence of endogenous enzyme was reflected
in a 2.5-fold increase in kinase activity in response to cGMP.
Homogenates transfected to express PKG-GFP exhibited constitutive
activity that could be only slightly increased by incorporation of cGMP
in the assay. As would be expected by the lack of a catalytic region,
overexpression of G1
R-GFP did not produce any basal kinase activity,
but notably this construct was able to block the cGMP-stimulated
activity of the endogenous kinase. In the absence of regulatory
regions, the GFP-G1C protein exhibited a constitutive activity that was unaffected by the presence of cGMP.

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Fig. 1.
Properties of GFP fusion proteins encoding
full-length and truncated PKG1 .
The amino acid sequence homology is compared for PKA type 1
regulatory and catalytic genes and type 1 PKG. Identical residues
are indicated as dark shading. The fusion proteins were made
with GFP (light shaded area) at the COOH terminus of
full-length PKG, with the regulatory regions corresponding to R1 or
with the catalytic regions corresponding to C1 . Domains with
reported functions such as nucleotide binding, PKG dimerization
(variable), and catalysis are indicated above.
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Fig. 2.
Expression of the PKG fusion proteins in
HEK-293 cells. PKG fusion constructs were transiently transfected
into HEK293 cells. A, protein expression was determined by
Western blotting with anti-PKG (left panel) and anti-GFP
(right panel) antibodies. B, the catalytic
properties of the PKG fusion proteins in homogenates from transfected
HEK-293 cells were examined using in vitro kinase assays as
detailed under "Experimental Procedures." The PKG-specific
phosphotransferase activity in the absence (open bars) or
the presence of 10 µM 8-Br-cGMP (closed bars)
was determined. The error bars represent S.E. from three
independent experiments, each with duplicate samples.
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The amino-terminal 100 residues of type 1 PKG encode a leucine zipper
motif that has been demonstrated as essential for homodimerization as
well as interaction with specific GKAPs (22-24). Within this region
there is also a pseudosubstrate sequence that is thought to interact
with the active site of the kinase. Experiments were performed to
determine whether the truncated regulatory domain could dimerize with
endogenous PKG and whether the catalytic domains could interact with
the regulatory region. To facilitate immunoprecipitations, the GFP
component of the PKG fusion proteins was replaced with FLAG epitope
(Fig. 3). In these experiments the
FLAG-G1
R protein, presumable through leucine zipper interactions,
was able to interact with cotransfected PKG-GFP, indicating proper
folding of these proteins. The regulatory domains were also found to
bind to the catalytic regions as measured by cotransfection of either
FLAG-G1C with G1
R-GFP or by FLAG-G1C with G1
R-GFP (Fig.
3A). These experiments suggested that the PKG fusion
proteins containing the amino-terminal regulatory regions should be
able to form dimers with endogenous PKG. To examine this possibility,
increasing amounts of FLAG-G1
R were transiently transfected into
HEK-293 cells followed by precipitation of the expressed regulatory
region with FLAG antibody (Fig. 3B). The endogenous PKG in
the precipitates was subsequently determined by quantitative Western
blotting with anti-PKG antibody. Since this antibody was raised against
a COOH-terminal peptide, it does not recognize the expressed truncation
in which the COOH-terminal region was deleted (see Fig. 2A).
These experiments revealed that the regulatory region expressed alone
could indeed bind to endogenous PKG as an 80-kDa band. When plotted as
a function of the amount of FLAG epitope, it was found that the
relative amount of endogenous PKG found in the precipitates decreased
with increased expression of the regulatory region (Fig.
3C). Based upon this finding it was hypothesized that lower
concentrations of G1
R protein formed heterodimers with endogenous
PKG, but higher expression levels resulted in a greater proportion of
homodimers composed of truncated regulatory region only.

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Fig. 3.
Interaction of PKG fusion proteins.
A, the interaction of the regulatory region of
PKG1 as a FLAG epitope-tagged fusion protein (FLAG-G1 R) with GFP
fusion proteins containing the catalytic domains (GFP-G1C) or
full-length PKG1 (PKG-GFP) was assessed by immunoprecipitation and
Western blotting with anti-GFP antibodies. The ability of FLAG-tagged
catalytic regions of PKG (FLAG-G1C) to precipitate the regulatory
regions of PKG1 as a GFP fusion protein (G1 R-GFP) is also shown.
B, the ability of transiently transfected G1 R to dimerize
with endogenous type 1 PKG in HEK-293 cells was examined. The FLAG
epitope-tagged G1 R was precipitated from cells transfected with
increasing amounts of this construct, and the pellets were examined by
Western blotting with anti-PKG COOH-terminal antibodies (upper
panel) or with anti-FLAG antibodies (lower panel). C,
the relative quantity of PKG as a function of expressed G1 R was
determined by densitometric analysis of the respective autoradiographs.
Data are representative of similar results obtained in two independent
experiments.
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Visualization of PKG1
Fusion Proteins in Vivo--
Although
widely appreciated as cytosolic, the intracellular localization of type
1 PKG has gained little attention. The experiments described here
demonstrate that the domains of the G1
R-GFP and PKG-GFP fusion
proteins involved in dimerization were functional, indicating that
these proteins would also be likely to interact with endogenous GKAPs.
Furthermore, as suggested by the in vitro kinase assays,
these fusion proteins should behave similarly to inactive and active
PKG (respectively). We set out to examine the intracellular
localization of the GFP fusion proteins by transiently transfecting
several cell types (Fig. 4). When
transfected to express either PKG-GFP or G1
R-GFP, HEK-293 cells
showed fluorescence throughout the cytosol, and in many cells there was
significantly more fluorescence of both constructs in the perinuclear
region. Importantly, even at high levels of expression, neither
construct was detected in the nucleus (<1%). A striking feature of
the HEK-293 cells expressing the G1
R-GFP protein was the presence of
pseudopodial extensions with a concentration of fluorescence at the
distal end. In several instances filaments were observed, which when examined using time-lapse video microscopy were very dynamic. In
contrast, the HEK-293 cells expressing the constitutive PKG-GFP protein
had fewer pseudopodia, and these structures were quiescent and were
devoid of visible filaments.

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Fig. 4.
Visualization of the PKG fusion
proteins in transfected cells. The GFP fusion proteins were
transiently transfected into HEK-293 (A, B), A549
(C, D) and HeLa (E, F), and
the GFP in the living cells was observed by epifluorescence microscopy.
Expression of the G1 R-GFP construct is shown in A,
C, and E and the PKG-GFP construct in
B, D, and F. The A549 cells in
C and D are shown using simultaneous phase
contrast and epifluorescence to visualize the cell perimeter. The
arrows in C indicate a lamellapod. The
bar in each panel represents 25 µm.
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Two epithelial cell lines, A549 (type ll human lung) and HeLa (human
cervical), were also transiently transfected to express the GFP fusion
proteins. Similar to the fibroblasts, in both epithelial cell lines the
G1
R-GFP and PKG-GFP proteins were observed throughout the cytoplasm
and were rarely present in the nucleus (Fig. 4). Interestingly, there
was a pronounced localization of G1
R-GFP at the leading edge of many
cells, and in some cells the G1
R-GFP protein was observed on
filaments resembling stress fibers. Similar to the pseudopodia of the
HEK-293 cells, time-lapse video microscopy revealed a dynamic movement
of these regions relative to the cell body with a rolling, retrograde
movement of the GFP. The PKG-GFP protein did not localize to the
membrane in any cell line tested but instead was more concentrated in a
perinuclear region away from the plasma membrane and cortical zones.
Notably, many cells transfected to express PKG-GFP appeared to be more
rounded with less prominent extensions.
Expression of the EGFP protein alone in either HEK-293 or A549 cells
was identical with the fluorescence uniformly distributed throughout
the cell without any distinguishing structures. Like PKG-GFP, the
GFP-G1C protein has constitutive kinase activity, but in contrast is
devoid of regulatory regions involved in dimerization and anchoring to
GKAPs. When either the HEK-293 or the A549 cells were transiently
transfected to express GFP-G1C, the fluorescence was present throughout
the cytosol but was much more concentrated in the nucleus (Fig.
5). Cells expressing this construct were also more rounded and had less pseudopodial extensions compared with
EGFP vector transfected cells. The lack of dynamic regions in the
GFP-G1C transfected cells was similar to those expressing PKG-GFP
except that the former were typically smaller and contained less
cytosol. The GFP-G1C construct was shown to interact with a FLAG
epitope-tagged construct encoding the regulatory regions of PKG1
(Fig. 3A). This interaction was confirmed in living cells, as coexpression of FLAG-G1
R was able to prevent the concentration of
the cotransfected GFP-G1C protein from entering the nucleus.

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Fig. 5.
Localization of GFP-G1C to the
nucleus. A549 cells (A, B) or HEK-293
cells (C, D) were transiently transfected
followed by visualization of expressed GFP in live cells using
epifluorescence microscopy. Cells were transfected with EYFP-C1 vector
(A), with a cDNA encoding the COOH-terminal catalytic
regions of PKG1 fused to GFP (GFP-G1C; B, C),
or were cotransfected with the GFP-G1C construct and the
NH2-terminal regulatory regions of PKG1 (FLAG-G1 R;
D). The fields shown are representative of observations from
at least two experiments.The bar represents 25 µm.
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Specific Interaction of Inactive PKG1
with F-actin--
The
appearance of spontaneous, prominent lamellopodia in A549 cells
prompted us to use this cell line for further study. The location of
G1
R-GFP in the live cells suggested that this catalytically inactive
truncation of PKG might associate with the microfilament component of
the cytoskeleton. To test this, cells that were transiently transfected
to express G1
R-GFP were subsequently fixed, and the F-actin was
stained using rhodamine-phallicidin (Fig.
6). These experiments revealed
colocalization of G1
R-GFP with F-actin in membrane ruffles and in
the active membrane regions found at the leading edge. In some of the
cells G1
R-GFP clearly colocalized with stress fibers proximal to
these active membrane regions, and occasionally it was observed in the
focal adhesion complexes. When transfected to express the constitutive
PKG-GFP or the free catalytic half of PKG (not shown), A549 cells were
more rounded in morphology, and there was a marked overall loss of
F-actin and stress fibers. Furthermore, in these cells, focal contacts and active membrane regions observed in the mock-transfected or the
G1
R-GFP transfected cells were rarely observed. The F-actin in the
cells expressing constitutive PKG-GFP localized predominantly to the
cell cortex and randomly throughout the cytoplasm. In support of the
visualization of PKG-GFP in live cells, in these fixed cells the fusion
protein was more centrally located, discretely separate from the plasma
membrane, and did not associate with the existing actin filaments.

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Fig. 6.
Localization of inactive PKG to filamentous
actin in A549 cells. The ability of the GFP fusion proteins
to colocalize with filamentous actin in A549 cells was assessed by
fixing transiently transfected cells and staining with
rhodamine-phallicidin. The panels on the left
show rhodamine fluorescence, the middle panels show GFP
fluorescence, and the panels on the right are
overlays with colocalization visualized as yellow. The top
panels (A-C) show a cell expressing G1 R-GFP and
reveal a typical active membrane region at the leading edge.
D-F, also transfected with G1 R-GFP, show
localization to stress fibers (arrows). The cells shown in
G-I were typical of those transfected to express PKG-GFP.
The bar represents 25 µm.
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Effect of PKG Fusion Proteins on VASP Phosphorylation--
The
VASP family members are structurally similar proteins that are
considered to be important regulatory components of the actin
cytoskeleton in most cell types (30). Reflecting the ability of
VASP members to bind focal adhesion and actin
cytoskeleton-associated proteins, including vinculin, zyxin, profilin,
and actin itself, these proteins localize to focal adhesions, stress
fibers, and dynamic membrane regions of the leading edge (30-36). The
prototypic member of this family is the VASP, and its phosphorylation
by PKA and PKG has been well documented (37, 38). Having examined the
catalytic properties of the PKG fusion proteins in vitro
using a peptide substrate, it was of interest to determine the effects of the proteins described here on the phosphorylation of VASP in
vivo. Furthermore, since the localization G1
R-GFP in epithelial cells paralleled that reported for VASP, we also sought to determine whether this well characterized substrate for PKG might be involved in
localizing the G1
R-GFP protein in vivo.
Initial studies involved transfection of both HEK-293 and A549 cells
with a cDNA encoding murine VASP, tagged at the amino terminus with
the FLAG epitope (21). Phosphorylation of the transfected VASP was
measured as a shift in electrophoretic mobility on Western blots probed
with anti-FLAG antibodies (Fig. 7). These experiments confirmed the presence of endogenous PKG in the HEK-293 cells but revealed its absence in the A549 cells, as there was no
cGMP-induced shift in VASP mobility in the latter. Cotransfection of a
cDNA encoding wild-type human PKG1
produced a basal level of
VASP phosphorylation in both cell types. In addition, exogenous expression of wild-type PKG enabled the A549 cells to respond to cGMP
treatment with a shift in VASP mobility and greatly augmented the
magnitude of VASP phosphorylation in the HEK-293 cells. Cotransfection of a previously described catalytic mutant of PKG1
(T516A) did not
produce any background VASP phosphorylation or confer cGMP responsiveness to the A549 cells. Surprisingly, transfection of HEK-293
cells with the T516A mutant produced a high background level of VASP
phosphorylation, similar in magnitude to that of cGMP stimulated cells
transfected with VASP alone. A similar result was obtained by
transfection of a truncated mutant of PKG that expressed only the
COOH-terminal catalytic regions (data not shown). The electrophoretic
shift of VASP produced by either the T516A mutant or by stimulation
with 8-Br-cGMP was similarly due to phosphorylation, since treatment of
the precipitated VASP with protein phosphatase 2A (39) elicited a
pattern of VASP migration corresponding to the basal state (Fig.
7C).

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Fig. 7.
Phosphorylation of vasodilator-stimulated
phosphoprotein in transfected cells. A549 epithelial cells
(A) and HEK-293 fibroblasts (B, C)
were transiently transfected with a cDNA encoding FLAG
epitope-tagged murine VASP together with either wild-type or mutant
PKG1 as indicated above. After 24 h, the transfected cells were
either left untreated or stimulated for 20 min with 100 µM 8-Br-cGMP as indicated. The phosphorylation state of
the expressed VASP was measured by Western blotting with anti-FLAG
antibodies as detailed under "Experimental Procedures." The
lower panels are parallel Western blots probed with anti-PKG
antibodies (A and B). The effect of
phosphorylation on the electrophoretic mobility of VASP was determined
by treating expressed VASP with protein phosphatase 2A
(PP2A) (C) as detailed under "Experimental
Procedures." Results shown are representative of at least two
experiments.
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Because of the existence of endogenous PKG, HEK-293 cells were
subsequently used to assess the effects of G1
R-GFP and PKG-GFP on
VASP phosphorylation. In these experiments, treatment of the cells with
50 µM 8-Br-cAMP was found to cause a much more dramatic shift in the mobility of VASP on PAGE gels than similar concentrations of 8-Br-cGMP. Despite the relatively small shift in VASP induced by
treatment of the cells with 8-Br-cGMP, there was a pronounced labeling
of VASP with an anti-phospho-Ser239 antibody that was
undetectable in the cAMP-treated cells (Fig. 8). These findings support several
previous studies indicating that Ser239 is a preferred
phosphorylation site for PKG but produces a lesser mobility shift,
whereas modification of Ser157, which is preferred by PKA,
results in a larger electrophoretic shift (37, 38). When cells were
cotransfected with increasing amounts of FLAG-G1
R, the ability of
cGMP to phosphorylate VASP at Ser239 was greatly
diminished. In contrast, the shift in VASP mobility induced by
treatment with cAMP was not affected by expression of equal amounts of
FLAG-G1
R. Identical results were obtained using the G1
R-GFP
construct (data not shown). This effect paralleled the ability of this
construct to inhibit the cGMP-stimulated phosphorylation of BDPEtide by
endogenous PKG in vitro. In support of in vitro kinase assays, transfection of HEK-293 cells to express PKG-GFP resulted in a marked (~60%) shift of the VASP on PAGE gels in the
absence of exogenously added cGMP. Furthermore, this constitutive level
of activity was enhanced by treatment of the cells with cGMP (Fig.
9). In cotransfection experiments this
fusion protein was also able to diminish the phosphorylation of VASP by
the cGMP-independent activity of the PKG-GFP protein.

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Fig. 8.
Specific inhibition of cGMP-stimulated
VASP phosphorylation by G1 R-GFP in
vivo. HEK293 cells were transiently transfected to
express FLAG-VASP and increasing quantities of FLAG-G1 R as indicated
below. The cells were stimulated with 8-Br-cAMP or 8-Br-cGMP (as
indicated above) for 20 min. The cell extracts were then analyzed by
Western blotting with anti-FLAG antibodies (upper panel) and
anti-VASP(Ser239(P)) (lower panel) as detailed
under "Experimental Procedures." The identity of the detected bands
is indicated on the right. Blots shown are representative of
at least three independent experiments.
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Fig. 9.
Constitutive phosphorylation of VASP on
Ser239 by PKG-GFP. HEK-293 cells were transiently
cotransfected to express FLAG-VASP with either vector alone (basal) or
with PKG-GFP (as indicated above). The effect of cGMP treatment (100 µM, 20 min) or expression of G1 R-GFP (as indicated
below) on VASP phosphorylation was determined by Western blotting with
anti-FLAG antibodies (upper panel) or with
anti-VASP(Ser239(P)) antibodies (middle panel).
To confirm expression of the GFP fusion proteins the homogenates were
also analyzed by Western blotting with anti-GFP antibodies (lower
panel). Results shown are representative of two independent
experiments.
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Interaction of PKG with VASP in Vivo--
The ability of the
G1
R-GFP fusion protein to inhibit the cGMP-independent
phosphorylation of VASP by PKG-GFP suggested a competitive mechanism.
The possibility that the PKG might localize to VASP in vivo
was tested by cotransfection of the PKG fusion proteins with FLAG-VASP
into A549 cells followed by fixation and staining with anti-FLAG
antibodies (Fig. 10). In these studies the exogenously expressed VASP protein was found to localize to focal
adhesions, membrane ruffles, and stress fibers as has been reported
previously (30-32). Although there was significant cytosolic staining,
the G1
R-GFP protein was found to colocalize with the VASP in active
membrane regions, but interestingly, it was only found in a subset of
the focal adhesions. As detailed earlier, the PKG-GFP protein is
constitutively active and its expression resulted in a general decrease
in the amount of cellular F-actin. When cotransfected with VASP, the
VASP also showed almost exclusively cytosolic staining, as did the
PKG-GFP. Similar results were obtained by cotransfection of VASP with
FLAG-G1C (data not shown). These data suggested that the GFP fusion
proteins are always associated with VASP, but as a function of
catalytic activity are able to alter their intracellular location. To
add support to the immunohistochemical observations, the GFP fusion
proteins were cotransfected with VASP followed by immunoprecipitation
of the VASP (Fig. 11). When probed with
anti-GFP antibodies it was found that both G1
R-GFP and PKG-GFP were
found in the pellets, although a greater percentage of the former was
present. These studies confirmed that both PKG-GFP and G1
R-GFP could
physically associate with VASP.

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Fig. 10.
Colocalization of GFP fusion proteins with
VASP in vivo. A549 cells were transiently
cotransfected to express FLAG-VASP and either G1 R-GFP (B,
D, F) or PKG-GFP (A, C, E).
After 36 h, cells were fixed and stained for VASP expression with
anti-FLAG antibodies followed by Texas Red-conjugated anti-mouse IgG
secondary antibodies as detailed under "Experimental Procedures."
The localization of VASP is shown in the upper panels
(A, B) as red fluorescence, the
middle panels show GFP fluorescence, and the lower
panels are overlays with colocalization visualized as
yellow. Representative cells from duplicate transfections
are shown. The bar represents 25 µm.
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Fig. 11.
Coprecipitation of PKG fusion proteins with
VASP. HEK-293 cells were transiently cotransfected with FLAG-VASP
and GFP fusion proteins as indicated. The VASP was immunoprecipitated
from cell extracts and the pellets subjected to Western blotting with
anti-GFP antibodies (upper panel) or anti-FLAG antibodies
(middle panel) to detect the GFP fusion proteins and VASP,
respectively. The lower panel shows the expression of the
GFP fusion proteins in the cell extracts prior to immunoprecipitation.
Gels shown are similar to those produced by at least three independent
experiments.
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DISCUSSION |
To examine the functions of specific regions of PKG1
in living
cells, we have created green fluorescent fusion proteins. Constructs
were created that express the full-length enzyme (PKG-GFP), the
amino-terminal regulatory region (G1
R-GFP), or the COOH-terminal catalytic region (GFP-G1C). It was found that when the amino-terminal regulatory half of the enzyme was replaced by GFP, the remaining catalytic half exhibited constitutive, cGMP-independent kinase activity. Surprisingly, addition of GFP to the COOH terminus of full-length PKG also resulted in constitutive activity. The behavior of
this construct might be explained by the ability of the GFP to
sterically hinder interactions (and consequently inhibition) between
the catalytic and the regulatory regions. This hypothesis is supported
by the observation that cGMP was able to augment the kinase activity,
indicating that this enzyme is in a dynamic state. When expressed
alone, the GFP-G1C protein concentrated in the nucleus. Since the size
of this protein was 70 kDa, entry into the nucleus is not likely to
occur by random diffusion. These data thus support the existence of a
functional nuclear localization sequence (NLS) that has previously been
reported to reside within the active site of this enzyme (20).
Interactions between the regulatory and catalytic parts of PKG1
were
found here to be strong enough to support coimmunoprecipitation. When
coexpressed, the regulatory regions (FLAG-G1
R) prevented GFP-G1C
entry into the nucleus. This observation might be explained by blockade
of the NLS by interactions between G1
R (such as the pseudosubstrate domain) with the active site of GFP-G1C to create a molecule resembling inactive PKG. However, the PKG-GFP protein did not enter the nucleus, despite constitutive activity (suggesting that the NLS is unmasked). Moreover, since the GFP did not block the active site of the PKG-GFP protein as detected by the ability to phosphorylate substrates, it
might be inferred that the NLS must also be exposed. This indicates that the presence of the regulatory domains prevents nuclear
translocation of PKG1
by other mechanisms, possibly by interaction
with anchoring proteins that reside within the cytosol.
The G1
R-GFP protein was able to inhibit the kinase activity of
endogenous PKG when overexpressed in HEK-293 cells. This property was
apparent both in vitro and in vivo, demonstrating
the potential utility of this construct in future studies aiming to
determine functions for PKG. Possible mechanisms for the dominant
negative effect include increased availability of the inhibitory
pseudosubstrate motif, as well as cGMP sequestration by excess
nucleotide binding domains. In support of the former mechanism,
however, overexpression of this construct was also able to block VASP
phosphorylation by the cGMP-independent activity associated with the
PKG-GFP protein. A somewhat paradoxical finding was that transfection
of HEK-293 cells with a catalytic mutant of PKG1
was able to
significantly increase the basal PKG activity as measured by
phosphorylation of VASP. This high background was also reported in
homogenates of HEK-293 cells containing the same T516A mutant as
measured by in vitro kinase assays with peptide substrate
(29). Since this activity was not apparent in the PKG-deficient A549
cells, it is likely that the mutant catalytic domains were able to
partially activate endogenous PKG. This can be explained if the T516A
mutant was able to compete with endogenous wild-type catalytic regions for binding to the pseudosubstrate domains of the holoenzyme, as has
been suggested to occur with isolated peptides corresponding to amino
acids 546-576 of the enzyme (40). This idea is supported by the
observed interaction between the catalytic half of PKG (either FLAG-G1C
or GFP-G1C) with the regulatory half of PKG in immunoprecipitation
studies. The inhibitory properties reported for the T516A mutant of
PKG1
are likely explained as mentioned above, by its ability to
behave as a cGMP sink when overexpressed. These studies indicate that
the T516A mutant of PKG1
should not be used as a dominant negative
enzyme to investigate PKG function in future studies.
Type-1 PKG is widely recognized as predominantly cytosolic, but the
recent identification of several GKAPs suggests that this enzyme might
exist in discrete cellular compartments (25, 41). Low expression levels
of PKG relative to other kinases make specific visualization in
situ difficult, and few reports have described the intracellular
localization (27, 28). In studies shown here, dimerization of fusion
proteins containing the amino-terminal regulatory regions (FLAG-G1
R,
G1
R-GFP), and heterodimer formation between FLAG-G1
R and
endogenous PKG, demonstrated that the leucine zipper domain is
functional in these fusion proteins. Because the amino-terminal leucine
zipper domain has also been shown to mediate the interaction of PKG
with several GKAPs, the G1
R-GFP protein would be expected to
localize to intracellular regions in a manner expected for inactive PKG
(since this construct does not express the catalytic regions of the
enzyme). Similarly, because the PKG-GFP protein also contains the
leucine zipper domain but is constitutively active, it is reasonable to
assume that this protein would mimic activated PKG in its intracellular
distribution. The G1
R-GFP protein was found to be associated with
F-actin in dynamic membrane regions and stress fibers in several cell
types. This contrasted with the localization of the PKG-GFP protein, which did not associate with the membrane, and appeared to reduce the
amount of F-actin and dynamic membrane regions in transfected cells.
This difference in localization is a direct result of enzyme activity,
since the fluorescence in cells transfected with PKG(T516A)-GFP more
closely resembled that of the G1
R-GFP (data not shown). These
observations support recent findings where investigators studying VASP
found that long term activation of PKG lead to loss of stress fibers
and focal adhesions with a concomitant localization of VASP to more
centralized regions (42). In the same study the authors demonstrated
that phosphorylation of VASP was central to this process. It was shown
here for the first time that both the active and inactive forms of PKG
fusion protein colocalize with VASP, suggesting that PKG activation
leads to removal of a PKG·VASP complex from the dynamic
membrane regions. More work is required to determine the precise
binding site for PKG on VASP. Immunoprecipitation studies shown here
did not detect a significant decrease in PKG fusion protein binding to
phosphorylated VASP when compared with unphosphorylated VASP,
suggesting binding to alternative sites. VASP family members
have recently been shown to have a negative regulatory role in
chemotaxis, possibly by inducing the formation of stable contacts with
the substratum (30, 42, 43). In addition to the regulation of
chemotaxis, the relocalization of PKG·VASP complexes upon activation
of PKG might therefore affect other cellular processes, as substrates at these new locations would be available for modification.