Phosphorylation-mediated Activation and Translocation of the
Cyclic AMP-specific Phosphodiesterase PDE4D3 by Cyclic
AMP-dependent Protein Kinase and Mitogen-activated
Protein Kinases
A POTENTIAL MECHANISM ALLOWING FOR THE COORDINATED REGULATION OF
PDE4D ACTIVITY AND TARGETING*
Hanguan
Liu
and
Donald H.
Maurice
§¶
From the Departments of § Pathology and
Pharmacology & Toxicology, Queen's University, Kingston,
Ontario K7L 3N6, Canada
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ABSTRACT |
In this study, we describe a novel mechanism by
which a protein kinase C (PKC)-mediated activation of the
Raf-extracellular signal-regulated kinase kinase (MEK)-extracellular
signal-regulated kinase (ERK) cascade regulates the activity and
membrane targeting of members of the cyclic AMP-specific
phosphodiesterase D family (PDE4D). Using a combination of
pharmacological and biochemical approaches, we show that increases in
intracellular cAMP cause a protein kinase A-mediated phosphorylation
and activation of the two PDE4D variants expressed in vascular smooth
muscle cells, namely PDE4D3 and PDE4D5. In addition, we show that
stimulation of PKC via the associated activation of the Raf-MEK-ERK
cascade results in the phosphorylation and activation of PDE4D3 in
these cells. Furthermore, our studies demonstrate that simultaneous activation of both the protein kinase A and PKC-Raf-MEK-ERK pathways allows for a coordinated activation of PDE4D3 and for the translocation of the particulate PDE4D3 to the cytosolic fraction of these cells. These data are presented and discussed in the context of the activation of the Raf-MEK-ERK cascade acting to modulate the activation and subcellular targeting of PDE4D gene products mediated by cAMP.
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INTRODUCTION |
Cyclic nucleotide phosphodiesterases
(PDEs)1 catalyze the
hydrolysis of adenosine cyclic 3',5'-monophosphate (cAMP) and of guanosine cyclic 3',5'-monophosphate (cGMP) to their respective 5'
nucleoside monophosphates and play a central role in regulating cyclic
nucleotide-mediated cell signaling (1-5). PDEs form a multigene
family, with individual members classified using several criteria
including substrate selectivity, inhibitor sensitivity, and molecular
sequence (1-5). Individual members of each individual PDE type are
encoded by as many as four different genes, each of which can in turn
give rise to variants by alternate splicing of mRNA or the use of
alternate promoters (2-5).
Recently, significant progress has been made in elucidating some of the
mechanisms regulating the activity and expression of some of the PDE.
In this regard, one family that has received a significant amount of
attention has been the cAMP-specific, Rolipram-inhibited PDE4 (2, 4,
5). In human, rat and mouse, four distinct genes encode PDE4
(PDE4A, PDE4B, PDE4C, and PDE4D), with each, as a result of alternate splicing or the
use of alternate promoters, potentially giving rise to multiple enzyme variants. As a result of early work by several laboratories, it has
been established that several PDE4 variants can be expressed in
individual tissues and that these genes are regulated by
transcriptional and/or posttranslational mechanisms (2, 4). Thus,
prolonged increases in cAMP in cells can cause marked increases in the
expression of certain PDE4 variants and a PDE-mediated desensitization
to the effects of activators of adenylyl cyclase (6-16). Although the
cAMP-mediated expression of individual PDE4 genes can be cell type-specific, the generality of this observation and the cellular mechanisms that allow for the selectivity are not known. PDE4 variants
are also regulated by protein phosphorylation (reviewed in Ref. 5).
Thus, a protein kinase A (PKA)-mediated phosphorylation and short-term
activation of a specific PDE4 variant, PDE4D3, has been reported
(6-8). Although PKA-mediated phosphorylation was shown to selectively
activate a subset of PDE4D3 with high affinity for the inhibitor
Rolipram, the molecular basis of this selectivity is unknown at the
present time. Phosphorylation of PDE4B2 by mitogen-activated protein
kinase (MAPK) has also been reported, although this phosphorylation did
not alter the enzymes activity or sensitivity to inhibitors (17). More
recently, expression of PDE4A and PDE4D gene products in heterologous
expression systems has shown that certain variants of PDE4A and PDE4D
are targeted to selected membrane fractions (Ref. 18 and reviewed in
Ref. 5). The relevance of these findings to the subcellular
distribution of PDE4 variants expressed endogenously in nontransformed
cells and the impact of selected subcellular expression of these
enzymes on their activity and sensitivity to inhibitors have not yet
been systematically or rigorously investigated. However, given that targeting of PKA to membrane compartments via specific anchoring proteins (19) has been shown to play a central role on the function of
this protein in cells, a similar impact of selective expression of PDE4
variants may perhaps be anticipated.
Presumably due to the lack of vasorelaxant effects of PDE4 inhibitor
(20), very few studies have investigated the PDE4 gene products
expressed in vascular tissues and the mechanisms regulating their
activity and expression. In a previous study, we demonstrated that
prolonged activation of adenylyl cyclase in aortic vascular smooth
muscle cells (VSMCs) caused up-regulation of total cAMP PDE activity,
with at least 60% of this effect due to increases in PDE4 (15, 16). In
these earlier studies, we also demonstrated that the cAMP-mediated
increase in PDE4 activity was partially responsible for a cAMP-mediated
desensitization to activators of adenylyl cyclase in these cells (15,
16). More recently, we have reported that PDE4 inhibitors were potent
regulators of VSMC migration and could potentiate the effects of
inhibitors of other cAMP PDEs on VSMCs (21), a result similar to that
previously reported for relaxation of blood vessel (22).
In this study, we have investigated the role that protein kinase C
(PKC)-mediated activation of the Raf-MEK-ERK cascade plays in
regulating the activity and subcellular targeting of the PDE4D variants
expressed in VSMCs. Using a combination of pharmacological and
biochemical approaches, we have delineated the signaling pathways that
allow for the coordinated activation of these PDE4D variants by
activators of PKC and PKA. Our data are consistent with a direct role
for PKA in activating PDE4D3 and PDE4D5 and a role for PKC, via its
effects on the Raf-MEK-ERK cascade, in the activation and translocation
of the membrane associated fractions of PDE4D3.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture reagents (Dulbecco's modified
Eagle's medium, calf serum, HEPES, penicillin/streptomycin, Hanks'
balanced salt solution (HBSS), and trypsin-EDTA) were from Life
Technologies, Inc. Radioactive products (5'-[14C]AMP,
[3H]cAMP, and [32P]orthophosphoric acid)
were from NEN Life Science Products. Phorbol 12-myristate 13-acetate
(PMA), 4
-phorbol-12,13-didecanoate (4
PDD), angiotensin II
(AngII), rapamycin, LY 294002, SB202190, Ro 20-1724, Ro-318220,
bisindolylmaleimide I, and PD98059 were from Calbiochem-Novachem Corporation, Ontario, Canada. Forskolin, 8-BrcAMP were from Research Biochemicals International (Natick, MA), whereas Tris-HCl, HEPES, benzamidine, EDTA, EGTA, dithiothreitol, phenylmethylsulfonyl fluoride,
Triton X-100, and Tween-20 were from ICN Biomedicals Inc. (Costa Mesa,
CA). Ionomycin was from Sigma. Leupeptin was from Boehringer Mannheim.
Affi-Gel 601 and the column supports were from Bio-Rad. ECL Western
blot detection kit was purchased from Amersham Pharmacia Biotech. The
BCA protein assay kit and bovine serum albumin were from Pierce. All
other chemicals were of reagent grade and purchased from Fisher. PDE4D
variants expressed in VSMCs were detected using monoclonal antibody
61D10E. This PDE4D-specific monoclonal antibody was purified from mouse
ascites by protein A affinity chromatography monoclonal antibody and
supplied to us by Cathy Farrell, Sharon Wolda, and Ken Ferguson of ICOS Corporation (Bothell, WA). Activated ERK1 and ERK2 were detected using
a monoclonal antibody that specifically reacts with the phosphorylated
forms of these proteins (Promega).
Cell Culture--
Primary cultures of rat aortic VSMCs were
established following isolation from rat aortae as described previously
and the identity of the VSMCs was confirmed using
-actin staining
(15, 16). VSMCs were routinely cultured in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum, 8 mM
HEPES, 100 units/ml penicillin, and 100 µg/ml streptomycin at
37 °C in a 95% air-5% CO2 humidified atmosphere. Cells
were passaged by washing once with HBSS (without Ca2+ or
Mg2+) and then incubating with 1% trypsin-EDTA for 2 min
to detach cells and resuspended in growth medium. To maintain cell
stocks, 75-cm2 flasks were seeded with 106
cells and 20 ml of medium per flask. For all experiments, cells were
used between passages 5 and 10.
Treatment of Cultured Rat Aortic VSMCs with Pharmacological
Agents--
In these experiments, 3 × 105 cells were
seeded in 25-cm2 flasks, and the experiments were initiated
when the cells reached confluence (3-4 days). Culture medium was
removed and replaced with 5.0 ml of fresh culture medium supplemented
with either (i) forskolin (0.1-100 µM), (ii) PMA (0.1-1
µM), (iii) angiotensin II (1 µM), (iv)
ionomycin (1-100 µM), or (v) vehicle (0.1% dimethyl sulfoxide (Me2SO)) and incubated for various times. After
treatment, cells were washed once with 6 ml of HBSS (with
Ca2+ and Mg2+) and harvested in 1 ml of lysis
buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM
MgCl2, 5 mM benzamidine, 1 mM EDTA,
1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 1%
Triton X-100. Cells were removed from the flask by scraping. Cellular debris and unlysed cells were removed by centrifugation at 1000 × g for 5 min at 4 °C. The 1000 × g
supernatant was transferred to microtubes and stored at 4 °C until
assayed for cAMP PDE activity (see below). For some experiments,
cytosolic and particulate fractions were prepared. In these instances,
VSMCs were lysed as described above, except that Triton X-100 was
excluded from the lysis buffer, and the 1000 × g
supernatant was subjected to a further centrifugation at 100,000 × g for 1 h at 4 °C.
Assay of cAMP Phosphodiesterase Activity--
Cyclic nucleotide
phosphodiesterase activity was assayed by a modification of the method
of Davis and Daly (23). Reactions were carried out in a total volume of
100 µl containing 5 µmol of Tris-HCl (pH 7.4), 0.5 µmol of
MgCl2, 10 nmol of EGTA, and 0.1 nmol of
[3H]cAMP (55000-60000 dpm). Following a preincubation
period of 2 min at 30 °C, a sample of VSMC homogenate (5 µg of
protein) was added, and the reaction was allowed to proceed at 30 °C
for 30 min. The reaction was terminated by addition of 50 µl of 0.5 M ice-cold EDTA (pH 7.4). Recovery marker (0.1 ml of
5'-[14C]AMP, 1800 dpm) and 0.3 ml of a HEPES-NaCl buffer
(0.1 M NaCl, 0.1 M HEPES, pH 8.5) was added to
each sample prior to purification of the product of the reaction,
5'-[3H]AMP. 5'-[3H]AMP and
5'-[14C]AMP were recovered by chromatography using a
polyacrylamide-boronate gel column (Affi-Gel 601, Bio-Rad; bed volume,
1 ml). Samples were applied following prewashing of the columns with 8 ml of HEPES-NaCl buffer. After four additional washes of the columns with 2 ml of HEPES-NaCl and equilibration of the columns with 1 ml of
0.05 M sodium acetate (pH 4.8), the 5'-AMP was eluted with
4 ml of 0.05 M sodium acetate. The recovered
5'-[3H]AMP was quantified by liquid scintillation
counting, corrected for recovery of 5'-[14C]AMP, and
normalized to the total protein used in the assay, and the total
activity was expressed as pmol min
1 mg
1 of protein.
Immunoblotting--
Rat aortic VSMC cultures incubated with
compounds of interest were homogenized in a buffer consisting of 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2,
0.1 mM EGTA, 5 mM benzamidine, 1 µg/ml
aprotinin, and 1 µg/ml leupeptin (Buffer A). These samples (5-20
µg of total homogenate protein or of protein from isolated
subcellular fractions) were subjected to SDS-PAGE. Following
electrophoresis, proteins were transferred to nitrocellulose membranes
(Bio-Rad), and the membranes were blocked by incubation with TBST (20 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20)
supplemented with 5% powdered nonfat milk for 1 h. Blots were
incubated with an appropriate dilution of primary antibodies for 1-2 h
and rinsed three times with TBST. Rinsed blots were incubated with
horseradish peroxidase-conjugated goat anti-mouse IgG for 1 h,
then rinsed with TBST, and immunoreactivity was detected by
chemiluminescence as per the manufacturer's recommendations (Amersham
Pharmacia Biotech).
Metabolic Labeling with
[32P]Orthophosphate--
Rat aortic VSMCs were seeded in
T75 boxes (Corning) and cultured as described above. At confluence, the
growth medium was replaced with phosphate-free minimal essential medium
containing 20 mM HEPES (pH 7.4) and carrier-free
[32P]orthophosphate (0.2 mCi/ml), and cells were
incubated for 2 h. During the last 0.5 h of the metabolic
labeling, PMA (100 nM), FSK (100 µM), or
Me2SO was added. At the end of this treatment, cells were
washed twice with HBSS; harvested in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS,
100 µg of phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1%
Nonidet P-40, and 0.5% sodium deoxycholate; and homogenized. After
centrifugation at 1000 × g for 5 min, supernatants
were incubated batch-wise for 30 min with DEAE-Sepharose buffered with
200 mM sodium acetate, pH 6.5. The resin was washed twice
with the same buffer, and the adsorbed proteins were eluted with 600 mM sodium acetate, pH 6.5. In order to remove nonspecific
immunoreactive proteins, the partially purified samples were incubated
with 2.5 µg/ml of an irrelevant monoclonal antibody (anti-elastase,
mouse IgG2b) for 2 h at 4 °C and then with protein G beads
(Amersham Pharmacia Biotech) for 30 min and centrifuged (1000 × g for 15 min). The supernatant were transferred to fresh
vials and incubated with 2.5 µg/ml of the PDE4D-specific monoclonal
antibody (61D10E) for 16 h at 4 °C. Again, precipitation was
achieved using protein G beads. The immunoprecipitates were washed five
times and resuspended in 1% SDS in phosphate-buffered saline, diluted
in SDS-PAGE sample buffer, and separated by 7.5% SDS-PAGE. Proteins
were blotted onto an Immobilon membrane, and the radioactive bands were
visualized and quantitated with an Instant Imager (Packard Instrument
Co.).
Protein Assays--
Protein was determined using the BCA protein
assay system from Pierce, according to the manufacturer's protocol,
with bovine serum albumin as the standard.
Statistical Analysis--
Numerical data (PDE activities) and
densitometric determinations of PDE4 phosphorylation status are
presented as mean ± S.E. of at least three independent
experiments. Immunoblots that are shown are representative of results
obtained in at least three individual experiments. Statistical
differences between cAMP PDE activities were determined using the
Student's t test for either paired or unpaired samples,
with p < 0.05 considered significant.
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RESULTS |
Incubation of VSMCs with Forskolin, PMA, or Angiotensin II
Increases PDE4 Activity--
Incubation of cultured VSMCs with the
activator of adenylyl cyclase, forskolin, caused a time- and
concentration-dependent increase in cAMP PDE activity
(Table I). Similarly, incubation of these
cells with PMA or with the vasoactive agent AngII caused time and
concentration dependent increases in cAMP PDE activity in these cells,
although the absolute magnitude of the increase brought about with
these agents was less marked than was obtained with forskolin (Table
I). Coincubation of PMA or AngII with forskolin resulted in an additive
increase in cAMP PDE activity when compared with the effects of the
individual agents used alone (Table I). The inactive analogues of
forskolin, 1,9-dideoxyforskolin, and PMA,
4
-phorbol-12,13-didecanoate (4
PDD), had no effect on total cAMP
PDE activity or on the fraction attributable to PDE4 (not shown). As
reported previously, PDE4 activity accounts for approximately 60% of
total cAMP PDE activity in these cells (15), and based on the effects
of selective PDE4 inhibition, increases in this fraction of the total
activity accounted for more than 90% of the total increase under our
experimental conditions (Table I). The increases in PDE4 activity
caused by either forskolin, PMA, or AngII or combinations of these
agents were not sensitive to cycloheximide or actinomycin D.
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Table I
Incubation of VSMC with forskolin, PMA, and AngII increases PDE4
activity
VSMCs were incubated with forskolin (100 µM), PMA (100 nM), or AngII (100 nM) for 30 min. Following
the incubation, cells were rinsed with HBSS, homogenized in lysis
buffer containing 1% Triton X100, and centrifuged at 1000 × g for 10 min. PDE activity was determined as described under
"Experimental Procedures," and cilostamide (1 µM) or
Ro, 20-1724 (10 µM) was used to determine PDE3 and PDE4
activities, respectively. Values are mean ± S.E. from four
determinations.
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Incubation of VSMCs with Forskolin, PMA, or AngII Results in
Phosphorylation of PDE4D Variants--
Because incubation of VSMCs
with forskolin, PMA, or AngII increased PDE4 activity, and
cAMP-mediated, PKA-dependent activation of a PDE4 variant
(PDE4D3) had previously been reported (6-8), we undertook to determine
which PDE4D gene products were expressed in these cells and determine
whether phosphorylation of these enzymes was involved in the increased
PDE4 activity caused by forskolin, PMA, AngII, or combinations of these
compounds. Immunoblot analysis of lysates of VSMCs with a
PDE4D-selective monoclonal antibody identified two individual
anti-PDE4D immunoreactive species with electrophoretic mobilities by
SDS-PAGE characteristic of previously described PDE4D variants (Fig.
1). The smaller more abundant PDE4D
species migrated with an observed molecular mass of 95 ± 2 kDa by
SDS-PAGE, a size similar to that of the human recombinant PDE4D3 (Fig.
1). Based on these characteristics, we identified this protein as
PDE4D3. The larger, less abundant PDE4 species migrated with an
observed molecular mass of 105 ± 3 kDa (Fig. 1), a mobility
consistent with PDE4D5 (18). Because this immunoreactive protein was
significantly smaller than the other known PDE4D species, PDE4D4, which
has been shown to migrate with an observed molecular mass of
approximately 119 kDa (18), we identified the larger VSMC PDE4D variant
as PDE4D5 (5, 18). Consistent with this identification, polymerase
chain reactions carried out using oligo(dT)18-primed VSMC
mRNA as a template and PDE4D3- and PDE4D5-specific oligonucleotide
primers allows the amplification of both PDE4D3 and PDE4D5 (not shown).
When cells were lysed in the absence of detergent, PDE4D3 was found
primarily in the supernatant fraction, with approximately 17% present
in the 100,000 × g pellets (Fig. 1). In contrast,
PDE4D5 was present in both the supernatant and particulate fractions in
roughly equal amounts (Fig. 1). Although the amount of PDE4D5 detected
in our experiments was variable, it was never present at more than 15% of the level of PDE4D3. The variability in the levels of PDE4D5 detected in our studies may be related to our observation that the
fraction of PDE4D5 expressed in the particulate fraction was not
efficiently solubilized with Triton X-100, the detergent used in most
of our studies (not shown).

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Fig. 1.
Subcellular localization of VSMC PDE4D3 and
PDE4D5. Confluent VSMCs were homogenized and centrifuged at
1000 × g for 10 min at 4 °C. Pellet and supernatant
fractions of the 1000 × g supernatant were obtained by
differential centrifugation at 100,000 × g for 1 h at 4 °C. After centrifugation, equivalent amounts (20 µg) of
each the pellet and supernatant fractions were electrophoretically
resolved by SDS-PAGE and blotted to nitrocellulose membranes as
described under "Experimental Procedures." Immunoblots were probed
with a PDE4D-selective monoclonal antibody (61D10E), and PDE4D3 and
PDE4D5 were visualized by ECL as per the supplier's recommendation
using a horseradish peroxidase-conjugated goat anti-mouse IgG, as
described under "Experimental Procedures."
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In most previous reports, phosphorylation of PDE4D3 has been studied by
quantitating the fraction of PDE4D3 that exhibited a retarded migration
by SDS-PAGE, an event that is detected as an upward shift in the
electrophoretic mobility of PDE4D3. Using this approach, all of our
data were consistent with a PKA-mediated phosphorylation of PDE4D3 in
response to incubation of VSMCs with forskolin. Thus, incubation of
VSMCs with forskolin or with a membrane-permeant analogue of cAMP,
8-Br-cAMP, caused a time- and concentration-dependent shift
in the electrophoretic mobility of PDE4D3 in these cells (Figs.
2 and 3),
an effect that was maximal following treatment of cells with 100 µM forskolin for 20 min. Incubation of VSMCs with
forskolin also caused a similar shift in the electrophoretic mobility
of the PDE4D5 expressed in these cells; however, owing to the low
abundance of this protein in VSMCs, and its limited extraction from the
particulate fraction with Triton X-100, this effect was more subtle and
only detected in some experiments (for example, see Fig.
4). In none of the 30 experiments in
which the effects of forskolin or 8-Br-cAMP were determined was either
agent capable of causing a shift of all of the PDE4D3 present in the
cell lysates to the slower migrating species. In fact, as determined by
densitometry, the maximum amount of PDE4D3 shifted following incubation
of VSMCs with 100 µM forskolin for 20 min (maximal
conditions) was 82 ± 3%. Incubation of VSMCs with PMA (Fig. 4)
or AngII (not shown) also resulted in a time- and
concentration-dependent shift in the electrophoretic
mobility of PDE4D3. Although the shift of PDE4D3 caused by PMA or AngII was qualitatively similar to that caused by forskolin, the time course
of the PMA-induced shift in PDE4D3 migration was very different. Thus,
whereas the effects of forskolin were rapid in onset and saturated
after only 20 min, the PMA-induced shift in mobility of PDE4D3 occurred
more slowly and only reached a maximum after 1-2 h (Fig. 4). Also,
incubation of VSMCs with PMA or AngII caused a much smaller fraction of
the total PDE4D3 to shift. In the 17 experiments in which this was
measured, incubation of VSMCs with a maximal concentration of PMA (100 nM) caused 17 ± 4% of the PDE4D3 to shift to the
slower migrating species. Interestingly, co-incubation of VSMCs with
forskolin (100 µM) and PMA (100 nM) resulted
in a significant increase in the amount of PDE4D3 that was shifted to
the more slowly migrating species, relative to the effect of either
agent alone (Fig. 4). Indeed, in the 10 experiments in which this was
measured, incubation of these cells with both 100 µM
forskolin and 100 nM PMA for 30 min caused a shift of
almost all of the PDE4D3 (95 ± 4%) to the slower migrating
species. In our experiments, incubation of VSMCs with PMA was not seen
to cause a change in the electrophoretic mobility of PDE4D5, although a
shift in a small percentage of the PDE4D5 present in these cells would
most likely have gone undetected in our experiments.

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Fig. 2.
Time-dependent, forskolin-induced
phosphorylation of VSMC PDE4D3 and PDE4D5. Confluent VSMCs were
incubated with fresh culture medium supplemented with 100 µM forskolin for 0, 2.5, 5, 10, 20, and 30 min. At the
end of the incubation period, VSMCs were washed with HBSS and lysed in
ice-cold lysis buffer (see under "Experimental Procedures"). VSMC
lysates were centrifuged at 1000 × g for 5 min, the
supernatant fractions were separated by SDS-PAGE and analyzed by
immunoblot with a PDE4D-specific monoclonal antibody (61D10E), and
PDE4D3 and PDE4D5 were visualized by ECL as per the supplier's
recommendation using a horseradish peroxidase-conjugated goat
anti-mouse IgG as described under "Experimental Procedures." The
percentage of PDE4D3 phosphorylated at different times was determined
by scanning densitometry of the shifted band relative to the intensity
of all of the PDE4D3 present in that lane using Corel Photo-Paint 7.0 software as per the manufacturer's recommendations.
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Fig. 3.
Concentration-dependent,
forskolin-induced phosphorylation of VSMC PDE4D3. Confluent VSMCs
were incubated with fresh culture medium supplemented with forskolin
(10-100 µM) or 8-Br-cAMP (1 mM) for 30 min.
At the end of the incubation period, VSMCs were washed with HBSS and
lysed in ice-cold lysis buffer (see under "Experimental
Procedures"). VSMC lysates were centrifuged at 1000 × g for 5 min, and the supernatant fractions were separated by
SDS-PAGE and analyzed by immunoblot with a PDE4D-specific monoclonal
antibody (61D10E) as described in the legend to Fig. 2.
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Fig. 4.
PMA- and forskolin-mediated VSMC PDE4D3
phosphorylation. Confluent rat aortic VSMCs were incubated with
fresh culture medium supplemented with PMA (100 nM) for 0, 0.5, 1, 2, 4, or 16 h or in the presence of forskolin (100 µM) with or without PMA (100 nM) for 0.5 h.
At the end of these incubations, VSMCs were washed with HBSS and lysed
in ice-cold lysis buffer (see "Experimental Procedures"). VSMC
lysates were centrifuged at 1000 × g for 5 min, and
the proteins in the supernatant fractions were electrophoretically
resolved by SDS-PAGE. Resolved proteins were transferred to
nitrocellulose, and PDE4D variants were visualized and quantitated as
described in the legend to Fig. 2.
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In order to more directly test whether the forskolin- or PMA-mediated
shifts in the electrophoretic mobility of PDE4D3 and PDE4D5 were the
result of phosphorylation of these proteins and to determine the
relationship between this event and the increase in PDE4 activity that
results from these treatments, we carried out two separate series of
experiments. First, we treated lysates isolated from control or
drug-treated cells with calf intestinal alkaline phosphatase (CIAP).
These experiments were carried out because we reasoned that if the
observed shifts in mobility were related to phosphorylation of these
proteins, removal of the phosphate should return the more slowly
migrating proteins to their original mobility and normalize the
activity to unstimulated levels. Our data are entirely consistent with
this (Figs. 5 and
6). Thus, CIAP treatment of an aliquot of
lysates generated from cells incubated with forskolin, PMA, or Ang II
alone or these agents in combination caused a downward movement of all
shifted bands present in the isolates. Consistent with a role of the
phosphorylation in the increased PDE4 activity, CIAP also reversed the
forskolin- or PMA-induced increases in PDE4 activity in the isolates.
Thus, treatment of VSMC lysates generated from VSMCs incubated with 100 µM forskolin or with 100 nM PMA with CIAP
reduced the drug-mediated increases in PDE4 activities from 139 ± 4% and 118 ± 4% of control values to 104 ± 5% and
97 ± 5%, respectively, of levels obtained when lysates obtained
from control untreated cells were incubated with CIAP. Second, we
metabolically labeled VSMCs with [32P]orthophosphate and
immunoprecipitated PDE4D immunoreactive proteins following incubation
of the metabolically labeled cells with forskolin, PMA, or
Me2SO, the drug vehicle. Fig. 6 shows results of such an
experiment. Thus, although no 32P-containing
PDE4D-immunoprecipitated proteins were detected in immunoprecipitates
of VSMCs incubated with Me2SO, an approximately 94-kDa
radiolabeled anti-PDE4D-immunoprecipitated protein was detected in
pellets obtained from lysates of VSMCs incubated with either forskolin
or PMA. The electrophoretic mobility of the labeled anti-PDE4D-immunoprecipitated protein was consistent with it being PDE4D3, the major PDE4D present in these cells, and the major protein
with a mobility shift following incubation of VSMCs with forskolin,
PMA, or AngII. Similar amounts of PDE4D immunoreactive protein were
immunoprecipitated under these conditions (Fig. 6B). The
amount of PDE4D3 expressed endogenously in VSMCs (approximately 3-5
ng/mg of protein) made any further analysis of the phosphorylated immunoprecipitated protein impossible.

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Fig. 5.
CIAP treatment of aortic VSMC lysates
reverses the forskolin-, PMA-, or AngII-induced shifts in PDE4D3.
Confluent rat aortic VSMCs were incubated for 1 h with fresh culture
medium supplemented with either vehicle (Me2SO), PMA (100 nM), or forskolin (100 µM) or a combination
of forskolin and PMA or AngII. Following these treatments, VSMCs were
washed with HBSS and lysed in ice-cold lysis buffer (see under
"Experimental Procedures"). VSMC lysates were centrifuged at
1000 × g for 5 min, and the supernatant fractions were
incubated with CIAP or with the CIAP reaction buffer alone at 25 °C
for 45 min. Following the incubation, the reaction mixtures were
separated by SDS-PAGE and transferred to nitrocellulose, and PDE4D
variant status was analyzed by immunoblotting as described in the
legend to Fig. 2.
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Fig. 6.
In vivo phosphorylation of PDE4D3
in aortic VSMCs incubated with forskolin or PMA. Individual
confluent flasks (75 cm2) of aortic VSMCs were incubated
with fresh phosphate-free culture medium supplemented with 0.2 mCi/ml
[32P]orthophosphate for 2 h. During the last
0.5 h of this incubation, PMA (100 nM), forskolin (100 µM), or the drug vehicle (Me2SO) were added.
Treated VSMCs were rinsed with HBSS and lysed in ice-cold
immunoprecipitation buffer, and the lysates were centrifuged at
1000 × g for 5 min (see under "Experimental
Procedures"). Following adsorption of the supernatant fractions to
DEAE-Sepharose and washing of the matrix with lysis buffer supplemented
with 300 mM sodium acetate, PDE4 activity was eluted with
lysis buffer supplemented with 750 mM sodium acetate.
Eluted PDE4 fractions were immunoprecipitated with a PDE4D-specific
monoclonal antibody (see under "Experimental Procedures").
Immunoprecipitated proteins were electrophoretically resolved by
SDS-PAGE and transferred to Immobilon membranes, and the radioactive
bands were visualized (A) and PDE4D was immunoblotted
(B) as described under "Experimental Procedures."
|
|
Activation of PKA and of the Raf-MEK-ERK Cascade in VSMCs Causes
PDE4D3 Phosphorylation--
Consistent with a role for PKA in the
effect of forskolin, incubation of VSMCs with 8-Br-cAMP caused a
similar shift in the mobility of PDE4D3 (Fig. 3), whereas addition of
the inactive forskolin analogue 1,9-dideoxyforskolin did not result in
a retarded electrophoretic mobility for PDE4D3 (not shown). In
addition, preincubation of VSMCs with the highly selective PKA
inhibitor, H89 (24), completely abolished the forskolin-mediated shift in the mobility of this protein (Fig. 7).
In contrast to its antagonism of the effects of forskolin, H89 did not
reduce the phosphorylation of PDE4D3 caused by either PMA or AngII (not
shown), a result consistent with the involvement of another kinase
catalyzing the phosphorylation of PDE4D3 brought about by incubation of
cells with these agents. Consistent with a role for PKC in mediating the phosphorylation of PDE4D3 by PMA or AngII, down-regulation of PKC
following a 16-h preincubation period with PMA (25) inhibited phosphorylation of PDE4D3 by either PMA (Fig. 4) or AngII (not shown),
and incubation of the VSMCs with the inactive phorbol ester, 4
PDD,
did not affect the electrophoretic mobility of PDE4D3 (not shown). Also
consistent with a role for PKC in these effects, inclusion of either
Ro-318220 (not shown) or bisindolylmaleimide I (Fig.
8), two highly selective PKC inhibitors
(26, 27), completely blocked the PMA- or AngII-induced phosphorylation
of PDE4D3. Consistent with PKC not being involved in the effects of
forskolin in these cells, bisindolylmaleimide I had no effect on
forskolin-mediated phosphorylation of PDE4D3 (Fig. 8). Because several
recent reports have shown that activation of PKC by PMA can lead to a
PKC-dependent activation of ERK1 and ERK2 (25, 28) and that
some of the effects of PMA have been shown to be dependent on
activation of these kinases, their involvement in the PMA- and
AngII-mediated phosphorylation of PDE4D3 was investigated. Because no
specific ERK1/ERK2 inhibitors exist, for these studies, we used a
selective inhibitor of MEK (PD98059) (29), the upstream kinase
responsible for the activation of ERK1 and ERK2 in cells. Consistent with a central role for this MAPK pathway in mediating the PMA- or AngII-induced phosphorylation of PDE4D3, PD98059
inhibited both the PMA-induced phosphorylation of both ERK1 and ERK2
(Fig. 9) and the phosphorylation of
PDE4D3 caused by these agents (Fig. 10). In our studies, PD98059 had no
effect on the shift in PDE4D3 induced by forskolin, a result excluding
a role for this pathway in mediating the effects of this agent. In all
of our experiments, ERK1 and ERK2 activation by PMA or AngII was
reduced in the presence of forskolin (Fig. 9), an effect presumably
related to PKA regulation of an upstream event, perhaps activation of
Raf-1 (25, 28). Inhibitors of other kinase cascades that could
potentially be affected by activation of PKC in our cells had no effect
on the PMA-induced phosphorylation of PDE4D3, either in the absence or presence of forskolin. Thus, neither rapamycin, LY 294002, SB202190, nor herbimycin A, potent and selective inhibitors of the p70S6 kinase,
phosphatidylinositol 3-kinase, p38 MAPK, or protein tyrosine kinases,
respectively, had any effect on PMA- or AngII-induced phosphorylation
of PDE4D3 (not shown). Similarly, because increases in Ca2+
have been reported following activation of the Raf-MEK-ERK cascade in
cardiac myocytes (30), a role for
Ca2+-dependent kinases (31) in the effects of
PMA was investigated using ionomycin. Addition of ionomycin to VSMCs
had no effect on total cAMP PDE activity or on the fraction attributed
to PDE4 (not shown).

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Fig. 7.
Inhibition of PKA activity in
forskolin-treated VSMCs abolishes PDE4D3 phosphorylation.
Confluent rat aortic VSMCs were incubated with fresh culture medium
supplemented with either vehicle (Me2SO) or H89 (1-10
µM) for 20 min. After this preincubation period,
forskolin (100 µM) was added, and VSMCs were incubated
for a further 30 min. Treated VSMCs were washed with HBSS and lysed in
ice-cold lysis buffer (see under "Experimental Procedures"). VSMC
lysates were centrifuged at 1000 × g for 5 min, and
PDE4D in the supernatant fractions was detected by immunoblotting as
described in the legend to Fig. 2.
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Fig. 8.
Inhibition of PKC activity in PMA-treated
VSMCs abolishes PDE4D3 phosphorylation. Confluent rat aortic VSMCs
were incubated with fresh culture medium supplemented with either
vehicle (Me2SO) or with bisindolylmaleimide I
(Bis-1) (2.5 µM) for 20 min. Following this
preincubation period, PMA (100 nM), forskolin (100 µM), or both agents were added, and the cells were
incubated for a further 30 min. Treated VSMCs were washed with HBSS and
lysed in ice-cold lysis buffer, and PDE4D in the VSMC supernatant was
visualized by immunoblotting as described in the legend to Fig.
2.
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Fig. 9.
Inhibition of mitogen-activated protein
kinase kinase (MEK) activity in PMA-treated VSMCs abolishes
ERK1 and ERK2 phosphorylation. Confluent rat aortic VSMCs were
incubated with fresh culture medium supplemented with either vehicle
(Me2SO) or PD98059 (1 µM) for 20 min.
Following this preincubation period, PMA (100 nM),
forskolin (100 µM), or both agents were added to VSMCs,
and the incubation was allowed to proceed for a further 0.5 or 1 h. At the end of the incubation period, VSMCs were washed with HBSS and
lysed in ice-cold lysis buffer, and lysates were centrifuged at
1000 × g for 5 min. Supernatant fractions were
separated by SDS-PAGE and analyzed by immunoblot with an antibody that
specifically recognizes the activated forms of ERK1 and ERK2
(Anti-ACTIVETM MAPK pAb, Promega), and the activated
proteins were visualized as described under "Experimental
Procedures."
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Fig. 10.
Inhibition of mitogen-activated protein
kinase kinase (MEK) activity in PMA-treated VSMCs abolishes PDE4D3
phosphorylation. Confluent rat aortic VSMCs were incubated with
fresh culture medium supplemented with either vehicle
(Me2SO) or PD98059 (1 µM) for 20 min.
Following this preincubation period, PMA (100 nM),
forskolin (100 µM), or both agents were added, and the
incubation was allowed to proceed for a further 30 min. At the end of
the incubation period, VSMCs were washed with HBSS and lysed in
ice-cold lysis buffer, and the PDE4D in the 1000 × g
supernatant was visualized as described in the legend to Fig. 2.
|
|
Coordinated Actions of Forskolin and PMA Cause Translocation of
PDE4D3--
In our experiments, incubation of VSMCs with either PMA or
AngII led to the phosphorylation of a maximum of 20% of total PDE4D3, whereas treatment with forskolin caused a maximum of 80% of the PDE4D3
to be phosphorylated. Because these percentages of PDE4D3 were roughly
equal to the fractions of PDE4D3 detected in the 100,000 × g particulate and supernatant fractions of these cells, respectively, we investigated the possibility that PMA and forskolin could have subcellular selective effects. Our data from these experiments are consistent with this. Thus, whereas immunoblots of
unfractionated VSMC lysates incubated with forskolin (100 µM) or PMA (100 nM) alone or in combination,
identified the previously described pattern of PDE4D3 phosphorylation
(Fig. 11), immunoblot analysis of
resolved particulate and supernatant fractions showed that the PDE4D3
in these fractions was differentially affected. Thus, whereas treatment
of VSMCs with forskolin for 30 min resulted in the phosphorylation of
more than 80% of the supernatant PDE4D3, less than 40% of the
particulate PDE4D3 was phosphorylated under these conditions (Fig. 11).
Similar results were seen when cells were incubated with forskolin for
periods between 5 min and 1 h (not shown). Incubation of VSMCs
with PMA also resulted in a lower percentage of particulate PDE4D3
phosphorylation, an effect that was most marked when VSMCs were
incubated simultaneously with both PMA and forskolin (Fig. 11). Indeed,
when both agents were added together, a very marked reduction in total
PDE4D3 was observed in the particulate fraction of treated cells (Fig.
11). Based on densitometric analysis, approximately 70% less PDE4D3 was present in the particulate fraction of VSMCs treated with both
forskolin and PMA than when samples derived from forskolin-treated cells were analyzed (Fig. 11). Because pharmacological agents that discriminate between the different PDE4 variants do not exist, a
strategy of selective pharmacological inhibition of the different PDE4
variants present in VSMC particulate fractions was not possible. Therefore, to more directly assess the effects of our treatments on
PDE4D3 activity in the particulate fraction of VSMCs, we used a
strategy of selective PDE4D immunoprecipitation. Our data from these
experiments are consistent with a forskolin- or PMA-mediated activation
of particulate PDE4D and with a translocation of PDE4D from this
fraction upon co-incubation with both these agents (Table II). Thus, incubation of VSMCs with
either forskolin or PMA for 30 min resulted in an 8- or 4-fold increase
in the PDE4 activity present in PDE4D immunoprecipitates obtained from
the particulate fractions, respectively; when cells had been incubated
with both forskolin and PMA for 30 min (Table II), there was a marked
reduction in the immunoprecipitatable PDE4D activity in this fraction.
In fact, only slightly more (approximately 40%) PDE4D activity was present in the particulate fraction of cells incubated with both these
agents than was obtained in immunoprecipitates of control untreated
cells. Interestingly, not all of the PDE4 activity present in the
particulate fraction was immunoprecipitated with the anti-PDE4D antibody in our studies. Because there was no PDE4D3 detectable by
immunoblotting in the particulate fraction following PDE4D immunoprecipitation (not shown), these data are consistent with the
presence of another PDE4 variant in this fraction. However, because the
particulate PDE4 activity not immunoprecipitated by the PDE4D antibody
was equivalent between fractions prepared from forskolin and forskolin
plus PMA-treated VSMCs, we can conclude that the loss of PDE4 activity
in this fraction is entirely attributable to the loss of PDE4D (Table
II and Fig. 11). Although preliminary data obtained in our laboratory
are consistent with the presence of both PDE4A and PDE4B variants in
the VSMCs particulate fractions isolated from untreated cells (not
shown), the identity of the PDE4 variant activated by forskolin but not
translocated in response to PMA (Table II) will require further
work.

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Fig. 11.
Phosphorylation and translocation of PDE4D3
in VSMCs treated with PMA and forskolin. Confluent rat aortic
VSMCs were incubated with fresh culture medium supplemented with either
vehicle (Me2SO), PMA (100 nM), forskolin (100 µM), or both agents for 30 min. At the end of the
incubation period, VSMCs were washed with HBSS and lysed in ice-cold
lysis buffer that did not contain Triton X-100. VSMC lysates were
centrifuged at 1000 × g for 5 min, and the supernatant
fractions were further centrifuged at 100,000 × g for
60 min. Proteins in an aliquot of the 100,000 × g
particulate, 100,000 × g supernatant, and 1000 × g supernatant fractions were electrophoretically resolved,
transferred to nitrocellulose, and blotted with a PDE4D-specific
antibody as described in the legend to Fig. 2.
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Table II
Effects of incubation of VSMCs with forskolin and/or PMA on particulate
PDE4D
VSMCs were incubated with forskolin (100 µM), PMA (100 nM), or a combination of these agents for 30 min. Following
the incubation, cells were rinsed with HBSS, homogenized in lysis
buffer without detergent, and centrifuged at 1000 × g
for 10 min at 4 °C. Pellet fraction of the 1000 × g
supernatant was obtained by differential centrifugation at 100,000 × g for 1 h at 4 °C. PDE4D was immunoprecipitated
with a PDE4D-specific monoclonal antibody (see under "Experimental
Procedures"). PDE activity was determined as described under
"Experimental Procedures," and Ro, 20-1724 (Ro; 10 µM) was used to determine PDE4 activity. Values are
means ± S.E. from eight determinations.
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|
 |
DISCUSSION |
The PDE4D gene encodes two distinct short forms (PDE4D1 and
PDE4D2) and at least three long forms, PDE4D3, PDE4D4, and PDE4D5 (18).
Although all five PDE4D variants contain identical catalytic domains,
the three long forms also contain two amino-terminal regions that have
been shown to be highly conserved in all PDE4 genes, but not in other
PDE families. These regions have been termed upstream conserved regions
1 and 2 (UCR1 and UCR2) and have been postulated to play important
roles in both catalysis by these enzymes and their susceptibility to
inhibition with PDE4-selective agents, such as Rolipram (reviewed in
Ref. 5). Although PDE4D1 and PDE4D2 have been shown to be soluble
proteins (14, 16), when expressed in heterologous expression systems,
PDE4D3, PDE4D4 and PDE4D5 were recovered in both the supernatant and
particulate fractions (18). The data presented in this study identify
the PDE4D variants expressed in VSMCs as PDE4D3 and PDE4D5,
characterize the subcellular expression pattern of these enzymes,
describe a cAMP/PKA mediated regulation of both PDE4D variants, and
delineate a previously unreported role for the Raf-MEK-ERK cascade in
regulating the activity and the subcellular localization of PDE4D3.
Using a PDE4D-selective antibody, we identified PDE4D3 and PDE4D5 as
the PDE4D variants expressed in aortic VSMCs and showed that PDE4D5 was
expressed at approximately 10% the level of PDE4D3. Whereas PDE4D3 was
predominantly a soluble enzyme, with less than 10% expressed in the
particulate fraction, PDE4D5 was found in equal amounts in both the
soluble and particulate fractions of lysed cells. Because in our
studies virtually all of the PDE4D3 and less than 10% of the PDE4D5
expressed in the particulate fraction were solubilized with Triton
X-100, our data are consistent with the idea that PDE4D3 and PDE4D5 are
expressed in different compartments or that they localize to a similar
compartment as a consequence of different molecular interactions.
Further studies will be required to fully address this issue.
Consistent with earlier reports using several different cell types,
incubation of VSMCs with agents that elevated cAMP or with cAMP
analogues caused a time- and concentration-dependent increase in PDE4 activity (6, 7, 10-16). Although we have published
previously that long term treatments with agents that elevate cAMP
caused a transcriptionally regulated increase in PDE4 activity (15,
16), the effects described in this report, using short (<30 min)
treatment regiments, were not sensitive to cycloheximide or to
actinomycin D, demonstrating that de novo protein synthesis
was not required. In fact, several pieces of evidence were consistent
with a PKA-mediated phosphorylation of VSMC PDE4D3 and PDE4D5 being
responsible for the forskolin-induced increase in PDE4 activity in
these cells. First, incubation of VSMCs with forskolin resulted in the
phosphorylation of both proteins as determined by in vivo
labeling, shifts in their electrophoretic mobilities as well as the
effects of incubation of treated VSMC cell lysates with the phosphatase
(CIAP). In addition, direct evidence of a central role for PKA in these
effects was obtained using a PKA-selective inhibitor, H89 (24).
Although PDE4D5 is present at much lower abundance than PDE4D3, our
data identify VSMC PDE4D5 as a substrate for PKA. Although PDE4D5
encodes the serine residue previously shown to be required for
PKA-mediated phosphorylation and activation of PDE4D3 (Ser-54 in PDE4D3
and Ser-126 in PDE4D5; see Ref. 18), further work will be required to
identify the site(s) phosphorylated by PKA on this protein.
A novel finding of our work was that incubation of VSMCs with PMA or
with AngII increased PDE4 activity. Although PMA had been shown to
increase PDE4 activity in rat renal collecting tubule (30), the enzyme
involved and the mechanism that accounted for this activation were not
described. In these studies, we show that this activation is a
consequence of protein phosphorylation and that a
PKC-dependent activation of the Raf-MEK-ERK cascade catalyzes this phosphorylation-mediated activation of PDE4 activity. Thus, down-regulation of PKC activity (24, 27), addition of selective
PKC inhibitors (Ro-318220 or bisindolylmaleimide I), and addition of
the MEK inhibitor (PD98059) all abolished the PMA- and AngII-induced
phosphorylation of PDE4D3. Because neither the PMA pretreatment nor the
PKC or MEK inhibitors had any effect on the phosphorylation of PDE4D3
in response to addition of forskolin and because forskolin did not
activate either ERK1 or ERK2, our results confirm that PKC and the
Raf-MEK-ERK pathways were not involved in the phosphorylation of PDE4D3
and PDE4D5 caused by cAMP elevating agents. In a previous report,
phosphorylation of PDE4B2 within a MAPK consensus sequence was
reported, although this modification had no effect on enzyme function
(17). Increases in intracellular Ca2+ concentrations have
been shown to occur upon activation of the Raf-MEK-ERK cascade in
cardiac myocytes (31), and several Ca2+-activated kinases
have been described (32). In our experiments, addition of the calcium
ionophore ionomycin to VSMCs had no effect on PDE4 activity or on the
phosphorylation status of PDE4D3. Similarly, selective inhibitors of
either p70 S6-kinase, phosphatidylinositol 3-kinase, p38 MAPK, or
tyrosine kinases were without effect on either the forskolin- or
PMA-induced phosphorylation and activation of PDE4D3 in our cells. The
residue phosphorylated in PDE4D3 as a consequence of the PKC-mediated
activation of the Raf-MEK-ERK cascade described in this report is at
present unknown, as is the mechanism that allows for activation of
PDE4D3 in response to this phosphorylation event. However, because our
experiments have shown that the site(s) phosphorylated as a consequence
of the PKC-mediated activation of the Raf-MEK-MAPK pathway in these cells caused a shift-coupled activation of the enzyme, very similar to
that caused by PKA-mediated phosphorylation of Ser-54 (in PDE4D3), it
is perhaps reasonable to postulate that the site(s) will be in
relatively close proximity to the site phosphorylated by PKA. Based on
these considerations, we would propose that Ser-119, which is in an ERK
consensus sequence, could perhaps be an excellent candidate site for
this phosphorylation. Clearly, further experimentation will be required
to validate this supposition.
A completely novel finding that emerged from our studies relates to the
observation that the combined actions of PMA and forskolin or AngII and
forskolin caused a translocation of PDE4D3 from the particulate to the
soluble fraction of these cells, with a resulting decrease in
particulate PDE4 activity relative to that caused by forskolin alone.
This conclusion was supported by the loss of significant amounts of
phosphorylated PDE4D3 in the particulate fraction of PMA-treated VSMCs
and by the marked loss of PDE4D3 in the particulate fraction of VSMCs
incubated with both forskolin and PMA. Based on our PDE4D-specific
immunoblotting and immunoprecipitation studies, changes in PDE4D
activity and targeting entirely accounts for the PMA-induced increase
in particulate PDE4 activity, as well as the reduction in PDE4 activity
following the coordinated phosphorylation of particulate PDE4D3 in the
presence of both forskolin and PMA. Although further studies will be
required to fully assess the significance of the translocation event
described in this work, some of its implications are clear. First,
although the increase in soluble PDE4D3 activity caused by the
PKC-Raf-MEK-ERK cascade-induced translocation in these cells would most
likely be small, the loss of PDE4D3 in the particulate fraction could have very significant functional consequences. For example, if the
location of PDE4D3 coincided with that of cAMP effector proteins, such
as PKA, significant changes in cAMP-mediated signaling might be
anticipated following treatment of cells with activators of PKC and
PKA. In order to assess this, studies are under way in our laboratory
to determine whether activation of the Raf-MEK-ERK cascade could alter
the ability of PDE4 inhibitors to affect cell functions, such as
proliferation and migration. In this context, we recently reported that
PDE4 inhibitors were potent inhibitors of PDGF-BB-mediated VSMC
migration (21). Clearly, if differences in the ability of PDE4
inhibitors to antagonize migration induced by chemotactic factors was
shown to be dependent on the relative contribution of the Raf-MEK-ERK
cascade to the effects of the chemotactic factor studied, the mechanism
of PDE4D3 regulation described here might have significant functional
relevance in many cell types. Because our data show that not all of the
increase in particulate PDE4 caused by forskolin can be attributed to
PDE4D, and variants of PDE4A and PDE4B are also known to also have
amino-terminal MAPK consensus phosphorylation sites (5, 18), the
generality of this mechanism will need to be further investigated.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. S. Wolda, Cathy
Farrell, and K. Ferguson of ICOS Corp. for providing the
yeast-expressed PDE4D1 and PDE4D3, as well as the PDE4D-specific
monoclonal antibody used in these studies. We are also indebted to
Sandra Jimmo and Daniel Palmer for technical assistance and useful
advice during these studies.
 |
FOOTNOTES |
*
This work was supported by Grant T-3671 from the Heart & Stroke Foundation of Ontario.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: Research Career
Scientist in the Health Sciences. Pharmaceutical Manufacturers' Association of Canada-Health Research Foundation/Medical Research Council of Canada Depts. of Pathology and Pharmacology & Toxicology, Botterell Hall, A221, Queen's University, Kingston, Ontario K7L 3N6,
Canada. Tel.: 613-533-6000, Ext. 75089; Fax: 613-533-6412; E-mail
Mauriced{at}post.queensu.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
PDE, phosphodiesterase;
AngII, angiotensin II;
CIAP, calf intestinal
alkaline phosphatase;
ERK, extracellular signal-regulated kinase;
HBSS, Hanks' balanced salt solution;
MAPK, mitogen-activated protein kinase;
MEK, ERK kinase;
PAGE, polyacrylamide gel electrophoresis;
PKA, protein
kinase A;
PKC, protein kinase C;
PMA, phorbol 12-myristate 13-acetate;
VSMC, vascular smooth muscle cell.
 |
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