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INTRODUCTION |
The cGMP-dependent protein kinases
(PKG1 or cGK) are important
regulators of diverse physiological processes. In addition to the well
known role of PKG in platelet activation and vasodilation, these
enzymes have been implicated as important in cell motility, neutrophil
degranulation, osteogenesis, apoptosis, and gene expression (1-3).
Although the PKG substrates that are important to these processes are
not clear, the ability of PKG to regulate signal transduction pathways,
particularly the MAP-kinase pathways, calcium mobilization, and ion
channel function are likely to be central to many processes
(4-15).
Two genes encode three isoforms of PKG, of which type 1 produces two
splice variants. All isoforms are activated by micromolar increases in
cellular cGMP levels that bind to tandem cyclic nucleotide binding
sites in the regulatory domains (16-20). Binding of cGMP causes
conformational changes that lead to elongation of the enzyme, presumably enabling access of substrates to the active site. As with
most protein kinases in this group, PKG activation has been associated
with isoform-specific autophosphorylation resulting in different
properties of the enzyme, including increased sensitivity to cyclic
nucleotides and constitutive activity (21-28).
Elevation of cellular cGMP levels occurs in response to stimulation of
the natriuretic peptide receptors, which have cytosolic guanylyl-cyclase activity, or more commonly by activation of soluble guanylyl-cyclase by nitric oxide (4, 9, 29, 30). Numerous studies have
inferred activation of PKG in response to diverse ligands, presumably
downstream of nitric oxide production, although rarely have NO levels
or PKG activity been measured. The activation of PKG by endothelial
nitric-oxide synthase pathways can occur rapidly and is probably the
case in endothelial cells stimulated with vascular endothelial growth
factor (31, 32). However, stimulation of human neutrophils with
N-formyl-peptides or lipopolysaccharide also leads to a
rapid activation of PKG in the absence of endothelial or neuronal
nitric-oxide synthase (8, 33, 34).
The present work provides evidence that PKG is a substrate for protein
kinase C both in vitro and in vivo, and
phosphorylation results in PKG activation. This finding highlights a
novel signal transduction pathway that provides an alternative
cGMP-independent mechanism for the activation of PKG.
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EXPERIMENTAL PROCEDURES |
Tissue Culture and Reagents--
HeLa epithelial cells and
HEK-293 fibroblasts harboring the T-antigen were maintained at
37 °C, 5% CO2 in T25 flasks at subconfluent density.
All tissue culture media and reagents were from Invitrogen unless
otherwise indicated. Cells were grown in RPMI 1640 medium containing
10% fetal bovine serum, 200 µM L-glutamine,
10 IU/ml penicillin, 10 µg/ml streptomycin. Cells were expanded for
an experiment by trypsinization and dilution into either six-well plates or 10-cm dishes as appropriate. The expression constructs encoding the tagged dominant negative regulatory subunit of PKA and the
tagged VASP have been described previously (6, 35). All reagents and
chemicals were from Fisher unless otherwise indicated.
Production of PKG Expression Plasmids--
The cDNA encoding
full-length human PKG1
and the regulatory region of PKG1
fused at
the C terminus with a FLAG epitope have been described previously (36).
For the present studies, a FLAG epitope (DYKDDDDK) was added to the
amino terminus using PCR, and the tagged product was subcloned into the
pCDNA3 expression vector. There was no difference noted between
wild type and tagged PKG with respect to in vitro kinase
assays (see below) or phosphorylation of VASP (data not shown).
Generation of point mutations to convert the threonine residue at
position 58 of PKG1
to alanine or glutamate was accomplished using
QuikChangeTM according to the manufacturer's instruction using human
PKG1
in the pCDNA3 expression vector as template. The
foreword mutagenic primers were CACATCGGCCCCCGGGCCACCCGGGCGCAGGGC for
Thr58
Ala and CACATCGGCCCCCGGGAGACCCGGGCGCAGGGC
for Thr58
Glu. All constructs were sequenced to
verify correct substitution, and the recombinant proteins were tested
for expression by Western blotting with an anti-PKG C-terminal antibody
(see below).
Expression and Purification of PKG--
PKG1
cDNA was
transiently transfected into either HEK-293 cells or HeLa cells using
LipofectAMINE-PlusTM according to the manufacturer's instructions
(Invitrogen). Generally, cells were split the day before transfection
to low cell densities (30%), and the following morning they were
transfected. This low cell density was essential for experiments
involving cotransfection of VASP and PKG, because at higher cell
densities a high background phosphorylation of VASP was observed (data
not shown). Cells were incubated in the DNA/liposome mixture in
half-volume serum-free RPMI 1640, and after 3 h they were
supplemented with an equal volume of medium containing 10% serum for a
final concentration of 5% serum overnight. For some experiments,
5 h following the addition of DNA, the medium was replaced with
serum-free RPMI 1640 before incubation overnight. This method produced
~70% transfection of HEK-293 cells and 30% HeLa as measured using
enhanced green fluorescent protein vectors (BD Biosciences
Clontech; Palo Alto, CA).
In order to purify PKG1
, the FLAG-tagged fusion protein was
transiently expressed in HEK-293 cells. Typically, 1 µg of DNA/10-cm dish was used, and three dishes were transfected for each purification. On the morning following transfection, the cells were placed on ice,
and the medium was aspirated and replaced with 10 ml of ice-cold PBS.
All subsequent steps were performed on ice or at 4 °C. The cells
were scraped into the PBS and subsequently concentrated by
centrifugation. The cell pellets were resuspended (1 ml/10-cm dish) in
lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 1% Nonidet P-40, 150 nM NaCl) containing phosphatase
and protease inhibitor cocktails (Invitrogen). Lysis was for 20 min at
4 °C followed by clarification by centrifugation at 10,000 × g for 10 min. The expressed PKG was immunopurified in batch
using anti-FLAG-M2-agarose beads (Sigma). The extracts were pooled, and
200 µl of 50% (v/v) anti-FLAG-M2-agarose (Sigma) was added to the
extract and rocked for 1 h at 4 °C. The beads were washed three
times in lysis buffer and once in PBS. The PKG attached to the beads
was then eluted by resuspension in 100 µl of PBS containing 400 µM FLAG peptide (Calbiochem). After 15-min agitation at
4 °C, the beads were pelleted by centrifugation, and glycerol was
added (50%, v/v) to the supernatant containing the PKG and stored at
80 °C until needed.
Immunoprecipitation and Western Blotting--
For
immunoprecipitation, cells grown in six-well plates, or 10-cm dishes
were transfected with plasmids encoding FLAG-PKG or FLAG-G1
R as
detailed above. After experimental treatment of the cells, the plates
were placed immediately on ice, and the medium was replaced by ice-cold
PBS. For 10-cm dishes, the cells were scraped into the PBS and
harvested by centrifugation at 4 °C, and the pellet was resuspended
by repeat pipetting in 1 ml of lysis buffer. The cells grown in
six-well plates were washed once in PBS, and 0.5 ml of lysis buffer was
added directly to the wells. Cell lysis was by agitation for 30 min at
4 °C, and the extracts were clarified by centrifugation at
10,000 × g for 10 min. The supernatants were either
used directly for Western blotting by boiling an aliquot for 5 min in
PAGE sample buffer, or specific proteins were precipitated by adding 30 µl of 50% (v/v) anti-FLAG-M2-agarose beads. Precipitation was
performed by rocking for 1 h at 4 °C, followed by washing of
the beads three times in 1 ml of lysis buffer and once in PBS.
Precipitated proteins were eluted from the beads by adding 30 µl of
PBS containing 400 µM FLAG-peptide and incubation for 30 min on ice. As with the lysates, the eluted proteins were boiled in
SDS-PAGE sample buffer for 5 min.
Protein samples were separated routinely on 10% polyacrylamide
minigels and transferred to supported nitrocellulose. The blots were
blocked by incubating at room temperature for 30 min in PTS (PBS
containing 5% bovine serum albumin and 0.25% Tween 20). Blots were
subsequently probed either for 1 h at room temperature or overnight at 4 °C, followed by three 5-min washes in PTS buffer. Blots were then incubated for 1 h in peroxidase-conjugated
secondary antibodies, and after extensive washing, the proteins were
visualized using chemiluminescence according to the manufacturer's
instruction (Pierce).
Phosphorylation of PKG--
The phosphorylation of PKG in
vitro was assessed using either purified FLAG-PKG immobilized on
anti-FLAG-M2-agarose beads or PKG1
purified from SF9 cells
(Calbiochem). A typical reaction contained 10 µl of PKG and 30 µl
of kinase reaction buffer (20 nM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol,
15 mM magnesium chloride, 10 µM ATP, 10 µCi
of [
-32P]ATP). Reactions contained in addition either
10 µM 8-Br-cGMP or 0.1 µg of human recombinant PKC
(Calbiochem), which was activated by 0.5 mM
Ca2+ and 80 nM PMA. Reactions were incubated
for periods up to 2 h at 30 °C and stopped by 10-fold dilution
in ice-cold PBS. The immobilized PKG was washed three times with
ice-cold PBS and then boiled 5 min in SDS-PAGE sample buffer.
Phosphorylation of PKG was assessed by SDS-PAGE and autoradiography of
the dried gels.
For measurement of PKG phosphorylation in vivo, HEK-293
cells grown in six-well plates were transiently transfected to express FLAG-PKG1
as detailed above. Cells were washed twice in
phosphate-free Dulbecco's modified Eagle's medium and incubated for
3 h in Dulbecco's modified Eagle's medium containing 1 mCi/ml
[32P]PPi (Amersham Biosciences).
Following loading of the cells, the medium was removed and replaced
with prewarmed Dulbecco's modified Eagle's medium with or without
activators or inhibitors as appropriate. After 1 h of incubation,
the cells were harvested, and the PKG was immunoprecipitated as
detailed above. Phosphorylation of the PKG was assessed by SDS-PAGE and autoradiography.
Measurement of PKG Activation--
HEK-293 cells were
transfected with wild type FLAG-PKG, and following overnight starvation
they were stimulated with 100 µM 8-Br-cGMP or 100 nM PMA for 1 h. Cells were harvested, and the transfected PKG was purified by immunoprecipitation as detailed above.
Quantitation of the PKG was performed by densitometric analysis of
Coomassie BlueTM-stained SDS-polyacrylamide gels in which
the FLAG-PKG was compared with a PKG1
standard purchased from
Calbiochem. Reactions (25 µl) contained 5 µl of PKG (~50 ng), 100 µM BPDEtide, 0.1 µM H-89 (Calbiochem), and
10 µM 8-Br-cGMP where indicated. Reactions were incubated
for 10 min at 30 °C in PCR tubes and stopped by placing on ice.
Duplicate 12-µl aliquots were spotted on numbered P81 phosphocellulose filters and washed five times in 0.75% phosphoric acid, 2 min in acetone. The phosphate transferred to the peptide on the
filters was measured by scintillation counting. For each sample,
duplicate reactions were performed, and each experiment was repeated at
least three times.
Antibody Production--
Antibodies were produced in rabbits
using standard methods for immunization and serum preparation. The
antigens were 10-15-mer peptides containing an amino-terminal cysteine
residue that was used for coupling to keyhole limpet hemocyanin using
the Imject maleimide-activated mcKLH kit (Pierce). A peptide
corresponding to the C-terminal 10 amino acids of type 1 PKG was used
to generate the anti-PKGct antibodies, which recognize the carboxyl
terminus of both PKG1
and PKG1
. The peptide sequences used to
generate the anti-phospho-Thr58 antibodies was
CAIGPRT*TRAQGISAEP (where the asterisk denotes a
phosphorylated residue). The sera obtained from inoculated rabbits were
tested for antibody titer using a standard enzyme-linked immunosorbent
assay against the antigenic peptide.
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RESULTS |
Activation of PKC by PMA Leads to PKG-dependent VASP
Phosphorylation--
VASP is a well characterized substrate for the
cyclic nucleotide-dependent protein kinases and can be
phosphorylated on several residues by both PKA and PKG (37).
Phosphorylation of VASP specifically on serine 157 causes a shift in
mobility on SDS-polyacrylamide gels, which is widely used as a measure
of kinase activation in vivo. In our studies of VASP
phosphorylation, an electrophoretic shift of VASP was observed in
HEK-293 cells in the presence of the phorbol ester PMA. In cells
treated with 100 nM PMA, phosphorylation of VASP was
detected as early as 15 min with a maximum at 1 h (Fig.
1A). The phosphorylation of
VASP observed following the addition of PMA was confirmed in Western
blots probed with phosphospecific antibodies directed against both
serine 157 and serine 239, which are the preferred sites for PKA and
PKG, respectively (data not shown). As an analog of diacyl-glycerol,
PMA binds to a subset of PKC isoforms, leading to their activation. The
importance of PKC in the PMA-stimulated phosphorylation of VASP was
examined using the specific PKC inhibitors Gö-6983 and
Rö-32-0432 (Fig. 1B). In these experiments, both
inhibitors were able to reduce the PMA-induced shift of VASP to basal
levels.

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Fig. 1.
VASP is phosphorylated in response to PMA in
HEK-293 cells. HEK-293 cells were transfected to express FLAG-VASP
and were treated with 100 nM PMA. VASP phosphorylation was
analyzed at different intervals by Western blotting (A). The
slower mobility of the phosphorylated form of VASP is indicated
(VASP-P). The importance of PKC in the PMA-induced VASP
mobility shift was assessed in FLAG-VASP-expressing HEK-293 cells. The
cells were incubated for 15 min in the PKC inhibitors Rö-32-0650
(1 µM) and Gö-6930 (1 µM) before
stimulating with 100 nM PMA for 1 h (B).
Western blotting then assessed the effect of PKC inhibition on VASP
phosphorylation. The Western blots shown are representative of three
independent experiments.
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The phosphorylation of VASP on serine 157 suggested the involvement of
the cyclic nucleotide-dependent protein kinases downstream of PKC activation in these cells. In order to examine the involvement of PKG and PKA, HeLa cells were used, since they lack detectable PKG
(Fig. 2). Stimulation of HeLa with 100 µM cAMP produced a significant shift in VASP on
polyacrylamide gels, as was expected due to the ubiquitous expression
of PKA. That PKA was responsible for the cAMP-induced VASP shift in
HeLa cells was confirmed by the lack of effect of cAMP in cells
transfected to express a mutant regulatory subunit that behaves as a
dominant negative for PKA (
R1
). In contrast, VASP did not shift
in HeLa treated with either 100 µM 8-Br-cGMP or with 100 nM PMA. Although overexposure of Western blots detected a
faint band at the size of PKG, the lack of effectiveness of cGMP on
VASP phosphorylation confirmed the absence of functional PKG in these
cells. Transfection of HeLa to express modest levels of PKG1
(0.1 µg/well of a six-well plate) conferred VASP responsiveness (mobility
shift) in response to both 8-Br-cGMP and PMA. Of interest,
co-expression of
R1
did not inhibit but notably enhanced the
effectiveness of both cGMP and PMA to stimulate a VASP mobility shift.
These data indicated that PKG but not PKA was important to VASP
phosphorylation in cells treated with PMA.

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Fig. 2.
VASP phosphorylation in response to PMA
requires PKG. The importance of the cyclic
nucleotide-dependent protein kinases downstream of
PMA-stimulated VASP phosphorylation was assessed in HeLa cells
transfected to express FLAG-VASP alone (A), with PKG1
(B), or with MYC- R1 (C). Cells were
stimulated with 100 µM 8-Br-cAMP (30 min), 100 µM 8-Br-cGMP (30 min), or 100 nM PMA (1 h) as
indicated. VASP phosphorylation was subsequently assessed by Western
blotting with anti-FLAG antibodies (upper panel).
The bottom panel shows expression of PKG in
parallel blots probed with anti-PKGct antibodies. The data shown were
reproduced in at least three separate experiments.
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Phosphorylation of PKG1
by PKC in Vivo and in Vitro Leads to
Enzyme Activation--
Since current literature revealed no precedence
for a role of PKG downstream of PKC, experiments were designed to
further characterize this relationship. The work with HeLa cells
suggested activation of PKG in response to treatment with PMA but did
not suggest a mechanism. To address this issue, HEK-293 cells were transfected to overexpress FLAG-PKG1
and treated with 8-Br-cGMP or
PMA (Fig. 3). Following stimulation, the
PKG was immunoprecipitated from the cells and examined for activity
in vitro using BPDEtide as substrate. The PKG from
unstimulated cells exhibited an ~2-fold increase in activity in the
presence of cGMP. In cells treated with 8-Br-cGMP, the washed PKG
demonstrated identical enzyme activity in vitro to that
obtained from unstimulated cells. This was consistent with previous
reports demonstrating rapid inactivation of PKG1
upon removal of
cGMP. In contrast, the PKG obtained from cells that had been treated
with PMA had a much higher basal activity than controls, reaching
~75% of maximum activity of control PKG activated by cGMP. The high
basal activity of the PKG from PMA-treated cells was further enhanced
in the presence of cGMP, producing more activity than PKG from either
unstimulated or cGMP-treated cells. These results demonstrated that PKG
activation by PMA involved a cGMP-independent modification of PKG1
,
since this stable activation did not occur when the cells were
stimulated with cGMP.

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Fig. 3.
Activation of PKG in vivo by
PMA occurs by mechanism that does not involve cGMP. HEK-293 cells
were transfected to express FLAG-PKG1 and subsequently stimulated
with either 100 µM 8-Br-cGMP or 100 nM PMA
for 45 min. The PKG in the cells was harvested by immunoaffinity
purification, and the enzyme activity was measured by in
vitro kinase assays using peptide substrate as detailed under
"Experimental Procedures." PKG activity of the purified enzyme was
assessed in the absence (open bars) or the
presence (filled bars) of cGMP. The Western blot
probed with anti-PKGct antibodies (below), shows equal
aliquots of the purified PKG used in the assay. The error
bars reflect the S.E. for three experiments. IP,
immunoprecipitation; IB, immunoblot.
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Threonine 58 of PKG1
Is a Target for PKC
Phosphorylation--
The apparent independence of cGMP and the
relatively stable activation of PKG1
in response to PMA treatment of
cells suggested that PKG might be a substrate for direct
phosphorylation by PKC. To test this notion, FLAG-PKG-expressing
HEK-293 cells were loaded with [32P]PPi
and stimulated with PMA. The PKG in these cells was subsequently immunoprecipitated, and the phosphorylation state was determined by
SDS-PAGE and autoradiography. Compared with untreated cells, incubation
of cells with PMA caused a dramatic increase in phosphate incorporation
into PKG (Fig. 4A). This
phosphorylation of PKG in vivo was not due to
autophosphorylation, since similar treatment with 100 µM
8-Br-cGMP for 1 h had no effect on PKG phosphorylation. In support
of a role for PKC in the phosphorylation of PKG, both PKC inhibitors
that were able to block the PMA-stimulated shift in VASP mobility
(Gö-6983 and Rö-32-0432), were also effective at blocking
PMA-stimulated phosphorylation of PKG. Confirmation that PKC could
directly phosphorylate PKG was obtained using purified components in
kinase assays in vitro (Fig. 4B). In these
experiments, incubation of PKG with purified PKC
resulted in a
dramatic increase in phosphate incorporation, which contrasted with
poor incorporation of phosphate in reactions containing cGMP but
lacking PKC.

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Fig. 4.
Phosphorylation of PKG by PKC in
vivo and in vitro. The ability of PMA
to stimulate phosphorylation of PKG in vivo was determined
in HEK-293 cells loaded with [32P]PPi
(A). The labeled cells were subsequently stimulated with 100 µM cGMP or with 100 nM PMA in the
presence or absence of the PKC inhibitors (1 µM
concentration of either Gö-6983 or Rö-32-0432) as
indicated. The PKG in the cells was immunoprecipitated with PKGct
antibodies, and phosphorylation of the PKG was assessed by SDS-PAGE and
autoradiography. B, the ability of purified PKC to
phosphorylate PKG in vitro was determined using
immunopurified FLAG-PKG1 as detailed under "Experimental
Procedures." The immobilized PKG was incubated in reaction buffer
containing either 25 µM 8-Br-cGMP or 0.1 µg of PKC
for various times as indicated. After washing, the phosphorylation of
PKG was determined by SDS-PAGE and autoradiography. A reaction
containing empty beads and PKC was used as a control. The
lower blot in each panel shows Western
blots (WB) probed for PKG to determine loading of PKG in the
reactions. Results shown are representative of three independent
experiments.
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The heterologous phosphorylation of PKG1
by PMA-responsive isoforms
of PKC is a new phenomenon that prompted further investigation of the
target residues. Amino acid sequence analysis of PKG1
revealed 11 potential PKC recognition sites (data not shown). One of these sites
localized to the inhibitory loop of PKG1
(Thr58),
adjacent to the pseudosubstrate domain (Fig.
5A). Notably, Thr58 is the first of tandem threonine residues, the latter
of which had previously been reported to be a site for
autophosphorylation in PKG1
(23, 24, 38). However, in these reports,
the numbering system used to identify amino acid residues omitted the
amino-terminal methionine such that the studies clearly highlighted
Thr59 as the phosphoacceptor residue, whereas the putative
PKC phosphorylation site found here was Thr58. In order to
examine the phosphorylation of Thr58, polyclonal antibodies
were produced in rabbits using a peptide antigen that contained
phosphorylated Thr58. These antibodies demonstrated
specificity using the peptide antigen in an enzyme-linked immunosorbent
assay but were ineffective when used to probe Western blots of cell
lysates from cells treated with PMA (data not shown). Using larger
quantities of purified PKG1
(Calbiochem), these antibodies were able
to specifically identify PKG1
that had been phosphorylated by PKC
in vitro but did not recognize similar amounts of PKG that
was either untreated or incubated in kinase reaction buffer containing
cGMP (Fig. 5B). In these experiments, the
anti-phospho-Thr58 antibodies also recognized a faint band
corresponding to PKC
, presumably the autophosphorylated form of this
enzyme.

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Fig. 5.
Threonine 58 of PKG1
is a substrate for PKC in vitro. Partial
amino acid sequence alignment of a portion of the regulatory regions of
PKG1 and PKG1 is shown (A). Identical amino acids are
shaded. The inhibitory pseudosubstrate sequences (PKG), and
a substrate recognition site for PKC (PKC) are indicated by
dashed boxes. The missing phosphoacceptor
residues in the pseudosubstrate regions are underlined, and
published autophosphorylation sites are circled.
Phosphorylation of PKG1 in vitro was assessed using pure
components with in vitro kinase assays as detailed under
"Experimental Procedures" (B). Reactions contained PKG
(100 ng) and either 25 µM cGMP or activated PKC (15 ng) and were incubated for the times indicated. Phosphorylation of
threonine 58 was subsequently assessed using Western blotting with
phospho-Thr58 polyclonal antibodies. Similar results were
obtained in three separate experiments.
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In order to examine the phosphorylation of Thr58 in
vivo, immunoprecipitates from HEK-293 cells transfected to express
FLAG-PKG1
were used, since the previous experiments indicated a
requirement for larger quantities of PKG and the slight
cross-reactivity of the anti-phospho-Thr58 antibodies to
autophosphorylated PKC. In these experiments, phosphorylation of
Thr58 was detected between 5 and 15 min following
stimulation of the cells with PMA and peaked at 1 h (Fig.
6A). In support of the idea
that phosphorylation of Thr58 in PKG1
might contribute
to enzyme activation, Thr58 phosphorylation detected on
Western blots slightly preceded the shift in electrophoretic mobility
of VASP in parallel experiments. The phosphorylation of
Thr58 by PKC and not by autophosphorylation was further
confirmed by the observation that the regulatory region (G1
R)
containing Thr58 in the absence of catalytic domains could
become phosphorylated in response to PMA stimulation of the cells (Fig.
6B).

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Fig. 6.
Threonine 58 of PKG1
is phosphorylated in PMA-treated cells. HEK-293 cells were
transfected to express either FLAG-PKG1 (A) or
FLAG-G1 R (B) and stimulated with 100 nM PMA.
At the indicated times (1 h in B), the tagged proteins were
immunoprecipitated (IP) with anti-FLAG-agarose beads and
subjected to Western blotting (IB) with
anti-phospho-Thr58 antibodies. The lower
panel in A shows a parallel experiment in which
cells were transfected to express FLAG-VASP, and the homogenates were
analyzed for phosphorylation-induced shift of VASP by Western blotting
at the indicated times. The lower panel in
B shows a Western blot in which part of the
immunoprecipitate was probed for PKG in order to determine protein
loading. Results shown were reproduced in three independent
experiments.
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Phosphorylation of Threonine 58 Is Essential but Not Sufficient for
PKG1
Activation by PKC--
The role of Thr58 in
activation of PKG1
by PKC was further addressed by creating
phospho-null (T58A) and phospho-mimetic (T58E) mutations in PKG1
,
which were immunopurified from transfected HEK-293 cells (Fig.
7A). In support of the T58E
mutation to mimic phosphorylation at this residue, the purified
PKG1
(T58E) mutant was strongly labeled by the
anti-phospho-Thr58 antibodies on Western blots. The
activation state of the purified PKG enzymes was assessed with in
vitro kinase assays using BPDEtide substrate (Fig. 7B).
In these experiments, the T58A mutant had similar enzymatic properties
to the wild type enzyme. In contrast, PKG1
containing the T58E
mutation was partially activated and exhibited a higher level of
phosphotransferase activity compared with wild type enzyme both in the
presence (20%) and absence (~2-fold) of cGMP.

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Fig. 7.
PKG1 (T58E) is
partially active in vitro. FLAG epitope-tagged
wild type (WT) and PKG1 in which threonine 58 was mutated
to alanine (T58A) or to aspartate (T58E) were purified from transfected
HEK-293 cells by immunoaffinity chromatography as detailed under
"Experimental Procedures." Aliquots of the purified proteins were
analyzed by Western blotting using PKGct antibodies,
phospho-Thr58 antibodies, or Coomassie BlueTM staining for
total protein as indicated (A). The phosphotransferase
activity of the purified enzymes was determined using in
vitro kinase assays with BPDEtide as substrate. Reactions were
performed in the absence (open bars) or the
presence (filled bars) of 25 µM
cGMP. The error bars shown in B
represent S.E. resulting from four separate experiments.
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To examine whether PKG1
(T58E) also exhibited activation in
vivo, this mutant was coexpressed with VASP in HeLa cells. These experiments revealed that the high basal activity of the purified T58E
mutant was not detectable in vivo using VASP mobility shift as a readout (Fig. 8). The lack of basal
activity of the T58E mutant in vivo was further confirmed
using the more sensitive anti-phospho-VASP239 antibodies (data not
shown). The previous work describing autophosphorylation of the
adjacent residue in PKG1
reported that modification of this residue
resulted in an increased sensitivity to cGMP compared with
unphosphorylated PKG (25, 26). In our experiments, when compared with
cells expressing wild type PKG1
, the T58E mutant was found to be
much more sensitive to activation by 8-Br-cGMP. In cells expressing
PKG1
(T58E), marked VASP phosphorylation was observed at
concentrations of 8-Br-cGMP as low as 1 µM (2-fold
increase over basal). In contrast, these concentrations of 8-Br-cGMP
had a minimal effect on VASP mobility in cells expressing similar
levels of wild type PKG. Moreover, the activation state of
PKG1
(T58E) measured by VASP mobility shift in vivo was
higher than wild type PKG1
at all concentrations of 8-Br-cGMP
tested.

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Fig. 8.
PKG1 (T58E)
activation is more sensitive to cGMP in vivo.
HeLa cells were cotransfected to express FLAG-VASP and either wild type
(WT) or PKG1 (T58E). The transfected cells were
subsequently incubated for 30 min with increasing concentrations of
8-Br-cGMP as indicated. Cell extracts were subjected to Western
blotting with anti-FLAG antibodies to detect VASP phosphorylation
(A). To facilitate comparison of the wild type and mutant
enzymes in vitro, the Western blots were quantitated, and
the relative VASP shift was expressed as the ratio of phosphorylated
and unphosphorylated forms (B). Results shown are
representative of two independent experiments.
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Whereas the activity of the T58E mutant was designed to
mimic phosphorylation of PKG by PKC, it was of interest to examine the
enzymatic properties of the T58A mutant, which is phospho-null with
respect to this residue. To address this issue, HEK-293 cells were
transfected to express PKG1
(T58A) and stimulated with PMA for 1 h. Wild type PKG1
derived from these cells revealed greater than
2-fold increase in basal activity (in the absence of cGMP) following
treatment of the cells with PMA (Fig.
9A). In contrast, the
PKG1
(T58A) mutant had similar basal activity in both untreated cells
and those that had been treated with PMA. Moreover, this level of
activity exhibited by the PKG1
(T58A) mutant was identical to wild
type PKG derived from unstimulated cells. Similar results were found
when immunopurified wild type and PKG1
(T58A) were phosphorylated
in vitro using purified PKC (Fig. 9B). However, in these studies, the effect of phosphorylation was more pronounced. Wild type PKG, which had been phosphorylated by PKC, was 3-fold more
active than nonphosphorylated enzyme. The basal activity of
PKG1
(T58A) following PKC treatment also increased but to a much
lesser extent than wild type PKG (~60%).

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Fig. 9.
Thr58 of PKG1
is required for activation of PKG by PKC. HEK-293 cells
cotransfected to express FLAG-tagged wild type (WT) or
PKG1 (T58A) were left untreated or were stimulated for 1 h with
100 nM PMA (A). The PKG was then immunopurified
with anti-FLAG-agarose and examined by in vitro kinase
assays using BPDEtide substrate. The effect of PKC phosphorylation
of PKG on the activity of the T58A mutant was assessed using purified
components as detailed under "Experimental Procedures"
(B). The phosphotransferase activity of the phosphorylated
enzymes was subsequently measured using in vitro kinase
assays with BPDEtide substrate in the absence (open
bars) or the presence (filled bars) of
25 µM cGMP. The error bars in both
experiments reflect the S.E. from three independent experiments.
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DISCUSSION |
The present study demonstrates the phosphorylation of PKG1
by
PMA-responsive isoforms of PKC. Moreover, phosphorylation of PKG1
by
PKC leads to PKG activation in the absence of cGMP. This is the first
report of a cGMP-independent mechanism for PKG activation and
highlights a novel signal transduction pathway with widespread implications for a variety of systems.
Phosphorylation and activation of PKG was demonstrated in
vitro using purified components, which indicates that this
phosphorylation is direct and is cGMP-independent. Using VASP
phosphorylation as an indicator, this phenomenon was also shown to
occur in vivo in response to PMA stimulation of HEK-293
cells with endogenous levels of PKG and PKC and also using HeLa cells
transfected to express modest levels of PKG. PKG1
has been shown to
autophosphorylate at an extremely slow rate (38). However, the results
shown here are unlikely to be due to autophosphorylation, since, unlike
PKG1
, this phenomenon does not lead to activation of PKG1
(27).
This notion is supported by experiments presented here in which
treatment of cells with 8-Br-cGMP did not lead to phosphorylation
in vitro or in vivo and did not lead to stable
activation in the time frame sufficient for effective activation by
PMA/PKC.
To further characterize the mechanism by which PKC phosphorylation can
lead to the activation of PKG1
, we have identified Thr58
as a PKC target in vivo and in vitro. Mutation of
this site to a phospho-null (T58A) indicated an important role for this
residue in activation of PKG by PKC, since this mutant was only
partially activated by PKC in vitro and not at all in
vivo. PKG1
harboring a phospho-mimetic T58E mutation was much
more sensitive to cGMP stimulation in vivo and was partially
active when purified. Similar to work shown here for
PKC-dependent phosphorylation of Thr58,
previous work has demonstrated that autophosphorylation of
Thr59 creates an enzyme that is sensitive to lower levels
of cGMP but does not lead to activation of the enzyme (26). The
phosphorylation of residues in other Ser/Thr kinases creates an
alteration in the shape of the inhibitory loop such that the
pseudosubstrate region no longer fits into the catalytic groove. Thus,
it is possible that phosphorylation of PKG at Thr58 or
Thr59 prohibits interaction of the inhibitory domain with
the catalytic cleft, thereby destabilizing the inactive form of the
enzyme. This hypothesis is supported by experiments shown here in which the T58E mutant was partially active when purified from cells and
required lower cGMP levels for activation in vivo.
It has been shown that there are other contact sites between the
regulatory and catalytic domains of PKG that stabilize the inactive
conformation in addition to the interaction between the catalytic cleft
and the pseudosubstrate domain (28, 39, 40). These sites remain obscure
but have been implicated as important to autoinhibition. Because PMA
treatment of cells was able to activate PKG, it is plausible that PKC
can phosphorylate more than one residue of PKG1
, and sites other
than Thr58 may therefore destabilize additional contact
sites, leading to enzyme activation. However, as shown here, neither
phosphorylation site alone is sufficient for activation. That other
residues on the PKG1
enzyme are targets for PKC phosphorylation
in vitro was verified by in vitro kinase assays
showing that the T58A mutant is still phosphorylated by PKC (data not
shown). The nature of these other site(s) will be potentially useful in
generating a mutant that cannot respond to PKC as a useful tool to
investigate the physiological responses of PKC mediated by PKG.
Although PMA was used to stimulate PKC in this report, it is unclear
which of the PMA-responsive PKC isoforms might be involved in
phosphorylation of PKG in vivo. In the present study, it was found that both PKC inhibitors Gö-6983 and Rö-32-0432 were
able to block both the PMA-stimulated VASP mobility shift and the
phosphorylation of PKG in vivo. These inhibitors exhibit
isoform specificity but have in common the ability to block only PKC
and PKC
. Because PKC
expression is more widespread, it is likely
that this isoform was responsible for observations in HEK-293 and HeLa
cells shown here. Since the substrate specificity of different PKC
isoforms is relatively conserved, it is also a possibility that
PMA-insensitive PKC isoforms might also function in this capacity in
cells (41). Identification of the specific PKC isoforms that
phosphorylate PKG in vivo should provide some insight into
which ligands might stimulate this pathway and what physiological
processes might be affected. PKC substrates are often determined by
subcellular colocalization with PKC, and many PKC isoforms move to the
plasma membrane upon activation (42). It has been reported recently that a relatively small subset of cellular PKG localizes to the plasma
membrane (36). If this membrane-bound PKG were specifically targeted by
PKC, this could provide both a mechanism of activation of PKG by PKC
and also serve to explain the lack of sensitivity of the
phospho-Thr58 antibody on Western blots containing whole
cell lysates as observed here.
In summary, we describe here for the first time a mechanism by which
PKG can be activated in the absence of cGMP. This phenomenon provides a
novel link between PKC- and PKG-mediated signal transduction pathways.
Future investigations are warranted to determine which upstream
pathways and PKC isoforms mediate this process, but certainly this
pathway has widespread implications for the regulation of diverse
cellular processes.