|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 5, 3310-3317, February 1, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, July 12, 2001, and in revised form, November 5, 2001
Nitric oxide and endogenous nitrovasodilators
regulate smooth muscle tone by elevation of cGMP and activation of
cyclic GMP-dependent protein kinase (PKG). The
amplitude and duration of the cGMP signal in smooth muscle is regulated
in large part by cGMP-specific cyclic nucleotide
phosphodiesterase (PDE5). Previous in vitro data
have suggested that both cAMP-dependent protein kinase and
PKG can regulate the activity of PDE5. To test if this type of
regulation is important in the intact cell, we have generated
phospho-PDE5-specific antisera and have utilized isolated smooth muscle
cells from mice having a disruption in the PKG I gene as well as cells
from normal human smooth muscle. The data show that in human smooth
muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of
PDE5, whereas no phosphorylation was seen in smooth muscle cells
isolated from mice in which the gene for PKG I had been disrupted. As
with the human cells, no phosphorylation was seen in the mouse cells in
response to 8-Br-cAMP. These results strongly suggest that a
major regulatory pathway for control of PDE5 phosphorylation and
activity in intact smooth muscle is via PKG-dependent
phosphorylation of PDE5. Finally, experiments with calyculin A and
okadaic acid suggest that PP1 phosphatase, the catalytic subunit of
myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and
its subsequent dephosphorylation by myosin phosphatase may be key steps
in the regulation of relaxation/contraction cycles of smooth muscle.
Smooth muscle cells
(SMCs)1 of the blood vessels,
uterus, bladder, gastrointestinal tract, and respiratory tract relax or
contract in response to hormonal or mechanical stimulation. Nitric
oxide, nitrovasodilators, and natriuretic peptides act as relaxants, regulating smooth muscle tone by direct activation of guanylyl cyclase,
leading to the elevation of cGMP and activation of
cGMP-dependent protein kinase (PKG). Similarly cAMP
(through Several different cyclic nucleotide phosphodiesterases (PDEs) can
hydrolyze cGMP and control the duration and amplitude of the cGMP
signal (3). A major cGMP-hydrolyzing enzyme expressed in all types of
smooth muscle is PDE5, and selective inhibitors of PDE5 can cause
smooth muscle relaxation. For example, sildenafil (Viagra®), a
specific PDE5 inhibitor, is effective in the treatment of male erectile
dysfunction because of its ability to potentiate the relaxant effect of
NO by enhancing cGMP accumulation in the corpus cavernosum (4).
The molecular mechanisms for regulation of PDE5 activity are not fully
understood, particularly in intact tissues. It is known from in
vitro studies that cGMP can be bound to noncatalytic binding sites
of PDE5 and also that phosphorylation of serine 92 is dependent on this
binding (5). Therefore, it is suspected that these events somehow
regulate PDE5 activity in the intact cell. This phosphorylation site is
conserved in bovine, human, canine, and rat isoforms, and several
groups have reported that PDE5 can be phosphorylated in
vitro by either PKG or PKA. For example using the purified bovine
lung enzyme, incorporation of 32P into PDE5 protein was
observed only in the presence of cGMP (6). Binding of cGMP to these
noncatalytic sites has been proposed to be a prerequisite for
phosphorylation in intact cells. However, no significant changes in PDE
activity were detectable in studies designed to directly test this
proposal. More recently, a 50-70% increase in the activity of
partially purified recombinant bovine PDE5 was reported after
phosphorylation in vitro (7).
In another study an indirect effect of PKA phosphorylation on PDE5
activity in guinea pig airway smooth muscle cells was proposed (8). It
was shown that the Because of these discrepancies a clear understanding of how PDE5
activity is regulated in intact cells has not been formed. A major
technical difficulty has been the absence of a sensitive and specific
method for assessment of PDE5 phosphorylation and activation. The
widely used method of 32P incorporation is greatly limited
in intact cells because of the low abundance of PDE5. It is also
necessary that antibodies used for such 32P incorporation
experiments be able to specifically and quantitatively immunoprecipitate PDE5 from crude extracts, a criterion that has been
hard to meet. Still by using such methods, a small activation of PDE5
has been reported in cultured rat smooth muscle cells treated with ANP
(9). However, questions remain about the specificity of the PDE5
antibody used in these studies. In addition, this method does not
provide information about which residue(s) on PDE5 are phosphorylated.
Here we report the application of phosphospecific (phosphoserine 92, bovine) PDE5 antibodies to determine the phosphorylation status of PDE5
in intact smooth muscle cells. These include human uterine and mouse
aortic smooth muscle cells. We also utilized intact tissues and
isolated cells from mice having disruptions in the PKG I gene. The data
strongly suggest that in the intact cell, cGMP-dependent
protein kinase but not cAMP-dependent protein kinase is a
major regulator of PDE5 phosphorylation and activity. We also provide
evidence that myosin phosphatase 1 (PP1) is likely to be a regulator of
the dephosphorylation of PDE5 in smooth muscle.
Reagents
Mono Q anion exchange columns (HR 5/5) were supplied by Amersham
Biosciences. Pepstatin, leupeptin, Nonidet P-40, and PP1 were obtained
from Roche Molecular Biochemicals. PKG I and the catalytic subunit of
PKA were obtained from Promega (Madison, WI). Heat-stable inhibitor of
PKA (PKI) was obtained from Biomol (Plymouth Meeting, PA). Calyculin A,
okadaic acid, and Dulbecco's modified Eagle's medium were obtained
from Life Technologies, Inc. (Gaithersburg, MD). Protein A-agarose and
protein G-agarose were purchased from Oncogene Research Products
(Cambridge, MA). SuperSignal® West Pico chemiluminescent substrate and
peroxidase-conjugated goat anti-mouse IgG were obtained from Pierce.
Isoelectric focusing ready gels and horseradish peroxidase-conjugated
goat anti-rabbit IgG were obtained from Bio-Rad. Centri-Sep columns
were from Princeton Separations (Adelphia, NJ). 3H-cAMP and
3H-cGMP were purchased from PerkinElmer Life Sciences.
Sildenafil was a gift from Pfizer. Brij 35 (protein grade detergent),
protein phosphatase 2A1 (PP2A1), and C-terminal (657) anti-protein
kinase G antibody were from Calbiochem. Mouse monoclonal antibody to VASP (specific for phosphoserine 239) and rabbit polyclonal antibody to
VASP were obtained from Alexis Biochemicals (San Diego, CA). All other
reagents were purchased from Sigma.
Cell Culture and Extract Preparation
Human uterine smooth muscle cells (Clonetics, San Diego, CA)
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% FBS and characterized as smooth muscle by expression of smooth
muscle Smooth muscle cells were harvested after washing three times with cold
PBS in homogenization buffer A (50 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, 1 mM dithiothreitol, 10 µg/ml
aprotinin, 5 µg/ml pepstatin, 20 µg/ml leupeptin, 1 mM
benzamidine, 0.2 mM sodium vanadate, 25 mM
sodium fluoride). The cells were homogenized by two brief sonications
(2-3 s) using a Virsonic 100 sonicator (Virtis Company, NY). Tissue
extracts were prepared in homogenization buffer A using a Polytron
homogenizer (Brinkmann Instruments) followed by two brief sonications.
The cell extracts were centrifuged at 16,000 or 100,000 × g for 20 min at 4 °C. The supernatant and pellet
fractions were assayed for PDE activity, and the supernatants were used
for anion-exchange chromatography.
In Vitro Phosphorylation and Dephosphorylation
For in vitro phosphorylation assays, cell extracts
were incubated for 30-60 min at 30 °C. The phosphorylation buffer
consisted of homogenization buffer A with 0.1 mM ATP, 10 mM MgCl2, and 1 µl of PKG (100-300 units
(Promega)/100 µl of final) or catalytic subunit of PKA (30-80 units
(Promega)/100 µl of final) with or without 2-10 µM
cGMP, in a total volume of 100 µl. At the end of the incubation 100 µl of the phosphorylated extract was applied to a Centri-Sep column
(previously reconstituted in homogenization buffer A) and quickly spun
to exchange buffers. For in vitro dephosphorylation assays,
cells were homogenized in homogenization buffer B (50 mM
Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM
dithiothreitol, 0.5 mM MnCl2, 0.03% Brij 35 (w/v) with protease inhibitors) and briefly sonicated. PP1 catalytic
subunit (0.1 µM or 0.5 µM) was added to the
extracts and incubated for 2 h at 36 °C. PP2A1 (5-20
milliunits/100 microliters of final) was incubated with the extracts
for 1.5 h at 30 °C. The reactions were terminated by the
addition of 2× SDS sample buffer to the samples and boiling for 5 min.
HPLC Chromatography
Cells from six 100-mm plates were homogenized in 3 ml of
homogenization buffer A, spun at 100,000 × g for
1 h, and loaded onto a Mono Q anion exchange column HR 5/5
(Amersham Biosciences). HPLC was conducted using conditions published
previously (11).
Antibody Production and Purification
Polyclonal Phosphospecific Antibody to PDE5--
A synthetic
phosphopeptide C-GTPTRKISASEFDR, corresponding to the N-terminal part
of bovine PDE5A1 (5) (aa 85-98, with a phosphorylated serine 92) or
human PDE5 (aa 95-108) (12), was conjugated to hemocyanin (an
N-terminal Cys residue was added to facilitate conjugation) and used as
immunogen for generating polyclonal phosphospecific PDE5 antibody
(QBC/BIOSOURCE International). For phospho-PDE5
antibody purification, the N-terminal fragment of bovine PDE5A1 (1-125
aa) was expressed in Escherichia coli as a
polyhistidine-tagged protein and purified on a Talon metal affinity
resin (CLONTECH). PDE5 recombinant protein (1-125
aa) was phosphorylated after a 60-min incubation in 100 µl of the phosphorylation buffer (homogenization buffer A with 0.1 mM
ATP, 10 mM MgCl2, and PKG (300 units
(Promega)/100 µl of final) or catalytic subunit of PKA (80 units
(Promega)/100 µl of final)). After phosphorylation proteins were
separated by isoelectric focusing and transferred to nitrocellulose.
The protein bands on the strips of nitrocellulose were visualized by
Ponceau S, cut from the blot, and used for immunoaffinity purification
of phospho-PDE5 antibody using standard protocols (13). Briefly, the
strips were blocked with 2% bovine serum albumin/PBS for 60 min and
incubated with serum (diluted 1:10 in PBS) at 4 °C overnight. After
washing the strips four times in 0.1% Tween/PBS, phosphospecific
antibody was eluted from the strips by incubation with 0.1 M glycine, 0.5 M NaCl, 1 mg/ml bovine serum
albumin, and 0.5% Tween-20, pH 2.5, for 1 min, immediately followed by
neutralization with 1 M Tris-HCl, pH 9.5.
Mouse Monoclonal PDE5 Antibody--
Purified PDE5 recombinant
protein (corresponding to bovine PDE5A1 aa 125-539) was used as an
antigen for production of mouse monoclonal antibodies in hybridoma cell
lines (Biologics Production Facility, Fred Hutchinson Cancer Research
Center, Seattle, WA). Mouse IgGs, secreted by the hybridomas grown in
culture, were used as a source of monoclonal antibody. The antibody
titer and specificity were tested for ability to either
immunoprecipitate PDE5 or detect it in Western blot analysis.
Monoclonal antibodies able to quantitatively immunoprecipitate human
and mouse PDE5 were used in this study.
Total PDE5 Antibody (Polyclonal) and PDE1C Antibody
(Polyclonal)--
Production and purification of the polyclonal
C-terminal PDE5 antibodies, raised against a synthetic peptide,
CRKNRQKWQALAEQQEK (bovine aa 836-852 and human aa 846-862), and
polyclonal PDE1C antibody, raised against a glutathione
S-transferase fusion protein from the C-terminal part of
PDE1C, were described previously (11).
Immunoprecipitation
Smooth muscle cells grown on 100-mm plastic cell culture plates
were washed three times with cold PBS. Cells were harvested in 500 µl
of immunoprecipitation buffer (IP buffer) containing 50 mM
Tris (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol,
150 mM NaCl, 1% Nonidet P-40, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 20 µg/ml leupeptin, 1 mM benzamidine,
0.2 mM sodium vanadate, and 25 mM sodium
fluoride. Cells were homogenized by two 2-3 s sonications using a
Virsonic 100 sonicator.
Lysates were incubated with phospho-PDE5 antibody or monoclonal PDE5
antibody overnight followed by incubation with protein A or protein
G-agarose beads and then separated into supernatant and pellet
fractions. The immunopellet was washed three times in IP buffer. PDE
activity was measured in samples before and after immunoprecipitation.
After the final wash, the immunopellets were either boiled in SDS
sample buffer for Western blot analysis or resuspended in the
homogenization buffer, pH 7.4, and subsequently assayed for PDE activity.
Western Blot Analysis
Samples were diluted in 2× SDS sample buffer, heated at
95 °C for 5 min, loaded onto an SDS-polyacrylamide gel (8%
acrylamide, 0.21% bisacrylamide), and electrophoresed. The separated
proteins were transferred onto nitrocellulose and immunostained with
isozyme-specific antibodies. The immunoreactivity was detected by
enhanced chemiluminescence using horseradish peroxidase-conjugated goat
anti-rabbit IgGs or goat anti-mouse IgGs and SuperSignal® West Pico
chemiluminescent substrate.
Isoelectric Focusing
Isoelectrofocusing (IEF) was performed under native conditions
using IEF ready gels, pH 5-8, in Mini-Protean II system with 1.2 mM phosphoric acid as the anode buffer and 20 mM lysine, 20 mM arginine as the cathode
buffer. Samples were diluted in 50% glycerol (v/v) and loaded onto the
IEF gel. IEF was run under constant voltage in three steps: 100 V for
1 h, 250 V for 1 h, and 500 V for 30 min. After focusing the
IEF gels were either stained with a staining solution or transferred to
nitrocellulose for Western blotting.
PDE Assays and Protein Determinations
Phosphodiesterase assays were carried out according to
established procedures (14). All assays were performed at 30 °C
using either 1 µM cAMP or 1 µM cGMP as
substrates in the presence of either 1 mM EGTA or 0.8 mM CaCl2 and 4 µg/ml calmodulin. Protein concentrations were determined using the Bradford method.
Characterization of Phosphospecific PDE5 Antibody--
A
phosphospecific PDE5 antibody was raised against a peptide containing
phosphoserine 92 (serine 92 in bovine PDE5). To determine the
specificity of the antibody it was necessary to generate samples of
PDE5 in both the dephosphorylated and phosphorylated states. A
completely dephosphorylated form of an N-terminal domain of bovine PDE5
(aa 1-125) that contained the phosphorylation site was expressed in
bacteria as a polyhistidine-tagged protein and purified to homogeneity
by metal-affinity chromatography. It was expected that because
bacterially expressed proteins are not likely to be phosphorylated this
method would provide a good control for specificity and sensitivity of
the antibody. However, to be sure the protein was fully in the
dephosphorylated form, the method of isoelectric focusing under native
conditions was employed. In these studies the PDE5 fragments were
resolved by isoelectric focusing into one major band with a pI of 6.9 and two minor bands. Phosphorylation of this protein either by PKG or
the catalytic subunit of PKA led to a shift of isoelectric points for
all bands (Fig. 1A). These
data verify that the bacterially expressed protein was in a
dephosphorylated state. The phosphorylation conditions used provided
complete phosphorylation of the PDE5 fragment, and no bands were left
in the positions detected before phosphorylation. The finding that the
bacterially expressed PDE5 N-terminal domain could be completely
phosphorylated and separated from dephospho forms was used as the basis
for affinity purification of phospho-PDE5 antibody. Crude serum was
purified on strips containing phospho-PDE5 according to the procedure
described in the previous section.
SDS-PAGE chromatography did not reveal any shift in
electrophoretic mobility between nonphosphorylated and phosphorylated forms of this protein (Fig. 1B). The specificity of the
purified phospho-PDE5 antibody was tested against the same proteins
using Western analysis. This antibody had a much higher sensitivity toward phosphorylated protein and could specifically recognize phosphorylated PDE5 but not dephosphorylated PDE5 analyzed at the ng
range of loaded protein (Fig. 1C).
PDE1C and PDE5 Are the Two Major cGMP-hydrolyzing Enzymes in Human
Uterine Smooth Muscle Cells in Culture--
Several PDE isozymes are
able to hydrolyze cGMP. To determine which ones were most likely to be
important in uterine smooth muscle, extracts from human uterine smooth
muscle cells were resolved by anion-change chromatography on a Mono Q
column, and PDE activities were analyzed at substrate concentrations of
1 µM. The Ca2+/CaM-stimulated activity eluted
in fractions 7-10 was identified as PDE1C using Western blot analysis.
PDE1C differs from other PDE1s in its ability to hydrolyze cAMP and
cGMP equally well. Immunoprecipitation of this activity with ACC-1
(mouse monoclonal IgG, which is reactive with all Ca2+/CaM
PDE1s) (11) revealed that PDE1C from these cells also had similar cAMP
and cGMP hydrolytic activities in the presence of Ca2+/CaM
(data not shown). The PDE5 activity, determined as cGMP hydrolytic activity measured in the presence of EGTA, was eluted in fractions 8-16 and partially overlapped with PDE1C in the HPLC profile. As shown
in Fig. 2, the PDE profile was found to
be similar to the profile obtained previously for human aortic smooth
muscle cells (11). However, the relative ratio between the peaks of activity corresponding to the two major PDE isoforms was somewhat different (Fig. 2). In uterine cells the level of PDE5 expression was
much higher, and PDE1C expression was lower than in aortic smooth
muscle cells. The presence of PDE1C in human uterine smooth muscle
cells supports the observation that this enzyme is specifically expressed in proliferating human smooth muscle cells but not in non-human smooth muscle cells.
PDE5 was the major cGMP-hydrolyzing enzyme in uterine smooth muscle
cells analyzed at 1 µM cyclic nucleotide concentration, although PDE1C probably also contributes to the total cGMP-hydrolyzing activity. To assess the relative ratio of these two PDEs in the crude
extract, PDE5 activity was precipitated from smooth muscle cell
extracts with an excess of mouse monoclonal antibody. By this estimate
about 75% of the total cGMP-hydrolyzing activity was contributed by
PDE5 as measured at 1 µM substrate. Similar results were
obtained by applying the PDE5-specific inhibitor, sildenafil. At 60 nM, sildenafil inhibited essentially all of the PDE5
activity with no significant effect on the Ca2+/CaM
activation of PDE1C (data not shown).
In Vitro Both PKA and PKG Can Phosphorylate PDE5 in Extracts of
Uterine Smooth Muscle Cells--
In vitro either PKG or the
catalytic subunit of PKA was effective in the phosphorylation of PDE5
in extracts of uterine smooth muscle cells. The degree of
phosphorylation was dependent on the concentration of cGMP present
during the phosphorylation step, reaching a maximum at 10 µM cGMP (Fig. 3).
Increasing the cGMP concentration to 25 µM did not lead
to a higher degree of phosphorylation. As control experiments,
PKA-induced phosphorylation of PDE5 in the presence of cGMP was
completely blocked by PKI, whereas PKG-induced phosphorylation was not
affected by the addition of PKI (data not shown). It has been shown
previously with purified proteins that phosphorylation of PDE5 is
promoted if cGMP occupies the noncatalytic binding sites (6). The
observation that the activity of the catalytic subunit of PKA could
significantly increase phosphorylation of PDE5 in the presence of cGMP
confirms this finding.
To measure cGMP PDE activity in the samples after phosphorylation,
quick-spin columns were used to exchange the buffer and remove excess
cGMP. Maximal activation of PDE5 by PKG or the PKA catalytic subunit
was 4-fold and corresponded to the samples with the highest
phosphorylation level. 8-Br-cGMP could stimulate phosphorylation of
PDE5 by either PKG or the catalytic subunit of PKA but only at high
concentrations. To reach the same level of phosphorylation elicited by
10 µM cGMP the concentration of 8-Br-cGMP needed to be
increased to 150 µM, presumably because it binds poorly
to the noncatalytic sites of PDE5 (6).
Activation of PKG, but Not PKA, Leads to Phosphorylation and
Activation of PDE5 in Intact Human Uterine Smooth Muscle
Cells--
For studies in intact cells the cell-permeant cyclic
nucleotide analogs 8-Br-cAMP and 8-Br-cGMP were used for activation of PKA and PKG. Phosphorylation of VASP, a well characterized substrate for both PKA and PKG, was also studied in these experiments as a
control for the effectiveness of the analogs (15). VASP is a 46/50-kDa
vasodilator-stimulated phosphoprotein that can be phosphorylated in
response to either cAMP-elevating agents (prostaglandins or forskolin)
or cGMP-elevating agents (nitric oxide donors). Phosphorylation of the
PKG-preferable site can be detected using a phospho-VASP monoclonal
antibody that reacts specifically with phosphoserine 239. PKG can also
phosphorylate another site (serine 157), which is the site
preferentially phosphorylated by PKA. Phosphorylation of serine 157 leads to a shift in the apparent molecular mass from 46 to 50 kDa on
SDS-PAGE. This shift is easily detectable by Western analysis using any
specific VASP antibody. Similarly, the PKG-preferable site, serine 239, could be also phosphorylated by PKA. Therefore, by using the
appropriate combination of serine 239 phosphospecific antibody and
total VASP antibody we could determine which of the two sites was
phosphorylated when either 8-Br-cAMP or 8-Br-cGMP was applied.
Addition of 1 mM 8-Br-cGMP to the human uterine smooth
muscle cells led to a gradual accumulation of the phosphorylated form of PDE5 starting from 10 min and reaching a maximum at 30 min (Fig.
4A). Phosphorylation remained
steady for up to 60 min of incubation time. Interestingly, the time
course of VASP phosphorylation was similar to the time course for PDE5
phosphorylation. Phospho-VASP appeared at approximately the same time
as phospho-PDE5, and both reached a maximum at 30 min. Detection of
phosphoserine 239 in the 50-kDa band was the result of phosphorylation
of the second PKG phosphorylation site (serine 157), leading to a shift
in band mobility.
The phosphospecific PDE5 antibody also was able to specifically
precipitate the phosphorylated form of PDE5 after cells were incubated
in the presence of 8-Br-cGMP (Fig.
5B). Using Western analysis it
was found that most of the phosphorylated PDE5 (80-85%) was pulled
down by the phosphospecific antibody, and only a small part of the
phosphorylated PDE5 was found in the supernatant after immunoprecipitation.
In control cells no PDE5 activity was detectable in the
immunoprecipitate after using phospho-PDE5 antibody (Fig.
5A). Western analysis also confirmed the high specificity of
the phospho-PDE5 antibody. In the immunopellet of control cells the
total PDE5 antibody did not detect any PDE5 after precipitation with
the phospho-PDE5 antibody, indicating that all PDE5 in unstimulated cells was present in the dephosphorylated form. To assess how much of
the total PDE5 was phosphorylated in cells treated with 1 mM 8-Br-cGMP, the amount of PDE5 left in the supernatant
after immunoprecipitation of control cells and cells stimulated with phospho-PDE5 antibody was compared. Using antibody reactive with all
forms of PDE5 it was estimated that 25-35% of the total PDE5 underwent phosphorylation.
To examine changes in PDE5 activity after phosphorylation a mouse
monoclonal PDE5 antibody was used to precipitate total PDE5 activity
(Fig. 6). This antibody quantitatively
precipitated the same amount of total PDE5 from the control and
stimulated cells as judged by immunoblot experiments with total
(C-terminal) PDE5 antibody. Again, phospho-PDE5 was detected only in
the immunopellet from the cells incubated with 8-Br-cGMP. As a result
of PDE5 phosphorylation, a 2-fold increase of PDE5 activity was
measured in the immunopellet from stimulated cells as compared with
control cells.
8-Br-cAMP did not have any significant effect on PDE5 phosphorylation,
although VASP was highly phosphorylated under these conditions (Fig.
4B). A total shift to the 50-kDa VASP band was observed,
indicating that complete phosphorylation of the PKA preferable site,
serine 157, had occurred. The PKG-preferable site, serine 239, was also
phosphorylated by PKA although with a short time delay (2-3 min after
phosphorylation of serine 157). Thus the absence of an 8-Br-cAMP effect
was not caused by the lack of activated PKA but rather an inability of
PKA to phosphorylate PDE5 in the intact cells.
Adding a combination of these two cyclic nucleotide analogs did not
reveal any additive or synergistic effect when either 0.2 mM or 1 mM 8-Br-cGMP was incubated with 1 mM 8-Br-cAMP (data not shown). This suggests that the
degree of PDE5 phosphorylation caused by 8-Br-cGMP was determined
primarily by the level of PKG activation.
Analysis of PDE5 Phosphorylation in Smooth Muscle Cells and Tissues
from PKG I-deficient Mice--
To discriminate between the effects of
PKA and PKG on PDE5 phosphorylation in intact cells, PKG I-deficient
smooth muscle cells were compared with wild-type cells. Cells derived
from the aortas of either mice having the PKG I gene disrupted or
control mice were analyzed in the first passage to minimize any changes in PKG expression (expression of this gene is known to be affected by
multiple passages of aortic smooth muscle cells). In aortic cells from
the PKG I
Addition of 8-Br-cGMP to the PKG I+/+ control cells led to
a significant phosphorylation of PDE5, comparable with the levels observed with human uterine smooth muscle cells (Fig.
7A). Similarly, treatment of
PKG I
As shown in Fig. 7A, activation of PKA by the addition of
8-Br-cAMP did not change the level of PDE5 phosphorylation in any mouse
aortic smooth muscle cells. Neither did treatment of the control cells
with a combination of both 8-Br-cAMP and 8-Br-cGMP induce additional
PDE5 phosphorylation. In fact, the level of phosphorylation appeared to
be reduced in comparison with the effect of 8-Br-cGMP alone.
Interestingly, treatment of PKG I
To further investigate which protein kinase is involved in PDE5
phosphorylation in vivo the endogenous level of phospho-PDE5 was measured in different tissues and compared with the same tissues from PKG I-deficient mice. In control mice the levels of endogenously phosphorylated PDE5 varied significantly between the tissues. For
example in mouse uterus phospho-PDE5 was easily detectable (Fig. 9). A
high level of endogenous phosphorylation also was seen in mouse lung,
but only small amounts of phospho-PDE5 were detectable in mouse aorta
(data not shown). In all tissues from PKG I-deficient mice, PDE5 was
almost entirely in the dephospho form (data not shown). These
experiments provide additional evidence that PDE5 phosphorylation
in vivo is predominately mediated through the cGMP/PKG I and
not through the cAMP/PKA pathway.
Myosin Light Chain Phosphatase, an Enzyme Controlling Smooth Muscle
Contraction and Relaxation, Can Regulate PDE5 Phosphorylation--
To
investigate the role of phosphatases in the regulation of the rate of
PDE5 phosphorylation, cells were incubated with serine/threonine phosphatase inhibitors. Treatment of cells with calyculin A for 30 min
increased PDE5 phosphorylation stimulated by 8-Br-cGMP (Fig.
8A). The highest level of PDE5
and VASP phosphorylation coincided with changes in cell morphology,
caused by calyculin A. After 30 min with 100 nM calyculin A
cells became rounded, presumably due to the effects of calyculin on
induction of actin polymerization (16).
At 100 nM calyculin A inhibits both protein phosphatase
type 1 and 2A. To differentiate which phosphatase was involved okadaic acid was used. At a low concentration (2 nM) okadaic acid
inhibits mostly PP2A, whereas at higher concentrations (1 µM) it inhibits both PP2A and PP1. As shown in Fig.
8A, okadaic acid was effective only at 1 µM as
a stimulator of PDE5 and VASP phosphorylation. Thus PP1 phosphatase
appears to be able to regulate the rate of PDE5 dephosphorylation.
To test the ability of PP1 to directly dephosphorylate PDE5 in
vitro, the experiments shown in Fig. 8B were performed.
Cell extracts containing phospho-PDE5 were incubated with different concentrations of the catalytic subunit of PP1. PDE5 was completely dephosphorylated by treatment with PP1 at 0.5 µM for
2 h. Treatments of cell extracts with different concentrations of
PP2A1 did have any effect on PDE5 dephosphorylation. No change in the
total amount of PDE5 was observed after these incubations.
In smooth muscle cells PP1 is the catalytic subunit of myosin light
chain phosphatase (MLCP), which provides a major control of smooth
muscle tone by regulating the dephosphorylation of myosin light chains.
Although it is possible that multiple protein phosphatases catalyze
PDE5 dephosphorylation, the phosphatase inhibitor studies in intact
cells along with the in vitro studies with purified phosphatases strongly suggest that PP1/MLCP is a major regulator of
PDE5 phosphorylation status. As such it represents an additional point
of cross-talk between the cGMP/PKG and the actin-myosin contractile systems.
PDE5 Is Phosphorylated in Intact Tissues (Mouse Uterus) upon
Activation of PKG--
As shown in the previous section, when mouse
uterus is rapidly removed from the animal and homogenized, PDE5 is
partially phosphorylated. Immunoprecipitation with the PDE5
phosphospecific antibody showed the presence of phospho-PDE5 hydrolytic
activity in this tissue (Fig.
9A). Three bands were detected
by Western blot analysis with the total PDE5 and phospho-PDE5
antibodies. This may suggest the presence of at least two different
splice variants of PDE5 in this tissue. However, the low molecular
weight band may be a result of partial proteolysis of the major PDE5 forms in mouse uterus. In cultured human uterine SMCs a small amount of
similar low molecular weight band was detectable in cell extracts only
after PDE5 underwent phosphorylation (Figs. 3 and 4). This suggests the
possibility that phospho-PDE5 is more accessible to proteolysis than
dephospho-PDE5.
To determine whether PDE5 present in the intact tissue can be
responsive to further PKG stimulation, mouse uterine rings were incubated in the presence of 1 mM 8-Br-cGMP. Activation of
PKG in this experiment led to a substantial additional phosphorylation of PDE5. A much higher level of PDE5 phosphorylation was detected using
Western analysis and immunoprecipitation (Fig. 9B). The intensity of all three bands increased after phosphorylation. These
data show that in intact tissues as well as in intact cells the
phosphorylation status of PDE5 is determined by the level of PKG activation.
PDE5 Is a Specific Substrate for PKG in Intact Smooth
Muscle--
The results presented in this paper demonstrate that the
phosphorylation status of PDE5 in intact cells is determined by the balance between cGMP-dependent protein kinase 1 activity
and myosin light chain phosphatase activity. In normal cultured smooth
muscle cells PDE5 is found to be almost completely in the
dephosphorylated form and can be rapidly phosphorylated when PKG is
activated. In intact tissues (mouse aorta, lung, and uterus) PDE5 is
partially phosphorylated in the basal state and can be further
phosphorylated by activation of PKG (mouse uterus). The partial
phosphorylation of the basal state is likely to explain, at least in
part, why a relatively low level of stimulation by PKG activation has
been seen in most previous studies of PDE5 activity.
Because both PKA and PKG can fully phosphorylate and activate PDE5
in vitro, it has not been clear which kinase signaling pathway is most important for activation of PDE5 in intact cells and
tissues. The cyclic nucleotide analog data in Figs. 4, 5, and 6
strongly suggest that the major regulatory pathway in intact smooth
muscle cells is via cGMP/PKG. This conclusion is reinforced by the
observation that in cells from mice in which the PKG I gene was
disrupted, no phosphorylation occurred even in the presence of high
8-Br-cGMP unless 8-Br-cAMP also was present (Fig. 7). In this latter
case only slight phosphorylation was seen. It is known that for
phosphorylation by either PKA or PKG to occur in vitro, cGMP
must be bound to the noncatalytic cGMP binding sites of PDE5.
Therefore, either 8-Br-cGMP also must have some ability to bind to the
noncatalytic sites of PDE5, or it can increase cGMP to some extent in
these intact cells.
A large number of proteins can be phosphorylated by PKG in
vitro, and most of these are also substrates for PKA. However, only a few physiologically relevant substrates for PKG have been shown
in intact cells. For example, in one study using expressed proteins in
HEK 293 cells the large conductance- and voltage-dependent Ca2+-activated K channel was shown to be phosphorylated on
serine 1072 by PKG, leading to hyperpolarization of the membrane (17). In another study using myometrial cells from pregnant human subjects, it was shown that PKG was more effective than PKA in increasing channel
opening (18). Recently a new protein, IRAG (IP3 receptor-associated cGMP kinase substrate), has been implicated as a target for PKG Ib
phosphorylation (19) where it may regulate IP3 mediated
Ca2+ release. Finally, MLCP is likely to be phosphorylated
and activated directly (20) or indirectly by PKG in intact cells (21).
Here we report that PDE5 also is a specific substrate for PKG in intact smooth muscle cells and tissues. Moreover, not only is PDE5 a PKG
substrate, but it is also a PKG regulator. Presumably, by controlling
the level and subcellular distribution of cGMP, PDE5 can regulate the
ability of PKG to phosphorylate specific proteins including the
in vivo PKG substrates mentioned above.
Because PDE5 is such a selective substrate for PKG, it also may turn
out to be one of the best markers for the activation of PKG. For
example, analysis of VASP phosphorylation at serine 239 has been used
as a method to measure PKG activity in intact cells (15). Our data show
that measurement of serine 92 phosphorylation on PDE5 utilizing
phosphospecific PDE5 antibodies presents another alternative for
quantitative analysis of PKG activation. This should be particularly
useful where the tissue profile for high level PDE5 expression overlaps
with PKG expression as in smooth muscle, platelets, and cerebellum.
The Role of PKG Phosphorylation of PDE5 in Smooth Muscle--
The
physiological function for the phosphorylation and activation of PDE5
is not yet established. It is expected that at a minimum PDE5 can
control the intracellular cGMP levels in close proximity to proteins
involved in cGMP-induced relaxation of smooth muscle. These include
MLCP and proteins associated with regulation of intracellular
Ca2+ concentration, e.g.
Ca2+-activated K channel and IRAG. In each of these cases,
activation of PDE5 may provide a negative feedback regulation of cGMP
and PKG when the intracellular concentration of cGMP reaches a high level. A model illustrating these pathways and feedback loops is shown
in Fig. 10.
One of the distinguishing features of smooth muscle is its ability to
go through numerous cycles of contraction and relaxation, determined by
changes in the phosphorylation status of myosin light chains (MLC)
(Fig. 10). Phosphorylation/dephosphorylation of myosin light chains is
under direct control of myosin light chain kinase and MLCP. As
shown in Fig. 8, PP1/MLCP is also involved in the dephosphorylation of
PDE5. Therefore, regulation of PDE5 dephosphorylation by myosin
phosphatase might be as important physiologically as regulation of PDE5
phosphorylation by PKG.
Clinically, continuous activation of the NO/cGMP pathway during chronic
administration of organic nitrates for treatment of coronary arterial
disease or pulmonary hypertension can lead to tolerance or
tachyphylaxis (22, 23). From the present data it would also be expected
to cause the accumulation of activated phospho-PDE5 in smooth muscle
cells and may therefore contribute to the development of tolerance.
In summary, the data show that PDE5 is phosphorylated in intact cells,
predominately by PKG. We would suggest that phosphorylation and
activation of PDE5 and its subsequent dephosphorylation by myosin
phosphatase may be a key step in the termination of relaxation (caused
by NO/cGMP) and preparation of smooth muscle for a next cycle of contraction.
We thank S. Martinez and X.-B. Tang
(Department of Pharmacology, University of Washington) for assistance
with the production of bovine PDE5 recombinant protein used for
generation of PDE5 monoclonal antibodies.
*
This work was supported by National Institutes of Health
Grants HL60178, HL4498, and DK21723. A preliminary account was
presented at the 12th Protein Kinase Symposium in 2000, Bad
Bruckenau, Germany.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:
Department of Pharmacology, Box 357280, University of Washington,
Seattle, WA 98195. Tel.: 206-543-4006; Fax: 206-685-3822; E-mail:
beavo@u.washington.edu.
Published, JBC Papers in Press, November 26, 2001, DOI 10.1074/jbc.M106562200
The abbreviations used are:
SMC, smooth muscle
cell;
PDE, cyclic nucleotide phosphodiesterase;
PDE5, cGMP-specific
PDE;
PDE1C, calmodulin-stimulated PDE;
PKG, cyclic
GMP-dependent protein kinase;
PKA, cyclic
AMP-dependent protein kinase;
VASP, vasodilator-stimulated
phosphoprotein;
PP1, protein phosphatase type I catalytic subunit;
PP2A1, protein phosphatase 2A1;
HPLC, high pressure liquid
chromatography;
MLCP, myosin light chain phosphatase;
PBS, phosphate-buffered saline;
IEF, isoelectrofocusing;
aa, amino
acids.
Regulation of cGMP-specific Phosphodiesterase (PDE5)
Phosphorylation in Smooth Muscle Cells*
,,¶
Department of Pharmacology, University of
Washington, Seattle, Washington 98195-7280 and the
§ Institut für Pharmakologie und Toxikologie,
Technische Universität München, Biedersteiner
Strasse 29, D-80802 München, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-adrenergic stimulation) also induces relaxation of the
same smooth muscle types. However, it has been reported recently that
the cGMP/PKG pathway is independent from the cAMP/PKA pathway as a
regulator of smooth muscle tone (1). Activation of PKG appears to
mediate all of the NO/cGMP relaxant effects in vascular smooth muscle
as disruption of the PKG I gene totally abolishes
NO/cGMP-dependent relaxation of smooth muscle in mouse
aorta, whereas in the same cells cAMP-dependent relaxation
remained intact. Defective responses also were found in intestinal
smooth muscle, where pyloric stenosis developed as a result of PKG I
disruption. Disruption of the PKG I gene also caused erectile
dysfunction in mice (2). In male mice the ability to reproduce was
greatly diminished, and no relaxation was induced by nerve stimulation
of the corpus cavernosum.
subunit of PDE6 and/or proteins immunologically
similar to it could decrease the level of PDE5 activation induced by
PKA. These authors suggested that activation of PDE5 by PKA may be a
result of phosphorylation of some cellular component that caused a
release of the subunit inhibitory effect on PDE5 activity. However,
no evidence for phosphorylation of PDE5 was provided.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin. PKG I-deficient mice were generated by homologous
recombination in embryonic stem (ES) cells. The 3' region of
exon 10 of the PKG I gene, which encodes part of the ATP-binding domain
essential for kinase activity, was replaced by a DNA cassette encoding
an internal ribosomal entry site, the CreERT recombinase, a
simian virus 40 polyadenylation signal, and a phosphoglycerate kinase
promoter-driven neomycin resistance gene. Correctly targeted ES cell
clones were identified by Southern blotting and injected into
blastocysts to generate chimeric mice that transmitted the modified PKG
I allele to the germ line. PKG I-deficient mice were phenotypically
indistinguishable from those reported previously (1). Mouse aortic
smooth muscle cells from wild-type and PKG I-deficient mice were
prepared by enzymatic digestion as described (10) and were used in the
first passage. All cells were maintained in a humid incubator at 5%
CO2 and 37 °C and were grown either on 6-well or 100-mm
plates. In all experiments cells were incubated in a cell culture
incubator at 37 °C after treatments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
Phospho-PDE5 antibody can specifically detect
phosphorylation of serine 92 PDE5. The N-terminal domain of PDE5
(1-125 aa) was phosphorylated by either PKG or the catalytic subunit
of PKA. Phosphorylated protein was separated by isoelectric focusing
under native conditions (pH 5-8) (A) or SDS-PAGE
(B) and transferred to nitrocellulose membrane. Membranes
were stained with Ponceau S to visualize proteins (A and
B) or treated with phospho-PDE5 antibody (C) for
Western analysis. The isoelectric points of the isoelectric focusing
standards (A) and the molecular weights
(Mr × 10 
3) of the protein
standards (B and C) are indicated on the
left side of each membrane and blot.
View larger version (19K):
[in a new window]
Fig. 2.
PDE5 is the major cGMP-hydrolyzing PDE
expressed in human uterine SMCs. HPLC separation of PDE activity
from human uterine smooth muscle cells in primary culture. The PDE
activity in each fraction was assayed with either 1 µM
cAMP or 1 µM cGMP in the presence of either 1 mM EGTA or 1.0 mM CaCI2 and 4 µg/ml calmodulin. PDE1C and PDE5 immunoreactivity was determined in
each fraction using Western analysis. The immunoblots with PDE1C and
PDE5 bands are shown above the corresponding HPLC fractions. PDE5 was
detected using a C-terminal polyclonal PDE5 antibody.
View larger version (48K):
[in a new window]
Fig. 3.
In vitro both PKG and PKA are able to
activate/phosphorylate PDE5 in the presence of cGMP. Extracts of
human uterine smooth muscle cells were incubated with either PKG or the
catalytic subunit of PKA in the phosphorylation buffer for 30-60 min
at 30 °C. After phosphorylation the extract was quickly passed
through a Centri-Sep column to exchange the buffer, samples were
prepared for Western analysis, and the rest was assayed for cGMP PDE
activity. PDE5 was determined as the portion of total cGMP-hydrolyzing
activity inhibited by 60 nM sildenafil. 100% activity is
defined as PDE5 activity of the sample, incubated without cGMP and PKG.
Mouse monoclonal phospho-VASP antibody was used to detect
phosphorylation of serine 239 on VASP and mouse monoclonal total VASP
antibody to detect a shift in its mobility.
View larger version (37K):
[in a new window]
Fig. 4.
In intact cells, activation of PKG but not
PKA leads to phosphorylation of PDE5. Either 1 mM
8-Br-cGMP (A) or 1 mM 8-Br-cAMP (B)
was added to the cultured human uterine smooth muscle cells. Cells were
harvested in homogenization buffer A at different times after addition
of PKA or PKG activators. After brief sonication samples were prepared
in SDS sample buffer for Western blot analysis.
View larger version (35K):
[in a new window]
Fig. 5.
Only dephospho-PDE5 is present in control
smooth muscle cells. In stimulated cells 25-30% of total PDE5
undergoes phosphorylation. Human uterine smooth muscle cells were
incubated with (B) or without (A) 1 mM 8-Br-cGMP for 60 min at 37 °C and were scraped
directly in IP buffer. Lysates were incubated with phospho-PDE5
antibody overnight followed by incubation with protein A-agarose
beads and then separated into supernatant and pellet. The immunopellet
was washed three times in IP buffer. PDE activity was measured in
samples before and after immunoprecipitation. In the same samples total
PDE5 and phospho-PDE5 were determined by Western blot analysis.
View larger version (25K):
[in a new window]
Fig. 6.
Phosphorylation of PDE5 leads to its
activation in human uterine smooth muscle cells. SMCs were
incubated with 1 mM 8-Br-cGMP for 60 min at 37 °C. After
incubation PDE5 was quantitatively immunoprecipitated using mouse
monoclonal PDE5 antibody. Immunoprecipitation experiments were
performed as in Fig. 5 except that protein G-agarose was used instead
of protein A-agarose.
/ mice the absence of PKG was confirmed by
Western blot analysis, whereas in the control cells PKG I bands were
easily detected. The levels of PDE5 expression in mouse aortic smooth
muscle cells, derived from either control or PKG I-deficient mice were
similar to each other.
/ cells with 8-Br-cGMP did not produce changes in
PDE5 phosphorylation, verifying that the mechanism of the 8-Br-cGMP
effect on PDE5 phosphorylation in the normal smooth muscle cells was
indeed through activation of PKG I. The higher phosphorylation level of
PDE5 in the control cells corresponded with higher enzymatic activity
(Fig. 7B). PDE5 activity was 2× greater in the immunopellet
of phosphorylated enzyme when total PDE5 monoclonal antibody was used
for immunoprecipitation, and correspondingly no changes in PDE5
activity were found in PDE5 from PKG I-deficient cells.
View larger version (37K):
[in a new window]
Fig. 7.
Analysis of PDE5 phosphorylation in PKG
I-deficient aortic smooth muscle cells. A, mouse aortic
smooth muscle cells from control mice (+/+) and PKG I-deficient mice
( 
/) were incubated with 1 mM 8-Br-cGMP (No.
1), 1 mM 8-Br-cAMP (No.
2), or both (No. 3) for 30 and 60 min.
B, changes in PDE5 activity in SMCs, incubated in the
presence of 1 mM 8-Br cGMP for 60 min at 37 °C. cGMP PDE
activity was measured in the immunopellets after total PDE5
immunoprecipitation from control mouse aortic smooth muscle cells and
PKG I-deficient smooth muscle cells.
/ cells with the same
combination of analogs (8-Br-cAMP and 8-Br-cGMP) caused a slight
phosphorylation of PDE5, suggesting that PKA can phosphorylate PDE5 in
the presence of a high level of cGMP even in the absence of PKG I. However, this effect is very much smaller than PKG-stimulated
phosphorylation of PDE5 in the control cells.
View larger version (31K):
[in a new window]
Fig. 8.
Protein phosphatase type 1, a catalytic
subunit of MLCP, is involved in regulation of PDE5 phosphorylation by
PKG. A, uterine smooth muscle cells were incubated with
1 mM 8-Br-cGMP in the presence of 20-100 nM of
calyculin A for 30 min or with 2 nM-1 µM
okadaic acid for 60 min at 37 °C. Cell morphology was assessed by
phase contrast microscopy. B, dephosphorylation of
phospho-PDE5 by PP1 and PP2A1 were measured in vitro. Cells
were incubated with 1 mM 8-Br-cGMP for 60 min at 37 °C
and then homogenized in homogenization buffer B. PP1 0.1 µM (lane 1) or 0.5 µM
(lane 2) was added to extracts of the cells and incubated
for 2 h at 36 °C. PP2A1 20 milliunits (lane
3) or 5 milliunits (lane 4) was
incubated with cell extracts for 1.5 h at 30 °C. SDS samples
were prepared at the end of incubation.
View larger version (34K):
[in a new window]
Fig. 9.
Activation of PKG in mouse uterus causes
significant phosphorylation of PDE5. Mouse uterine horns were
excised and placed into cold physiological salt solution. Rings (4-6
mm in width) were cut and incubated at 25 °C with or without 1 mM 8-Br-cGMP for 60 min. After incubation control uterine
rings (A) and uterine rings incubated with 1 mM
8-Br-cGMP (B), were homogenized by Polytron.
Immunoprecipitation was performed as in Fig. 5. Rabbit polyclonal VASP
antibody was used to detect total VASP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 10.
Schematic representation of the role of PDE5
in regulation of smooth muscle contraction. Dephosphorylation of
myosin light chains (MLC) by MLCP causes relaxation in smooth muscle by
disrupting actin-myosin formation. PKG-dependent phosphorylation
controls MLCP and PDE5 activities. This phosphorylation and
dephosphorylation of PDE5 by PKG and MLCP provide a point of cross-talk
between cGMP/PKG and the actin-myosin contractile systems.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Pfeifer, A.,
Klatt, P.,
Massberg, S., Ny, L.,
Sausbier, M.,
Hirneiss, C.,
Wang, G. X.,
Korth, M.,
Aszodi, A.,
Andersson, K. E.,
Krombach, F.,
Mayerhofer, A.,
Ruth, P.,
Fassler, R.,
and Hofmann, F.
(1998)
EMBO J.
17,
3045-3051[CrossRef][Medline]
[Order article via Infotrieve]
2.
Hedlund, P.,
Aszodi, A.,
Pfeifer, A.,
Alm, P.,
Hofmann, F.,
Ahmad, M.,
Fassler, R.,
and Andersson, K. E.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2349-2354 3.
Juilfs, D. M.,
Soderling, S.,
Burns, F.,
and Beavo, J. A.
(1999)
Rev. Physiol. Biochem. Pharmacol.
135,
67-104[Medline]
[Order article via Infotrieve]
4.
Boolell, M.,
Allen, M. J.,
Ballard, S. A.,
Gepi-Attee, S.,
Muirhead, G. J.,
Naylor, A. M.,
Osterloh, I. H.,
and Gingell, C.
(1996)
Int. J. Impot. Res.
8,
47-52[Medline]
[Order article via Infotrieve]
5.
McAllister-Lucas, L. M.,
Sonnenburg, W. K.,
Kadlecek, A.,
Seger, D.,
Trong, H. L.,
Colbran, J. L.,
Thomas, M. K.,
Walsh, K. A.,
Francis, S. H.,
and Corbin, J. D.
(1993)
J. Biol. Chem.
268,
22863-22873 6.
Thomas, M. K.,
Francis, S. H.,
and Corbin, J. D.
(1990)
J. Biol. Chem.
265,
14971-14978 7.
Corbin, J. D.,
Turko, I. V.,
Beasley, A.,
and Francis, S. H.
(2000)
Eur. J. Biochem.
267,
2760-2767[Medline]
[Order article via Infotrieve]
8.
Lochhead, A.,
Nekrasova, E.,
Arshavsky, V. Y.,
and Pyne, N. J.
(1997)
J. Biol. Chem.
272,
18397-18403 9.
Wyatt, T. A.,
Naftilan, A. J.,
Francis, S. H.,
and Corbin, J. D.
(1998)
Am. J. Physiol.
274,
H448-H455 10.
Kuhbandner, S.,
Brummer, S.,
Metzger, D.,
Chambon, P.,
Hofmann, F.,
and Feil, R.
(2000)
Genesis
28,
15-22[CrossRef][Medline]
[Order article via Infotrieve]
11.
Rybalkin, S. D.,
Bornfeldt, K. E.,
Sonnenburg, W. K.,
Rybalkina, I. G.,
Kwak, K. S.,
Hanson, K.,
Krebs, E. G.,
and Beavo, J. A.
(1997)
J. Clin. Invest.
100,
2611-2621[Medline]
[Order article via Infotrieve]
12.
Loughney, K.,
Hill, T. R.,
Florio, V. A.,
Uher, L.,
Rosman, G. J.,
Wolda, S. L.,
Jones, B. A.,
Howard, M. L.,
McAllister-Lucas, L. M.,
Sonnenburg, W. K.,
Francis, S. H.,
Corbin, J. D.,
Beavo, J. A.,
and Ferguson, K.
(1998)
Gene
216,
139-147[CrossRef][Medline]
[Order article via Infotrieve]
13.
Smith, D. E.,
and Fisher, P. A.
(1984)
J. Cell Biol.
99,
20-28 14.
Sonnenburg, W. K.,
Rybalkin, S. D.,
Bornfeldt, K. E.,
Kwak, K. S.,
Rybalkina, I. G.,
and Beavo, J. A.
(1998)
Methods
14,
3-19[CrossRef][Medline]
[Order article via Infotrieve]
15.
Smolenski, A.,
Bachmann, C.,
Reinhard, K.,
Honig-Liedl, P.,
Jarchau, T.,
Hoschuetzky, H.,
and Walter, U.
(1998)
J. Biol. Chem.
273,
20029-20035 16.
Patterson, R. L.,
van Rossum, D. B.,
and Gill, D. L.
(1999)
Cell
98,
487-499[CrossRef][Medline]
[Order article via Infotrieve]
17.
Fukao, M.,
Mason, H. S.,
Britton, F. C.,
Kenyon, J. L.,
Horowitz, B.,
and Keef, K. D.
(1999)
J. Biol. Chem.
274,
10927-10935 18.
Zhou, X. B.,
Wang, G. X.,
Ruth, P.,
Huneke, B.,
and Korth, M.
(2000)
Am. J. Physiol. Cell Physiol.
279,
C1751-C1759 19.
Schlossmann, J.,
Ammendola, A.,
Ashman, K.,
Zong, X.,
Huber, A.,
Neubauer, G.,
Wang, G. X.,
Allescher, H. D.,
Korth, M.,
Wilm, M.,
Hofmann, F.,
and Ruth, P.
(2000)
Nature
404,
197-201[CrossRef][Medline]
[Order article via Infotrieve]
20.
Surks, H. K.,
Mochizuki, N.,
Kasai, Y.,
Georgescu, S. P.,
Tang, K. M.,
Ito, M.,
Lincoln, T. M.,
and Mendelsohn, M. E.
(1999)
Science
286,
1583-1587 21.
Sauzeau, V., Le,
Jeune, H.,
Cario-Toumaniantz, C.,
Smolenski, A.,
Lohmann, S. M.,
Bertoglio, J.,
Chardin, P.,
Pacaud, P.,
and Loirand, G.
(2000)
J. Biol. Chem.
275,
21722-21729 22.
Omura, T.,
Matsumoto, T.,
Nakae, I.,
Takahashi, M.,
and Kinoshita, M.
(2001)
Clin. Exp. Pharmacol. Physiol.
28,
259-265[CrossRef][Medline]
[Order article via Infotrieve]
23.
De Garavilla, L.,
Pagani, E. D.,
Buchholz, R. A.,
Dundore, R.,
Bode, D. C.,
Volberg, M. L.,
Jackson, K. N.,
Pratt, P.,
and Silver, P. J.
(1996)
Eur. J. Pharmacol.
313,
89-96[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Pokreisz, S. Vandenwijngaert, V. Bito, A. Van den Bergh, I. Lenaerts, C. Busch, G. Marsboom, O. Gheysens, P. Vermeersch, L. Biesmans, et al. Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice Circulation, January 27, 2009; 119(3): 408 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-J. Zhang, K. B. Cahill, A. Elfenbein, V. Y. Arshavsky, and R. H. Cote Direct Allosteric Regulation between the GAF Domain and Catalytic Domain of Photoreceptor Phosphodiesterase PDE6 J. Biol. Chem., October 31, 2008; 283(44): 29699 - 29705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Wilson, H. S. Elbatarny, S. W. Crawley, B. M. Bennett, and D. H. Maurice Compartmentation and compartment-specific regulation of PDE5 by protein kinase G allows selective cGMP-mediated regulation of platelet functions PNAS, September 9, 2008; 105(36): 13650 - 13655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Heikaus, J. R. Stout, M. R. Sekharan, C. M. Eakin, P. Rajagopal, P. S. Brzovic, J. A. Beavo, and R. E. Klevit Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: INSIGHTS INTO NUCLEOTIDE SPECIFICITY, DIMERIZATION, AND cGMP-DEPENDENT CONFORMATIONAL CHANGE J. Biol. Chem., August 15, 2008; 283(33): 22749 - 22759. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. Bessay, M. A. Blount, R. Zoraghi, A. Beasley, K. A. Grimes, S. H. Francis, and J. D. Corbin Phosphorylation Increases Affinity of the Phosphodiesterase-5 Catalytic Site for Tadalafil J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 62 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Russo, P. Del Mese, G. Doronzo, L. Mattiello, M. Viretto, A. Bosia, G. Anfossi, and M. Trovati Resistance to the Nitric Oxide/Cyclic Guanosine 5'-Monophosphate/Protein Kinase G Pathway in Vascular Smooth Muscle Cells from the Obese Zucker Rat, a Classical Animal Model of Insulin Resistance: Role of Oxidative Stress Endocrinology, April 1, 2008; 149(4): 1480 - 1489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. N. Farrow, B. S. Groh, P. T. Schumacker, S. Lakshminrusimha, L. Czech, S. F. Gugino, J. A. Russell, and R. H. Steinhorn Hyperoxia Increases Phosphodiesterase 5 Expression and Activity in Ovine Fetal Pulmonary Artery Smooth Muscle Cells Circ. Res., February 1, 2008; 102(2): 226 - 233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Burnett Molecular Pharmacotherapeutic Targeting of PDE5 for Preservation of Penile Health J Androl, January 1, 2008; 29(1): 3 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zaccolo and M. A. Movsesian cAMP and cGMP Signaling Cross-Talk: Role of Phosphodiesterases and Implications for Cardiac Pathophysiology Circ. Res., June 8, 2007; 100(11): 1569 - 1578. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bruder, A. Schultz, and J. E. Schultz Characterization of the Tandem GAF Domain of Human Phosphodiesterase 5 Using a Cyanobacterial Adenylyl Cyclase as a Reporter Enzyme J. Biol. Chem., July 21, 2006; 281(29): 19969 - 19976. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Mullershausen, A. Lange, E. Mergia, A. Friebe, and D. Koesling Desensitization of NO/cGMP Signaling in Smooth Muscle: Blood Vessels Versus Airways Mol. Pharmacol., June 1, 2006; 69(6): 1969 - 1974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. MacPherson, T. D. Gillespie, H. A. Dunkerley, D. H. Maurice, and B. M. Bennett Inhibition of Phosphodiesterase 5 Selectively Reverses Nitrate Tolerance in the Venous Circulation J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 188 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mewe, C. K. Bauer, D. Muller, and R. Middendorff Regulation of Spontaneous Contractile Activity in the Bovine Epididymal Duct by Cyclic Guanosine 5'-Monophosphate-Dependent Pathways Endocrinology, April 1, 2006; 147(4): 2051 - 2062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Hofmann, R. Feil, T. Kleppisch, and J. Schlossmann Function of cGMP-Dependent Protein Kinases as Revealed by Gene Deletion Physiol Rev, January 1, 2006; 86(1): 1 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-H. Su and V. D. Vacquier Cyclic GMP-specific Phosphodiesterase-5 Regulates Motility of Sea Urchin Spermatozoa Mol. Biol. Cell, January 1, 2006; 17(1): 114 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Belmonte, C. Ticconi, S. Dolci, M. Giorgi, A. Zicari, A. Lenzi, E. A. Jannini, and E. Piccione Regulation of Phosphodiesterase 5 Expression and Activity in Human Pregnant and Non-pregnant Myometrial Cells by Human Chorionic Gonadotropin Reproductive Sciences, December 1, 2005; 12(8): 570 - 577. [Abstract] [PDF]
|
|
|
|
 |
|
 G. M. Pitari, R. I. Baksh, D. M. Harris, P. Li, S. Kazerounian, and S. A. Waldman Interruption of Homologous Desensitization in Cyclic Guanosine 3',5'-Monophosphate Signaling Restores Colon Cancer Cytostasis by Bacterial Enterotoxins Cancer Res., December 1, 2005; 65(23): 11129 - 11135. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wharton, J. W. Strange, G. M. O. Moller, E. J. Growcott, X. Ren, A. P. Franklyn, S. C. Phillips, and M. R. Wilkins Antiproliferative Effects of Phosphodiesterase Type 5 Inhibition in Human Pulmonary Artery Cells Am. J. Respir. Crit. Care Med., July 1, 2005; 172(1): 105 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. D. Barrett, D. J. Triggle, M. J.A. Walker, and D. H. Maurice Mechanism of Tissue-Selective Drug Action in the Cardiovascular System Mol. Interv., April 1, 2005; 5(2): 84 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Heintz, T. Koch, and A. Deussen Intact nitric oxide production is obligatory for the sustained flow response during hypercapnic acidosis in guinea pig heart Cardiovasc Res, April 1, 2005; 66(1): 55 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zoraghi, E. P. Bessay, J. D. Corbin, and S. H. Francis Structural and Functional Features in Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and Regulation J. Biol. Chem., March 25, 2005; 280(12): 12051 - 12063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Takimoto, H. C. Champion, D. Belardi, J. Moslehi, M. Mongillo, E. Mergia, D. C. Montrose, T. Isoda, K. Aufiero, M. Zaccolo, et al. cGMP Catabolism by Phosphodiesterase 5A Regulates Cardiac Adrenergic Stimulation by NOS3-Dependent Mechanism Circ. Res., January 7, 2005; 96(1): 100 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Mullershausen, M. Russwurm, D. Koesling, and A. Friebe In Vivo Reconstitution of the Negative Feedback in Nitric Oxide/cGMP Signaling: Role of Phosphodiesterase Type 5 Phosphorylation Mol. Biol. Cell, September 1, 2004; 15(9): 4023 - 4030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Blount, A. Beasley, R. Zoraghi, K. R. Sekhar, E. P. Bessay, S. H. Francis, and J. D. Corbin Binding of Tritiated Sildenafil, Tadalafil, or Vardenafil to the Phosphodiesterase-5 Catalytic Site Displays Potency, Specificity, Heterogeneity, and cGMP Stimulation Mol. Pharmacol., July 1, 2004; 66(1): 144 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Mullershausen, M. Russwurm, A. Friebe, and D. Koesling Inhibition of Phosphodiesterase Type 5 by the Activator of Nitric Oxide-Sensitive Guanylyl Cyclase BAY 41-2272 Circulation, April 13, 2004; 109(14): 1711 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bonnevier, R. Fassler, A. P. Somlyo, A. V. Somlyo, and A. Arner Modulation of Ca2+ Sensitivity by Cyclic Nucleotides in Smooth Muscle from Protein Kinase G-deficient Mice J. Biol. Chem., February 13, 2004; 279(7): 5146 - 5151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. Kostic, S. A. Andric, and S. S. Stojilkovic Receptor-Controlled Phosphorylation of {alpha}1 Soluble Guanylyl Cyclase Enhances Nitric Oxide-Dependent Cyclic Guanosine 5'-Monophosphate Production in Pituitary Cells Mol. Endocrinol., February 1, 2004; 18(2): 458 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Feil, S. M. Lohmann, H. de Jonge, U. Walter, and F. Hofmann Cyclic GMP-Dependent Protein Kinases and the Cardiovascular System: Insights From Genetically Modified Mice Circ. Res., November 14, 2003; 93(10): 907 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Munzel, R. Feil, A. Mulsch, S. M. Lohmann, F. Hofmann, and U. Walter Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase Circulation, November 4, 2003; 108(18): 2172 - 2183. [Full Text] [PDF]
|
|
|
|
 |
|
 F. J. Rivero-Vilches, S De Frutos, M Saura, D Rodriguez-Puyol, and M Rodriguez-Puyol Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-I{alpha} activation by NO/cGMP Am J Physiol Cell Physiol, October 1, 2003; 285(4): C891 - C898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Rybalkin, C. Yan, K. E. Bornfeldt, and J. A. Beavo Cyclic GMP Phosphodiesterases and Regulation of Smooth Muscle Function Circ. Res., August 22, 2003; 93(4): 280 - 291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shimizu-Albergine, S. D. Rybalkin, I. G. Rybalkina, R. Feil, W. Wolfsgruber, F. Hofmann, and J. A. Beavo Individual Cerebellar Purkinje Cells Express Different cGMP Phosphodiesterases (PDEs): In Vivo Phosphorylation of cGMP-Specific PDE (PDE5) as an Indicator of cGMP-dependent protein kinase (PKG) Activation J. Neurosci., July 23, 2003; 23(16): 6452 - 6459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sebkhi, J. W. Strange, S. C. Phillips, J. Wharton, and M. R. Wilkins Phosphodiesterase Type 5 as a Target for the Treatment of Hypoxia-Induced Pulmonary Hypertension Circulation, July 1, 2003; 107(25): 3230 - 3235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Manganiello Cyclic Nucleotide Phosphodiesterase 5 and Sildenafil: Promises Realized Mol. Pharmacol., June 1, 2003; 63(6): 1209 - 1211. [Full Text] [PDF]
|
|
|
|
|
|
J. D. Corbin, M. A. Blount, J. L. Weeks II, A. Beasley, K. P. Kuhn, Y. S. J. Ho, L. F. Saidi, J. H. Hurley, J. Kotera, and S. H. Francis [3H]Sildenafil Binding to Phosphodiesterase-5 Is Specific, Kinetically Heterogeneous, and Stimulated by cGMP Mol. Pharmacol., June 1, 2003; 63(6): 1364 - 1372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kimura, Y. Higashi, K. Hara, K. Noma, S. Sasaki, K. Nakagawa, C. Goto, T. Oshima, M. Yoshizumi, and K. Chayama PDE5 Inhibitor Sildenafil Citrate Augments Endothelium-Dependent Vasodilation in Smokers Hypertension, May 1, 2003; 41(5): 1106 - 1110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. G. Muradov, K. K. Boyd, S. E. Martinez, J. A. Beavo, and N. O. Artemyev The GAFa Domains of Rod cGMP-phosphodiesterase 6 Determine the Selectivity of the Enzyme Dimerization J. Biol. Chem., March 14, 2003; 278(12): 10594 - 10601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Mullershausen, A. Friebe, R. Feil, W. J. Thompson, F. Hofmann, and D. Koesling Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling J. Cell Biol., February 24, 2003; (2003) 200211041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yan, D. Kim, T. Aizawa, and B. C. Berk Functional Interplay Between Angiotensin II and Nitric Oxide: Cyclic GMP as a Key Mediator Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 26 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Francis, E. P. Bessay, J. Kotera, K. A. Grimes, L. Liu, W. J. Thompson, and J. D. Corbin Phosphorylation of Isolated Human Phosphodiesterase-5 Regulatory Domain Induces an Apparent Conformational Change and Increases cGMP Binding Affinity J. Biol. Chem., November 27, 2002; 277(49): 47581 - 47587. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |