Phosphorylation and Regulation of G-protein-activated
Phospholipase C-
3 by cGMP-dependent Protein Kinases*
Chunzhi
Xia
,
Zhenmin
Bao
§,
Caiping
Yue¶,
Barbara M.
Sanborn¶, and
Mingyao
Liu
From the
Department of Medical Biochemistry and
Genetics, Center for Cancer Biology and Nutrition, Institute of
Biosciences and Technology, Texas A & M University System Health
Science Center, Houston, Texas 77030, the § Department of
Biology, Ocean University of Qingdao, Qingdao 266003, Peoples
Republic of China, and the ¶ Department of Biochemistry and
Molecular Biology, University of Texas Houston Medical School,
Houston, Texas 77030
Received for publication, July 14, 2000, and in revised form, February 7, 2001
 |
ABSTRACT |
Among the drugs that are known to relax the
vascular smooth muscle and regulate other cellular functions,
-adrenergic agonists and nitric oxide-containing compounds are some
of the most effective ones. The mechanisms of these drugs are thought
to lower agonist-induced intracellular [Ca2+] by
increasing intracellular cAMP and cGMP, activating their respective
protein kinases. However, the physiological targets of cyclic
nucleotide-dependent protein kinases are not clear. The
molecular basis for the regulation of intracellular Ca2+ by
signaling pathways coupled to cyclic nucleotides is not well defined.
G-protein-activated phospholipase C (PLC-
) catalyzes the hydrolysis
of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol
and inositol 1,4,5-triphosphate, leading to the activation of protein
kinase C and the mobilization of intracellular Ca2+. In
this study, we shown that G-protein-activated PLC enzymes are the
potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-
2 and PLC-
3 in
vitro with purified proteins and in vivo with
metabolic labeling. Phosphorylation of PLC-
leads to the inhibition
of G-protein-activated PLC-
3 activity by 50-70% in COS-7 cell
transfection assays. By using phosphopeptide mapping and site-directed
mutagenesis, we further identified two key phosphorylation sites for
the regulation of PLC-
3 by PKG (Ser26 and
Ser1105). Mutation at these two sites (S26A and
S1105A) of PLC-
3 completely blocked the phosphorylation of
PLC-
3 protein catalyzed by PKG. Furthermore, mutation of these
serine residues removed the inhibitory effect of PKG on the activation
of the mutant PLC-
3 proteins by G-protein subunits. Our results
suggest a molecular mechanism for the regulation of G-protein-mediated
intracellular [Ca2+] by the NO-cGMP-dependent
signaling pathway.
 |
INTRODUCTION |
G-protein-mediated intracellular Ca2+ signaling plays
an important role in mediating the vascular smooth muscle tone and
other cellular functions. Contraction of the smooth muscle defines the lumen in blood vessels, airways, uterus, and bladder. An abnormal increase in smooth muscle tone has been implicated in the pathogenesis of hypertension, cardiogenic shock, and other motility-related disorders (1, 2). Relaxation of the smooth muscle can result from the
increase of intracellular cAMP and cGMP generated by the activation of
adenylyl cyclases and guanylyl cyclases, respectively (3-8). The
mechanism by which cyclic nucleotides (cAMP and cGMP) could relax the
smooth muscle is believed to involve the inhibition of intracellular
free Ca2+ concentration as a result of activating cyclic
nucleotide-dependent protein kinases (9). However, the
molecular target for the action of cyclic
nucleotide-dependent protein kinases
(PKA1 and PKG) in regulating
intracellular Ca2+ signaling is unclear.
Phosphoinositide-specific phospholipase C (PLC) is a family of key
enzymes in the generation and regulation of intracellular Ca2+ signaling, in cell growth, and differentiation (10).
Molecular cloning has identified 10 mammalian PLC isozymes that can be
grouped into three subfamilies (PLC-
, PLC-
, and PLC-
) on the
basis of their size, amino acid sequences, domain structure, and
activation mechanisms (11, 12). The various PLC enzymes appear to be activated by different receptors and mechanisms. The PLC-
subfamily (
1 and
2) is directly phosphorylated and
activated by tyrosine kinase-dependent signaling pathways
(12). The four isozymes of PLC-
are activated to various extents by
the G
q subfamily of G-proteins that couple seven
transmembrane receptors to intracellular signaling pathways (13-18).
Specific activation of PLC-
2 and PLC-
3 by the G-protein 
subunits of the G
i/o family of G-proteins has also been
reported (19-21). Therefore, functionally distinct G-proteins could
selectively couple different receptors to PI hydrolysis and
intracellular calcium release in the same cell.
There is a wide range of interactions, both between homologous
signaling pathways and between heterologous systems (22-25). These
interactions provide a mechanism that allows G-protein-coupled pathways
to influence each other's function and to modulate multiple receptor
signaling pathways. For example, there is increasing evidence for a
physiological interaction between the PI and cAMP signaling pathways.
Both IP3 and diacylglycerol, products of PLC activation,
affect cAMP formation. Calcium mobilized from intracellular stores by
IP3 forms a complex with calmodulin.
Ca2+-calmodulin can directly bind to certain forms of
adenylyl cyclases to activate them (26, 27). Diacylglycerol can also
activate protein kinase C that attenuates or potentiates
hormone-induced cAMP accumulation in different cell types. On the other
hand, there are also numerous reports that the hydrolysis of
phosphatidylinositol 4,5-bisphosphates (PIP2) is regulated
by cAMP and cAMP-dependent protein kinase (PKA). The
effects of cAMP on agonist-stimulated phosphatidylinositide production
range from stimulation to inhibition. cAMP or agents that stimulate
production of cAMP (forskolin, prostaglandins E1 and E2, theophylline,
prostacyclin, and
-adrenergic agents), inhibit
phosphatidylinositide breakdown and intracellular Ca2+
mobilization in a number of cells and tissues (3, 5, 28-38). However,
the molecular mechanism by which the cAMP pathway regulates the
PIP2 metabolism and intracellular Ca2+
mobilization is poorly understood. Our recent work (22, 39), together
with others (37), has suggested that PKA could phosphorylate G-protein-activated phospholipase C isozymes, regulating the
G-protein-mediated intracellular Ca2+ signaling.
Ca2+ and nitric oxide (NO) work together in the control of
cell homeostasis. NO has been recognized to account for the activities of the endothelial-derived relaxing factor by increasing intracellular cGMP concentration. In a number of cells, nitric oxide and atrial natriuretic factor inhibited the release of intracellular
Ca2+ from internal storage sites. The inhibitory effects of
these agents appear to be mediated by increasing intracellular cGMP and
by activating cGMP-dependent protein kinases (6, 9, 40-46). Potential PKG phosphorylation targets of intracellular Ca2+ signaling pathway include phospholipase C, G-proteins,
Ca2+-ATPase, phospholamban, IP3 receptor ion
channels, and myosin phosphatase (47). However, the molecular mechanism
of PKG phosphorylation in mediating intracellular Ca2+
release has not been defined. In most cases, the physiological targets
of PKG phosphorylation remain to be identified, and their function
needs to be clarified. Knowledge of the molecules involved in the
regulation of intracellular Ca2+ by
cGMP-dependent protein kinase could be of great importance in pharmacology (48). For example, the effect of the popular drug,
Viagra (sildenafil), is to increase intracellular cGMP concentration and lower intracellular Ca2+ levels via inhibiting a
specific phosphodiesterase in the vascular smooth muscle. In other
words, Viagra achieves its pharmacological effects of relaxing the
vascular smooth muscle through the increase of intracellular cGMP and
possibly cGMP-dependent protein kinase (9). However, the
molecular mechanism on how an increase in cGMP affects intracellular
Ca2+ and, consequently, the relaxation of the vascular
smooth muscle is still under active investigation. In this report, we
demonstrate that G-protein-activated PLC-
2 and PLC-
3 are directly
phosphorylated by PKG in vitro with purified proteins and
in vivo with metabolic labeling. Phosphorylation of PLC
blocks the activation of the proteins by G-protein subunits.
Furthermore, we identified two serine residues (Ser26 and
Ser1105) as the PKG phosphorylation sites in PLC-
3.
Mutation of these two sites completely abolished the phosphorylation of
the protein by PKG and the inhibitory effect of PKG on the activation
of PLC-
3 by G-protein subunits. Therefore, these data define a
potential cross-talk mechanism between G-protein-mediated intracellular Ca2+ release and the cGMP/PKG signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Materials--
PLC-
antibodies, immunoblotting, and
immunoprecipitation reagents were obtained from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). LipofectAMINE, DMEM, and all other
cell culture reagents were obtained from Life Technologies, Inc.
[3H]Inositol (22 Ci/mmol),
[32P]orthophosphate, and [
-32P]ATP (3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. The
PLC-
1 clone in baculovirus was obtained from Dr. P. Sternweis (University of Texas Southwestern Medical Center). cDNAs
encoding three different PKG isozymes (PKGI
, PKGI
, and PKGII)
were kindly provided by Dr. Suzanne Lohmann at the University
of Wurzburg, Germany) (70, 74).
Protein Purification and Site-directed
Mutagenesis--
His6-tagged PLC-
2 and PLC-
3 in
baculovirus (PharMingen, San Diego, CA) were constructed from the
respective PLC cDNA plasmids. The expressed proteins,
His6-PLC-
2, and His6-PLC-
3, were purified by chromatography over Ni2+-agarose, and Mono Q and
Mono S columns as described previously for PLC-
1 from the membrane
fraction of Sf9 cells (49). The purity of the proteins is more
than 98% as judged by SDS-PAGE. Site-directed mutations at
Ser26, Ser474, Ser1105 (Ser to Ala)
were generated by polymerase chain reaction or using the GeneEditor kit
(Promega, Madison, WI). All PLC mutant sequences were confirmed by DNA sequencing.
In Vitro Phosphorylation of PLC-
and Phosphopeptide
Mapping--
Purified His6-tagged recombinant PLC-
(
1,
2, and
3) were incubated with purified PKG enzymes or PKA
catalytic subunit at a molar ratio 50:1 in the presence of 1-10 µCi
of [
-32P]ATP and 100 µM ATP in a total
volume of 50 µl of kinase buffer (10 mM HEPES, pH 7.2, 10 mM MgCl2) for 30 min at 30 °C. For the time
course study, 2 µg of PLC-
proteins were incubated with PKG or PKA
for the indicated time. PKG enzymes were obtained from Promega or
kindly provided by Dr. S. Lohmann at the University of Würzburg,
Germany. Phosphorylation reactions were terminated by addition of 4×
SDS sample buffer and boiling for 5 min. Proteins were separated on 8%
SDS-PAGE gels, and the phosphorylated proteins were visualized by autoradiography.
Two-dimensional phosphopeptide mapping of PLC-
3 phosphorylated by
PKG or PKA in vitro was carried out with a Hunter thin layer
electrophoresis system (CBS Scientific Company, Del Mar, CA) according
to the manufacturer's instructions. Briefly, the phosphorylated PLC
proteins were separated by SDS-PAGE. PLC proteins were washed and
digested with trypsin for 24 h at 35 °C. The phosphopeptides were separated by two-dimensional chromatography.
Preparation of Smooth Muscle Cells and in Vivo 32P
Labeling--
Smooth muscle cells were isolated from the aorta of
Harlan Sprague-Dawley rats as described previously (50). Briefly, the aorta was incubated in collagenase (1.5 mg/ml) for 10 min at 37 °C
before removing the adventitia. The smooth muscle cells were maintained
in DMEM with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.2 mM glutamine. The cells were
grown to confluence and used until the third passage to prevent the loss of endogenous PKGs. Nearly confluent smooth muscle cells (10-cm
dish) were labeled with [32P]orthophosphate (0.33 mCi/ml)
in phosphate-free DMEM containing 10% dialyzed fetal calf serum for
6 h. After stimulation with the membrane-permeable cGMP analogue,
8-pCPT-cGMP (100-200 µM), cells were lysed in ice-cold
lysis buffer containing a mixture of protease and phosphatase
inhibitors (37). Cell extracts were centrifuged at 15,000 × g for 10 min at 4 °C. Proteins immunoprecipitated with
specific anti-PLC-
3 antibody were separated on a 7.5% SDS-PAGE and
analyzed by autoradiography. The amount of PLC-
3 protein was
normalized for sample loading by Western blot.
Cell Culture, Transfection, and PLC Activity Assays--
COS-7
cells were cultured and transfected as described previously (22).
Briefly, COS-7 cells (1 × 105 cells per well) were
seeded in 12-well plates in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum a day before transfection. The total
amount of DNA in all transfections was 1-1.2 µg per well. The
cytomegalovirus vector, pCIS, encoding
-galactosidase was used to
maintain the amount of DNA constant and the amount of a particular
cDNA in each set of experiments equal. cDNAs were mixed with
LipofectAMINE (Life Technologies, Inc.) in serum-free Opti-MEM (Life
Technologies, Inc.), and cells were transfected for about 5 h.
24 h post-transfection, cells were washed with phosphate-buffered
saline (PBS) and incubated in 0.5 ml of inositol-free DMEM medium
containing 10 µCi ml
1 of
myo-[2-3H] inositol (Amersham Pharmacia
Biotech) and 10% dialyzed fetal bovine serum. 24 h later, cells
were washed with PBS and incubated in inositol-free medium containing
10 mM LiCl for 30 min at 37 °C. For activation
cGMP-dependent protein kinases, 100 µM
8-pCPT-cGMP was added to the inositol-free LiCl medium. The cells were
lysed by adding ice-cold 10 mM formic acid and incubated on
ice for 30 min. Then the lysate was neutralized with 15 mM
NH4OH. Total inositol phosphates were separated by an anion
exchange column (AG 1-X8, Bio-Rad) as described. The eluted inositol
phosphates was mixed with 10 ml of scintillation mixture (BCS; Amersham
Pharmacia Biotech) and counted in a liquid scintillation counter.
 |
RESULTS |
Phosphorylation of PLC-
by PKG in Vitro and in Vivo--
We
have shown previously that PKA can directly phosphorylate PLC-
2 and
PLC-
3, blocking the activation of the enzymes by G-protein subunits
(22) (39). On the other hand, direct phosphorylation of PLC-
isozymes by cGMP-dependent protein kinases has not been reported, although it has been known that an increase in intracellular cGMP can inhibit the hydrolysis of phosphatidylinositide and the release of intracellular Ca2+. In order to understand the
regulation of G-protein-mediated PLC-Ca2+ signaling by
pathway coupled to intracellular cGMP, we extended our studies on the
phosphorylation of PLC-
isozymes by PKG both in vitro and
in the smooth muscle cells. As shown in Fig.
1A, incubation of purified
PLC-
isozymes with purified PKG demonstrated that both PLC-
2 and
PLC-
3 are good substrates for PKG, whereas PLC-
1 was not
phosphorylated by the kinase under the same conditions. To quantify
PKG-stimulated PLC-
3 phosphorylation, purified PLC-
3 was
phosphorylated by PKG in vitro. Fig. 1B shows the
time-dependent incorporation of 32P into
PLC-
3 protein catalyzed by PKA and PKG. Compared with the
phosphorylation of PLC-
3 by PKA (Fig. 1B, top), PKG
phosphorylation is slower and approached a plateau in about 60 min.
Estimation of the Km value for PKA is about 1 µM, whereas the Km value for PKG is
somewhat higher, ~12 µM when assayed with different concentrations of purified PLC-
3 protein. A maximum ratio of 1.3 mol
of phosphate/mol of PLC-
3 was determined by a filter binding assay
at the 60-min incubation point, suggesting two possible phosphorylation
sites in PLC-
3 protein.

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Fig. 1.
Phosphorylation of purified
PLC- isozymes by PKG in vitro.
A, purified His6-tagged PLC- 1, PLC- 2, and
PLC- 3 proteins were phosphorylated by PKGI (0.01 µg, 30 units,
Promega) for 30 min at 30 °C. PLC- 2 and PLC- 3, but not
PLC- 1, are good phosphorylation substrates for PKGI. A total of 2 µg of purified PLC- proteins were used in each reaction. 0.5 µg
of protein was loaded on the gel. Similar phosphorylation was observed
using PKGII (data not shown). B, time-dependent
phosphorylation of PLC- 3 in vitro. Reactions were
terminated at the indicated times (in minutes). Compared with PKA
phosphorylation of PLC- 3 (top), phosphorylation of
PLC- 3 by PKG is slower, reaching a plateau at ~60 min.
C, phosphorylation of PLC- 3 by three different PKG
isoforms. PLC- 3 was overexpressed in COS-7 cells and
immunoprecipitated by specific anti-PLC- 3 antibody. The precipitated
protein was phosphorylated by purified PKG isoforms (PKGI , PKGI ,
and PKGII) (0.05-0.1 µg/reaction) (73). Phosphorylation of
immunoprecipitates by preimmune serum (without PLC- 3 protein) ( )
and by specific anti-PLC- 3 antibody are shown and labeled for the
presence of PLC- 3 protein. Putative autophosphorylated PKGs were
detected along with phosphorylated PLC- 3 in the immunoprecipitation
complexes in the assay. Samples were analyzed by SDS-PAGE and
visualized by autoradiography.
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There are three different isoforms of PKG that differ in their
sequences, distribution, and membrane association (51). To examine
whether all three isozymes of PKG can phosphorylate PLC-
proteins,
we phosphorylated overexpressed PLC-
3 in the presence of PKGI
,
PLGI
, and PKGII, and we then immunoprecipitated PLC-
3 using
specific anti-PLC-
3 antibodies. As shown in Fig. 1C,
PLC-
3 was phosphorylated by all three isozymes of PKG in our
in vitro phosphorylation assays.
To test the notion that PLC-
2 and PLC-
3 are physiological targets
of cGMP-dependent protein kinases in the cell, we further analyzed whether activation of PKG can directly phosphorylate PLC-
isoforms in cells expressing these proteins. Cell extracts from
cultured smooth muscle cells were incubated in the presence of
[
-32P]ATP. As shown in Fig.
2A, stimulation with selective
PKG agonist, 8-Br-cGMP, greatly increased the phosphorylation
intensities of 130-140-kDa proteins. Immunoprecipitation with specific
antibodies against PLC-
2 and PLC-
3 indicated that both proteins
are phosphorylated by the stimulation of 8-Br-cGMP (Fig.
2B). Together these data suggested that PLC-
proteins
could be one of the physiological targets upon stimulation of smooth
muscle cell extracts by intracellular cGMP.

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Fig. 2.
Phosphorylation of PLC-
in smooth muscle cells. A, smooth muscle cell extracts
were prepared by centrifugation and incubated with
[ -32P]ATP at 37 °C for 10 min in the presence or
absence of the PKG activator, 8-Br-cGMP (1 µM). Then the
phosphorylated proteins were separated by SDS-PAGE and visualized by
autoradiography. EGTA (2 mM) was included in all reactions
to remove calcium, an activator of proteases and protein kinase C. B, PLC- 2 and PLC- 3 were phosphorylated in smooth
muscle cell extracts stimulated with 8-Br-cGMP (1 µM).
The phosphorylated cell extracts were immunoprecipitated with specific
anti-PLC- 2 and PLC- 3 antibodies. Precipitated PLC- 2 and
PLC- 3 proteins were separated by SDS-PAGE and visualized by
autoradiography. The specificity of the anti-PLC- 2 and anti-PLC- 3
antibodies was tested in vitro with purified PLC proteins
and in cell lysates. C, in vivo 32P
labeling of PLC- 3 isolated from cultured smooth muscle cells. VSMCs
from rat aorta between the third and fifth passage were used so that
endogenous PKG can be preserved. Western blot shows that PKG can be
detected up to passage level 7 in the VSMCs. The cells were
metabolically labeled with [32P]orthophosphate in
phosphate-free DMEM in the presence or absence of PKG activator,
8-pCPT-cGMP (100 µM) for 3-4 h. Then the PLC- 3 was
immunoprecipitated by specific anti-PLC- 3 antibody, and the proteins
were separated on SDS-PAGE. Phosphorylated PLC- 3 was visualized by
autoradiography. Similar results were obtained from three independent
experiments. As a control, we did not see any specific band
corresponding to the size of PLC- when precipitated with a preimmune
serum. Bottom panel, Western blot of PLC- 3 using specific
anti-PLC- 3 antibody, demonstrating an equal amount of protein was
loaded in the gel.
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To confirm that PLC-
3 is phosphorylated upon stimulation of
increased cGMP in the cell, cultured smooth muscle cells before passage
level 5 were metabolically labeled with
[32P]orthophosphate. The expression of PKG in VSMCs
before passage level 8 was confirmed by Western blot using specific
anti-PKG antibodies. PLC-
3 protein was immunoprecipitated by a
specific anti-PLC-
3 antibody after labeling of the cell. Stimulation
of PKG by the selective activator, 8-pCPT-cGMP, substantially increased the 32P incorporation into PLC-
3 protein (Fig.
2C), indicating that PLC-
3 is a potential physiological
target of cGMP-dependent protein kinases in the cell.
Inhibition of G-protein-activated PLC-
3 by PKG Isoforms--
To
test whether cGMP and PKG are involved in the agonist-stimulated and
G-protein-mediated PLC signaling pathway, we constructed mammalian
expression plasmids that encode the three different PKG isoforms. Then
we reconstituted the G-protein-activated PLC-Ca2+ signaling
pathway in COS-7 cells by introducing cDNAs encoding G-protein
subunits, PLC-
, and PKGs into the cells. Specific activation of
PLC-
3 by G-protein 
subunits was measured by the release of
inositol phosphates (IP3) (Fig.
3A). In the presence of PKG isozymes, the activation of PLC-
3 by G-protein 
subunits was substantially reduced upon stimulation of PKGs by the
membrane-permeable cGMP analogue, 8-pCPT-cGMP (Fig. 3A).
Since PLC-
3 is also activated by G
q, we examined the
PI turnover in COS-7 cells transfected with PLC-
3 and
G
q protein in the presence or absence of PKG. As shown
in Fig. 3B, transfection of empty vector, G
q,
PLC-
3, or PKG alone had little effect on basal PI turnover.
Cotransfection of G
q with PLC-
3 increased the
production of inositol phosphates by approximately 5-fold. On the other
hand, cotransfection and activation of PKG inhibited more than 60% of
the activation of PLC-
3 by G
q.

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Fig. 3.
Inhibition of G-protein-activated
PLC- by PKGs. A,
activation of PLC- 3 by G subunits was inhibited by three
different PKG isoforms (PKGI , PKGI , and PKGII) in COS-7 cells.
cDNAs transfected into the cells are listed below the
column. The total amount of cDNAs added was 1.2 µg per well. The
amount of cDNA for each gene was kept the same in each transfection
(0.3 µg/well). pCMV-lacZ was used to make the total amount of
cDNAs equal in each well (1.2 µg/well). B, PKG
inhibits G q-stimulated PLC- 3 activity in COS-7 cells.
In all transfection experiments PKG was activated in the cell by 100 µM 8-pCPT-cGMP for 30-60 min. Total amount of cDNA
for each gene transfected into COS-7 cells is 0.35 µg/well. Data
presented are the means ± S.E. (n = 3) of three
transfection repeats (wells). C, activation of PKG inhibits
agonist-induced PI turnover in COS-7. COS-7 cells were transfected with
M1 muscarinic receptor (0.3 µg) in the presence or absence of PKG
isozymes (0.6 µg). After 48 h of transfection,
[3H]inositol-labeled cells were incubated with 100 µM 8-pCPT-cGMP for 30 min. The cells were stimulated with
20 µM carbachol for 20 min (dark columns). The
control cells were treated with PBS (light column). Inositol
phosphates were determined as described (22, 39). All cDNAs are
under the control of the cytomegalovirus promoter. The expression level
of endogenous PLC- is not affected by the expression of PKG in the
assay conditions (data not shown). Data represent the average value of
triplicates. Error bars give the range of triplicates.
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To examine whether the agonist-induced IP3 synthesis is
indeed inhibited by the activation of PKG, we measured the production of IP3 induced by activation of the M1 muscarinic receptor
in COS-7 cells in the presence or absence of overexpressed PKG. COS-7 cells were transfected with the M1 muscarinic receptors coupled to
PLC-
through G
q protein. As shown in Fig.
3C, when the M1 receptor was transfected in COS-7 cells and
stimulated with its ligand, carbachol, the production of
[3H]IP3 was increased about 5-6-fold.
Cotransfection and activation of the PKG isoforms inhibited the
agonist-stimulated IP3 release by 30-40% in COS-7 cells
(Fig. 3C). Since COS-7 cells contain multiple PLC-
isoforms (
1,
2, and
3), the observed inhibitory effects may
underestimate the effects of PKG on the regulation of
receptor-stimulated G-protein-coupled IP3 signaling in the cell. Together, these data suggest that activation of PKG could phosphorylate PLC and inhibit the agonist-induced IP3
production in the cell.
PKG Phosphorylates Distinct Peptides in PLC-
3 Compared with PKA
Phosphorylation--
To examine whether PKG phosphorylates PLC-
3 at
the same sites as PKA phosphorylation, we phosphorylated purified
PLC-
3 protein with sequential addition of the two kinases (PKA and
PKG) in the presence of non-radioactive ATP first and then of
[
-32P]ATP. Initial phosphorylation of PLC-
3 by PKA
in the presence of non-radioactive ATP has limited effect on the
subsequent phosphorylation of the protein by PKG (Fig.
4A). However, initial
phosphorylation of PLC-
3 by PKG in the presence of non-radioactive
ATP prevents subsequent phosphorylation of the protein by PKA (Fig.
4B). These results indicate that PKG not only shares one or
more phosphorylation sites in PLC proteins with PKA, but also has
unique phosphorylation sites that are not blocked by initial PKA
phosphorylation of the protein in the presence of non-radioactive ATP.
On the other hand, the PKA phosphorylation sites in PLC-
are shared
by PKG, as shown by the inhibitory effect of initial PKG
phosphorylation on the subsequent PKA phosphorylation of the PLC-
protein (Fig. 4B).

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Fig. 4.
Sequential phosphorylation of
PLC- by PKA and PKG. A,
initial phosphorylation of PLC- 3 by PKA in the presence of unlabeled
ATP has little effect on the subsequent phosphorylation of the protein
by PKG in the presence of [ -32P]ATP, suggesting
different sites of phosphorylation in the protein by PKA and PKG.
Lane 1 (control), phosphorylation of PLC- 3 by PKG in the
presence of [ -32P]ATP for 60 min. Lane 2,
PLC- 3 was phosphorylated by PKA for 30 min in the presence of
unlabeled ATP, followed by the addition of PKG and
[ -32P]ATP for 60 min at 30 °C. B,
initial phosphorylation of PLC- 3 by PKG dramatically reduced the
subsequent phosphorylation of the proteins by PKA. Lane 1 (control), purified PLC- 3 protein (1 µg) was phosphorylated by PKA
for 30 min at 30 °C. Lane 2, the same amount of PLC- 3
protein was first phosphorylated by PKG for 30 min in the presence of
unlabeled ATP, followed by the addition of PKA and
[ -32P]ATP for another 30 min.
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To understand further the phosphorylation pattern of PLC-
protein,
we compared the phosphopeptide mapping of PLC-
3 protein phosphorylated by either PKA or PKG. As shown in Fig.
5, phosphopeptide mapping of in
vitro phosphorylated PLC-
3 by PKA (Fig. 5A) and PKG
(Fig. 5B) digested by trypsin showed largely similar
patterns of peptide phosphorylation. However, compared with PKA
phosphorylation, PKG has a unique phosphopeptide at position 1 in Fig.
5B. Both PKG and PKA have favored phosphorylation sites
(position 3 for PKG and position 2 for PKA in Fig. 5, A and
B). Together, these data suggest that PKG phosphorylates at
least one unique site in PLC-
3 protein compared with PKA.

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Fig. 5.
Two-dimensional phosphopeptide mapping of
PLC- 3 digested by trypsin. A,
PLC- 3 phosphorylated by PKA. B, phosphorylation of
PLC- 3 by PKG. PLC- 3 was purified from COS-7 cells by
immunoprecipitation. The protein was phosphorylated in vitro
by purified PKG (Promega). Phosphorylated proteins were separated by
SDS-PAGE and digested with trypsin (400 µg/ml). After two-dimensional
phosphopeptide mapping, phosphopeptides were visualized by
autoradiography. Compared with the phosphorylation of PLC- 3 by PKA
(A), PKG phosphorylates a unique site at point 1 in
B.
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Identification of the PKG Phosphorylation Sites in
PLC-
3--
Detailed characterization of the phosphorylation sites
in PLC-
isoforms is an essential step toward understanding the
mechanism of PLC-
activation and regulation by
cGMP-dependent protein kinase. In this report, we focus on
the identification of phosphorylation sites in PLC-
3. Three putative
PKG phosphorylation sites (Ser26, Ser474, and
Ser1105) were found in human PLC-
3 (Fig.
6A). Ser26 is
located in the putative PH domain of the protein. Ser474 is
in the catalytic domain (X box) of the protein. Ser1105 is
in the so-called G box region where G
q was found to
interact with in PLC-
1 protein (52). To determine which serine
residue is phosphorylated in the protein, we purified a mutant PLC-
3 protein (S1105A) from insect Sf9 cells. Mutation
at this site abolished the phosphorylation of PLC-
3 by PKA (Fig.
6B), consistent with the result reported previously (39).
However, the phosphorylation of purified mutant PLC-
3 protein
(S1105A) by PKG was only reduced by ~30% compared with the
phosphorylation of wild-type PLC-
3 protein (Fig. 6C),
suggesting PKG also phosphorylates additional sites, consistent with
the results of phosphopeptide mapping.

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Fig. 6.
Identification of PKG phosphorylation sites
in PLC- 3. A, domain structure
and putative phosphorylation sites of PLC- 3. The PH domain may play
a role in interacting with G and phospholipid substrates.
X and Y boxes are the catalytic domains of the
protein. G box is responsible for the interaction with
G q in PLC- 1. Three putative phosphorylation sites are
found in PLC- 3 as follows: Ser26 in peptide 1 (P1), Ser474 in peptide 2 (P2), and
Ser1105 in peptide 3 (P3). B,
mutation at Ser1105 to Ala completely abolished the
phosphorylation of the protein by PKA. Purified
His6-PLC- 3 was phosphorylated by PKA in
vitro. Phosphorylated protein was separated by SDS-PAGE and
visualized by autoradiography. C, phosphorylation of the
His6 PLC- 3 mutant (S1105A) protein by PKG. Mutation at
this site reduced the PKG-catalyzed phosphorylation of PLC- 3 by
about 30-40%. D, phosphorylation of mutant PLC- 3
proteins expressed and isolated from COS-7 cells. Mutation at
Ser26 and Ser1105 reduced the phosphorylation
of the protein by ~70% and 40%, respectively. Double mutation of
Ser26 and Ser1105 to Ala completely blocked the
phosphorylation of the protein by PKG. Mutation at Ser474
has little effect on the phosphorylation of the protein by PKG. Similar
results were obtained from three independent experiments. Bottom
panel, Western blot of PLC- 3 and mutants, showing equal amount
of PLC proteins were loaded in the gel.
|
|
To identify the remaining phosphorylation sites, we made three single
mutations at Ser26, Ser474, and
Ser1105 and one double mutation (S26A,S1105A).
cDNAs corresponding to the wild-type and the mutant PLC-
3 were
cotransfected into COS-7 cells, and the proteins were isolated and
purified by immunoprecipitation using specific antibodies against
PLC-
3. Then the purified proteins were phosphorylated in
vitro using purified PKG. As shown in Fig. 6D,
wild-type PLC-
3 was a good substrate of PKG, whereas mutation at
Ser26 dramatically reduced the phosphorylation of the
protein. Similar to our in vitro phosphorylation assay,
mutation at Ser1105 inhibited about 30-40% of the
incorporation of 32P into the protein. Double mutations at
serine residues 26 and 1105 (S26A,S1105A) almost completely abolished
the phosphorylation of the protein by PKG, indicating that
Ser26 and Ser1105 are the PKG phosphorylation
sites in PLC-
3. On the other hand, mutation at Ser474
has little effect on the phosphorylation of the protein by PKG. Similar
results were obtained when the mutant PLC-
3 genes were cotransfected
with PKG and metabolically labeled with
[32P]orthophosphate in the presence of the
membrane-permeable cGMP analogue, 8-pCTP-cGMP (data not shown). These
data suggest that serine 26 and serine 1105 are the two major
phosphorylation sites in PLC-
3 catalyzed by PKG. Sequence comparison
of the PLC-
isozymes reveals that Ser26 and
Ser1105 of PLC-
3 are not conserved among the PLC
enzymes. Therefore, the mode of regulation between different PLC-
isoforms (
1,
2,
3, and
4) could vary.
To confirm our observation that serines 26 and 1105 are functional
phosphorylation sites in the protein, we cotransfected the mutant
proteins with G-protein subunits into COS-7 cells and measured the
activation of the mutant proteins in the presence of activated PKG. As
shown in Fig. 7, A and
B, mutation at the serine residues (Ser26,
Ser474, and Ser1105) had no effect on the
activation of PLC-
3 by G-protein subunits in COS-7 cell transfection
assays. However, unlike the wild-type PLC-
3, cotransfection and
activation of PKG no longer elicited inhibitory effects on the
activation of the double mutant PLC-
3 protein (S26A,S1105A) by
G
q (Fig. 7A) and G
subunits (Fig. 7B). Mutation at either Ser26 or
Ser1105 alone had very limited effect on the inhibition of
PKG, whereas mutation at Ser474 has no effect on the
inhibition of PLC by PKG (Fig. 7, A and B).
Together, these data indicate that the two serine residues (Ser26 and Ser1105) play a key role in the
regulation of PLC-
3 enzyme by cGMP-dependent protein
kinase.

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|
Fig. 7.
Mutation at Ser1105 and
Ser26 abolished the inhibitory effect of PKG on the
activation of PLC- 3. A,
PLC- 3 and mutant proteins were activated by G q. The
inhibitory effect of PKG was affected by the mutation of PLC- 3
protein at Ser1105 and Ser26 or both, but not
by mutation at Ser474. cDNAs corresponding to
PLC- 3WT, PLC- 3 mutants, G q, and PKGI were
transfected into COS-7 cells. The production of 3H-labeled
inositol phosphates was measured as counts/min. Data presented are the
means of three repeat transfections (wells) ± S.E.
(n = 3). Plasmids transfected into the cells are listed
below the column. The total amount of cDNAs transfected
into COS-7 cells is 1.2 µg/well with 0.3 µg of cDNA for each
gene. B, effect of PKG on the G -stimulated activation
of PLC- 3 mutants. PLC- 3 and mutant plasmids were activated by
G 1 2 subunits in COS-7 cell transfection assays. Mutation at
Ser26 and Ser1105 to Ala completely removed the
inhibitory effect of PKG on the activation of PLC- 3 by
G 1 2. cDNAs transfected into COS-7
cells are labeled below the column. Total amount of
cDNAs (1 µg/well) transfected in each well are maintained the
same by adding pCIS-LacZ.
|
|
 |
DISCUSSION |
In the vascular smooth muscle, nitric oxide (NO) has been
recognized to account for the activities of the endothelially derived relaxing factor by increasing intracellular cGMP concentration via the
activation of guanylyl cyclases. NO inhibits IP3 generation and intracellular Ca2+ mobilization by increasing the
intracellular cGMP concentration. The downstream target for cGMP is
believed to be the PKGs, which mediate the effects of cGMP in a number
of cells or tissues, including the vascular smooth muscle and platelets
(9, 31, 40, 51, 53). Whereas an increase in cGMP level correlates well
with a decrease in intracellular Ca2+ concentration and the
relaxation of the vascular smooth muscle in a time- and
concentration-dependent manner, the molecular mechanisms for cGMP-PKG to regulate intracellular Ca2+ signaling are
not well defined. In this report, we demonstrate that PLC-
2 and
PLC-
3 are good substrates for cGMP-dependent protein
kinases, whereas PLC-
1 was not phosphorylated in the same assays.
These results correlate well with our cell transfection assays in which
activation of PLC-
2 and PLC-
3 by G-protein subunits is inhibited
by coexpression and activation of PKG isozymes (regulation of PLC-
2
by PKG has not been shown in this paper). By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key
phosphorylation sites (Ser26 and Ser1105) for
the regulation of PLC-
3 by PKG. Mutation at these sites of PLC-
3
dramatically reduced the inhibitory effect of PKG on the activation of
PLC by G-protein and the phosphorylation of the PLC protein by PKG.
These results indicate that PKG can directly phosphorylate PLC
isozymes (
2 and
3) and regulate the activation of
receptor-mediated G-protein-coupled phosphatidylinositol (PI) turnover
and intracellular Ca2+ release.
There are four isozymes of phospholipase C-
that are activated to
various extents by G
q proteins, which account for the pertussis toxin-resistant activation of PLC and intracellular Ca2+ increase. It has been shown that the C terminus of
PLC-
1 is critical for the activation of the enzyme by
G
q (52, 54). Deletion studies have identified a P box
region (Thr903 to Gln1030) and a G box domain
(Lys1031 to Leu1142) in the C terminus of
PLC-
1. The P box of PLC-
1 is important for the association of
PLC-
1 with cell membrane and the activation of the enzyme by
G
q, whereas the G box domain is involved in the
interaction with G
q subunits (52). We previously showed that phosphorylation of serine residues at the C-terminal domains of
PLC-
by PKA inhibited the G-protein-mediated PLC-
activity (22,
39). Phosphorylation at Ser1105 of PLC-
3 accounts for
the inhibitory effect of PKA on the activation of the protein by
G
q (39). In this report, we shown that PKG also
phosphorylated Ser1105 in PLC-
3. Phosphorylation of the
protein at serine 1105 could potentially reduce or block the
association of PLC-
with G-protein
subunit.
Specific activation of PLC-
2 and PLC-
3 by G-protein 
subunits accounts for the pertussis toxin-sensitive PLC
activation and Ca2+ release. The sites of interaction with
G-protein 
subunits in PLC-
3 are located in both the
N-terminal domain and in the catalytic X domain of the PLC-
protein
(55-57). The N-terminal first 100 amino acids are predicted to form a
pleckstrin homology (PH) domain (58). The PH domain isolated from
PLC-
binds to PIP2 and IP3, and deletion of
the PH domain from PLC-
inhibited anchoring of the protein to
PIP2-containing membrane but did not inhibit catalysis
(59-61). The role of PH domain in PLC-
isoforms is not clear.
Recent data indicate that a fragment encompassing the N-terminal PH
domain bound to G-protein 
subunits, and the PH domain of
PLC-
2 determines the binding and activation by G-protein 
subunits (55, 62). Therefore, phosphorylation of Ser26 in
PLC-
3 by cGMP-dependent protein kinase could affect the
interactions of the PLC protein with either G-protein 
subunits
or phospholipid in the membrane or with both. We are in the process of
testing this hypothesis.
Nitric oxide and cyclic GMP have emerged recently as a principal focus
in signal transduction, playing important regulatory roles in smooth
muscle relaxation and proliferation, inhibition of platelet
aggregation, neutrophil degranulation, and initiation of visual signal
transduction. A number of intracellular proteins are activated by an
increase in intracellular cGMP. Examples include cGMP-dependent protein kinases (PKGs), cyclic
nucleotide-gated ion channels, cGMP-binding phosphodiesterases, PKA,
and myosin phosphatase (47, 63, 64). In vascular smooth muscle cells, platelets, and neuronal cells, nitric oxide and atrial natriuretic factor inhibited the intracellular Ca2+ release induced by
activation of G-protein-coupled receptors. The inhibitory effects of
these agents appear to be mediated by increasing intracellular cGMP and
by activating cGMP-dependent protein kinases. Three
different isozymes of cGMP-dependent protein kinases
(PKGs), PKGI
, PKGI
, and PKGII, have been cloned and characterized
(51) (64). PKGI
and -I
are alternative spliced variants, highly
expressed in the vascular and gastrointestinal smooth muscle, lung,
cerebellum, platelets, and heart (51, 65-68). The PKGII isoform is a
membrane-associated protein, expressed in secretory epithelium and in
various regions of the brain (69-71). Our in vitro studies
have shown that all three PKG isoforms can directly phosphorylate
PLC-
3 and PLC-
2. Cotransfection and activation of the kinases in
COS-7 cells decreased the G-protein-activated IP3 release,
a result of blocking PLC-
activation. The precise physiological role
of PLC-
phosphorylation by cGMP-dependent protein
kinases may depend on the localization of the two proteins in the cell
or tissue.
A recent report demonstrates that cGMP kinase I
can form
signaling complex with IP3 receptor and IRAG (an
IP3R-associated cGMP kinase substrate) in microsomal smooth muscle
membrane to regulate intracellular calcium (72). The observation that
PLC-
3 and PKGs are immunoprecipitated together suggests an
interesting possibility that these proteins may form signaling complex
in the cell. The notion that PLC may form signaling complex with PKG
and other molecules is under investigation.
In summary, this work provides the first evidence that
G-protein-activated PLC-
isozymes are the potential physiological target for signaling pathways coupled to cGMP and
cGMP-dependent protein kinases. A working model (Fig.
8) is proposed in which nitric oxide and
other peptides, such as atrial natriuretic peptide and brain
natriuretic peptide, stimulate guanylyl cyclases, leading to the
production of cyclic GMP and the activation of
cGMP-dependent protein kinases. Phosphorylation of PLC
isozymes by PKG blocks the activation of the enzymes by G-protein
subunits, resulting in the inhibition of agonist-stimulated
intracellular Ca2+ release and the regulation of cellular
functions.

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|
Fig. 8.
Proposed model for the regulation of
G-protein-activated PLC-Ca2+ by signaling pathway coupled
to cGMP-dependent protein kinase. Receptors
(R) coupled to both G q and
G i/ can activate PLC- isozymes, leading to the
production of IP3 and diacylglycerol (DAG),
key second messengers in the release of intracellular Ca2+
and the activation of protein kinase C, respectively. Reagents, such as
nitric oxide (NO), atrial natriuretic peptide
(ANP), and brain natriuretic peptide (BNP), can
stimulate guanylyl cyclases, leading to the increase of intracellular
cGMP from GTP. One of the functions of cGMP is to activate
cGMP-dependent protein kinases that can directly
phosphorylate and inhibit G-protein-activated PLC- activity,
blocking the hydrolysis of inositol phosphates and the release of
intracellular Ca2+ and, consequently, regulating cellular
functions.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Melvin I. Simon (California
Institute of Technology, Pasadena, CA) for support and encouragement.
We thank Dr. Suzanne Lohmann (University of Würzburg, Germany)
for suggestions on the manuscript and for reagents.
 |
FOOTNOTES |
*
This work was supported in part by a Scientist Development
grant from the National American Heart Association, a Basil O'Connor Starter Scholar Research award from the March of Dimes Birth Defects Foundation (to M. L.), and National Institutes of Health Grant HD09618
(to B. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of
Biosciences and Technology, Texas A & M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7505; Fax: 713-677-7512; E-mail: mliu@ibt.tamu.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M006266200
 |
ABBREVIATIONS |
The abbreviations used are:
PKA, cAMP-dependent protein kinase;
PLC, phospholipase C;
PIP2, phosphatidylinositol 4,5-bisphosphate;
IP3, inositol 1,4,5-triphosphate;
PKG, cGMP-dependent protein kinases;
PAGE, polyacrylamide gel
electrophoresis;
PI, phosphatidylinositol;
DMEM, Dulbecco's
modified Eagle's medium;
PBS, phosphate-buffered saline;
NO, nitric
oxide;
PH, pleckstrin homology;
VSMCs, vascular smooth muscle
cells;
PAGE, polyacrylamide gel electrophoresis;
8-pCPT-cGMP, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate;
8-Br-cGMP, 8-bromo-cGMP.
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