From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, October 10, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Ethanol can enhance
Gs Intracellular signaling via cAMP generates downstream effects that
range from changes in the function of ion channels to changes in
intracellular energy metabolism to changes in gene transcription (for
review see Ref. 1). It is therefore not surprising that the generation
of intracellular cAMP is a tightly regulated process that involves the
It was therefore not entirely unexpected that the activity of the
various isoforms of AC were also found to be differentially sensitive
to the effect of ethanol (4). We and others have shown that ethanol
acutely potentiates G cAMP signaling has been shown to be important in the behavioral effects
of ethanol through studies with Drosophila (9) and mice (10,
11). Mutations in the components of the cAMP-generating or degrading
systems in Drosophila significantly altered the responses of
the fly to the intoxicating (incoordinating) effects of ethanol (9). A
similar relationship between ethanol-induced incoordination and changes
in cAMP generating systems was found in both mice and rats (10, 12).
Activation of cAMP generation in the brains of mice, by the use
of forskolin, altered the development of tolerance to the
sedative effects of ethanol (11). Clinical studies with humans have
also linked the cAMP signaling system to behavioral manifestations of
physical dependence on ethanol. Alcohol-dependent individuals, even when abstinent for substantive periods of time, have
been shown to have lower G-protein-activated AC activity in their
platelets than control subjects (13). More recently it was found that
low platelet AC activity was characteristic of individuals classified
as family history positive for alcoholism, regardless of whether the
subjects were themselves diagnosed alcoholics (14, 15). Because ethanol
has been demonstrated to enhance receptor/G-protein-coupled generation
of cAMP in platelets, the depressed AC activity in the platelets of
individuals at risk to develop alcoholism may parallel what is
occurring in the brain and may contribute to the addiction process
(16).
We have recently shown that in human erythroleukemia (HEL) platelet
precursor cells, which contain a preponderance of AC7 mRNA (17),
both ethanol and phorbol esters (PDBu and phorbol 12-myristate
13-acetate) could significantly potentiate AC activity generated in
response to the activation of the prostanoid receptor by the agonist
PGE1 (8). This potentiation of AC activity by either
ethanol or PDBu could be diminished by PKC inhibitors such as
staurosporine and chelerythrine (8). In the current work, we report on
a series of studies that determine the PKC isotype(s) involved in the
enhancement of G Materials--
[2-3H]Adenine was obtained from
Amersham Biosciences. 3-Isobutyl-1-methylxanthine, phorbol
12,13-dibutyrate (PDBu), staurosporine, chelerythrine, rottlerin,
thapsigargin, and Gö 6976 were purchased from
Calbiochem (La Jolla, CA). Anti-PKC monoclonal antibodies were obtained
from Transduction Laboratories (Lexington, KY). Dr. W.-J. Tang
(University of Chicago) kindly provided the anti-AC II family antibody,
C6C. PGE1 was obtained from the Cayman Chemical Company
(Ann Arbor, MI). Cicaprost was a generous gift from Schering AG
(Berlin, Germany). All of the other products were purchased from Sigma.
Concentrated solutions of drugs, including PGE1 and cicaprost, were prepared in Me2SO, and the final
concentration of Me2SO in the assay mixtures was never
greater than 0.8%. Control assays were always performed containing the
appropriate amounts of Me2SO. Dr. Daria Mochly-Rosen (18)
kindly provided the following PKC-derived inhibitory peptides: Construction of the T7-tagged AC7--
A BamHI site
was introduced upstream of the initiation codon of the human AC7
cDNA (19) by in vitro mutagenesis using an oligonucleotide, CGTGCCAAGGATCCGGAGGATGCCAG. The mutation
was confirmed by DNA sequencing. A 3.6-kb fragment containing the human
AC7 coding sequence was prepared from pBlueScript II SK-containing the
AC7 cDNA by digestion with BamHI and XbaI.
The fragment was inserted into a pcDNA3.1 His (Invitrogen)
mammalian expression vector after digestion of this vector with the
restriction enzymes BamHI and XbaI. The
constructed vector provides an N-terminal fusion of hexahistidine tag
and T7 epitope with AC7 under the control of the cytomegalovirus promoter.
The recombinant baculovirus for AC7 was constructed as follows.
cDNA for human AC7 (19) was subcloned into the BamHI and BglII sites of an Invitrogen pBlueBacHis2 vector (frame C).
Sf9 cells were cotransfected with the AC7 plasmid and wild-type
viral DNA, and positive recombinant viral plaques were isolated. When expressed in the insect cells, this vector generated a fusion protein
including AC7 that contains N-terminal (His)6, T7, and XpressTM epitopes. The fusion junction was verified by automated sequencing. The control vector was generated from the T7-AC7 vector by
creating a stop codon in the multiple cloning site of the vector and
the control vector produced only the fusion tag.
Cell Culture of HEL and HEK 293 Cells and Expression of Tagged
AC7 in HEK 293 Cells--
HEL cells (American Type Culture Collection,
Manassas, VA) were grown in suspension culture in RPMI 1640 medium
(Invitrogen) containing 10% heat-inactivated, charcoal-stripped fetal
calf serum (Gemini-Bio-Products, Calabasas, CA). HEL cells were
maintained at 37 °C (5% CO2) and used for experiments
at a density range of between 0.2 and 0.4 × 106
cell/ml. HEK 293 cells were grown in 20 mM HEPES-minimal
essential medium containing 10% fetal bovine serum at 37 °C
(5% CO2). pcDNA3.1-T7.His-AC7 and the control
pcDNA3.1-T7.His vector were transfected in HEK 293 cells using
Effectene reagent (Qiagen, Valencia, CA) while maintaining the cells in
the regular growth medium. After incubation with the transfection
reagents for 48 h, the cells were maintained in serum containing
minimal essential medium for another 24 h prior to harvesting the cells.
Expression of Tagged AC7 in Sf9 Cells--
AC7 was
expressed in Sf9 cells using the baculovirus expression system.
The Sf9 cells were cultured in a spinner vessel containing Grace's insect cell medium (Invitrogen) and 10% fetal bovine serum at
27 °C (Tissue Culture Core/University of Colorado Cancer Center). Sf9 cells (300 × 106) were then infected with
the positive recombinant baculovirus for 60 min in serum-free Grace's
insect medium followed by an additional 48 h incubation in
Grace's insect medium containing 10% fetal bovine serum.
AC7-expressing Sf9 cells were identified based on Western blot
analysis of total Sf9 cell protein using a monoclonal anti-T7
antibody (Novagen, Madison, WI). No signals in the molecular mass
range of full-length AC7 were detected by the anti-T7 antibody in
solubilized protein preparations from control vector-infected
Sf9 cells.
HEK 293, HEL, and Sf9 Cell Membrane Preparations--
For
the "back-phosphorylation" experiments, T7-tagged AC7-transfected
HEK 293 or control transfected HEK 293 cells were pretreated with drugs
or other reagents prior to harvesting as indicated in the figure
legends and the text. For harvesting, the cells were resuspended in
ice-cold membrane lysis buffer containing 20 mM
HEPES, pH 7.4, 2 mM Na2EDTA, 150 mM
NaCl, 0.2 mM Na3VO4, 10 mM
For the isolation of HEL or HEK 293 cell membranes in experiments not
involving back-phosphorylation, a minimum of 15 × 106
cells were washed twice with ice-cold phosphate-buffered saline. The
pelleted cells were resuspended at 5-6 × 106
cells/ml of lysis buffer containing 50 mM Tris, pH 7.6, 2 mM MgCl2, 0.1 mM
Na3VO4, 10 mM
Membranes from T7-AC7-expressing Sf9 cells or control
vector-expressing Sf9 cells were harvested by centrifugation
(2000 × g, for 5 min at 4 °C), suspended in 20 ml
(per 100 ml of culture pellet) of 20 mM HEPES, pH 7.8, 500 mM NaCl, 5 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, and protease inhibitors (as described
above). The samples were freeze-thawed twice (from liquid nitrogen into a 42 °C water bath), and the DNA was sheared by passing the
preparation through an 18-gauge needle four times. The cell debris was
removed by centrifugation at 500 × g for 10 min at
4 °C. The supernatant was then centrifuged at 100,000 × g for 40 min at 4 °C, and the membrane pellet was
resuspended in 20 mM HEPES, pH 7.8, 200 mM sucrose, 1 mM dithiothreitol plus protease inhibitors, as
described above. The suspension was then recentrifuged at 100,000 × g for 40 min at 4 °C, and the final pellet was
solubilized in 1.0% SDS and stored at Immunoprecipitation, Phosphorylation, and Back-phosphorylation of
AC7--
For the immunoprecipitation of T7-tagged AC7, all of the
steps were carried out at 4 °C, except when noted. In each sample, 150-200 µg of solubilized total Sf9 or HEK 293 cell membrane
protein (AC7 and control vector-transfected) in 1% SDS was diluted
with the IP buffer (40 mM Tris-HCl, pH 7.8, 100 mM NaCl, 5 mM Na2EDTA, 0.4 mM MgCl2, 2 mM methionine, 10 mM NaF, 1 mM Na3VO4, 20 mM sodium pyrophosphate, 25 mM
Na2-
For the phosphorylation by full-length recombinant PKC, T7-tagged
AC7-transfected HEK 293 cells or control transfected HEK 293 cells were
lysed in ice-cold membrane lysis buffer and a Triton X-100-insoluble plasma membrane fraction (representing a membrane microdomain thought to have characteristics similar to lipid rafts and
enriched in signaling molecules) was isolated. This membrane fraction
was isolated as described above under "HEK 293, HEL, and Sf9
Cell Membrane Preparations" with the following additional modification; in the final step the non-nuclear cell lysate was centrifuged at 90,000 × g in the presence of 0.5%
Triton X-100 for 60 min at 4 °C. This membrane pellet was
solubilized in 1% SDS, and the T7-tagged AC7 was immunoprecipitated
with an anti-T7 monoclonal antibody with the addition of PAA beads (as
described above). The immunoprecipitates from T7-AC7 and control
transfected HEK 293 cells were then incubated for 30 min at 30 °C
with recombinant PKC PKC Binding Assay--
Recombinant purified PKC
The Triton X-100-insoluble plasma membrane fraction (100 µg) from
control or AC7-transfected HEK cells was suspended in IP binding buffer
A (20 mM HEPES, pH 7.8, 100 mM NaCl, 1 mM Na2EDTA, 0.4 mM
MgCl2, 1 mM CaCl2, 2 mM
methionine, 1 mM Na3VO4, 20 mM sodium pyrophosphate, 25 mM
Na2- Western Blot Analysis--
For Western blotting, whole cell
Sf9, HEK 293 cell lysates, and HEL cell lysates (in 1% SDS)
were reduced and alkylated as described under "Immunoprecipitation,
phosphorylation, and back-phosphorylation of AC7" prior to gel
electrophoresis. Precast 8% Tris-glycine polyacrylamide gels
(Novex/Invitrogen) were used to separate the protein bands. After the
electrophoresis, the samples were transferred to MSI micronSep
nitrocellulose (Osmonics, Westborough, MA) using a Xcell II Blot Module
(Novex/Invitrogen) according to the manufacturer's recommendations.
The blots containing Sf9 or HEK 293 cell protein were blocked
with 3% nonfat dairy milk (NFDM) in TBS, pH 8.0, containing 0.1%
Tween 20 (TBST) for 1 h at room temperature and then incubated for
1 h in 3% BSA in TBST containing 1:3000 dilution of monoclonal
anti-T7 antibody (Novagen) or a 1:5000 dilution of the C6C anti-AC II
family antibody (21) and washed three times with 3% NFDM TBST. The
blots were then incubated for 1 h in 5% NFDM-TBST containing
1:30,000 dilution of goat anti-mouse IgG-horseradish
peroxidase-conjugated secondary antibody and subsequently washed three
times with TBST and twice with TBS. The blots containing HEL cell
protein were blocked with 5% NFDM-TBST for 1 h at room temperature, quickly rinsed in TBS, and then blotted overnight at
4 °C in 3% NFDM-TBST containing 1:1500 dilution of anti- Assay of Adenylyl Cyclase Activity in HEL Cells--
For whole
cell cAMP synthesis measurements, HEL cells were preloaded with 2 µCi/ml [2-3H]adenine in HEL culture medium for 6 h
at 37 °C as previously described (4, 22). At the end of the
incubation, HEL cells were pelleted by centrifugation (200 × g for 5 min), washed, and resuspended at a cell
concentration of 1 × 106/ml in serum-free RPMI 1640, without phenol red, supplemented with 20 mM HEPES, pH 7.4 (assay buffer). Aliquots (0.4 ml) of the cell suspension were added to
each well (24-well plates) and allowed to equilibrate for 30 min at
36 °C before the start of an assay. In experiments that utilized the
PKC-derived inhibitory peptides, the assay buffer was modified slightly
as described below to prevent premature reduction of the chimeric
peptide (the PKC-derived/antennapedia peptides contain a Cys-Cys
disulfide bond) prior to entry into the cells. Briefly, HEL cells were
extensively washed three times (to remove exogenous glutathione) and
resuspended at a cell concentration of 1 × 106/ml in
glutathione-free, serum-free RPMI 1640 supplemented with 20 mM HEPES, pH 7.4, and 0.05% BSA. PKC-derived inhibitory
peptides were added (final concentration of 2.0 µM) to
the cell suspension and incubated at 37 °C for 2 h prior to the
start of the experiment.
cAMP formation was measured by monitoring the conversion of
[3H]ATP to [3H]cAMP. The cells were treated
with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (400 µM), for 10 min prior to the addition of agonist. After
the addition of agonist (PGE1 or cicaprost), cAMP formation
was allowed to continue for 5 min before the reaction was terminated
with trichloroacetic acid (final concentration of 10%). Other
modulators of AC activity were added at concentrations and time points
as described in the text and figure legends. ATP and cAMP were
separated by sequential chromatography on Dowex 50 and neutral alumina
columns and quantitated using a Beckman LS 6000TA liquid scintillation
counter as previously described (4, 22). The amount of
[3H]cAMP produced in the assay was calculated and
expressed as a fraction of the available pool of [3H]ATP
as previously described (4, 22).
Fluorimetric Measurement of Intracellular Ca2+
Concentration, [Ca2+]i--
To measure
[Ca2+]i in HEL cell populations, 2 × 106 HEL cells were washed and resuspended in serum free
RPMI 1640 supplemented with 20 mM HEPES, pH 7.4, and 0.1%
BSA. The extracellular Ca2+ concentration in the RPMI 1640 assay buffer was 0.4 mM. The cells were then incubated with
6 µM Fura-2/AM for 45 min at room temperature to minimize
vesicle sequestration and efflux of Fura-2/AM. At the end of the
loading period, the medium containing Fura-2/AM and other drugs, if
present, was removed. The cells were pelleted and resuspended in assay
buffer containing 0.1% BSA and allowed to equilibrate for 5-10 min.
Cells at a density of 1 × 106 cells/ml were placed in
a thermostatted cuvette maintained at 37 °C and kept in suspension
with a magnetic stir bar. Fluorescence was measured using an SLM-Aminco
dual wavelength spectrofluorometer (excitation at 345 and 380 nm;
emission at 505 nm) with a time resolution of 0.5 s. The data were
stored on a NEC 286 computer, using software from SLM-Aminco (Urbana,
IL). Intracellular calcium concentrations were calculated from the
relationship [Ca2+]i = Kd × (r Adenovirus Propagation, Purification, and Infection of HEL
Cells--
The Ad5 DL312 replication deficient adenovirus was
propagated in HEK 293 cells as follows. Thirty T150 mm flasks of HEK
293 cells suspended in 5 ml of serum-free Dulbecco's modified Eagle's medium were infected using a solution containing diluted virus at a
multiplicity of infection of 10-25 (based on a plaque formation assay). After 60 min, 20 ml of 2% fetal bovine serum/Dulbecco's modified Eagle's medium was added to the cells, and they were allowed
to grow for an additional 48 h. The cells were pelleted and
combined with serum-free RPMI medium (10 mM HEPES, pH 8.0; final volume of 5 ml). The cell suspension was frozen (liquid nitrogen)
and then thawed three times to lyse the cells and release the
adenovirus. Adenovirus particles were purified from HEK 293 cell debris
as follows. The suspension containing virus was sonicated (low energy;
<70% of microtip limit) for 5 min on ice. Sodium deoxycholate was
added to the virus suspension (final concentration of 0.1%), and the
solution was then sonicated for 5 min on ice. An equal volume of
prechilled (
For infection, HEL cells were plated at 0.1-0.2 × 106 cells/ml in fresh RPMI 1640 culture medium 1 day prior
to the addition of adenovirus. The cells were pelleted and resuspended
in 5 ml of Opti-MEM, 20 mM HEPES, pH 7.4, and were infected
at a 5 × 104 virus particles/cell during a 3-h
incubation at 37 °C. The cells were then pelleted, washed, and
resuspended in RPMI 1640 culture medium and allowed to continue growing
for 2 days prior to the assay. In preliminary experiments, a
replication deficient adenovirus carrying the green fluorescent protein
was used to verify the ability of the virus to infect HEL cells (data
not shown).
Statistics--
All of the statistical analyses were performed
using the Sigmastat program (Jandel Scientific Software). The data are
presented as the means ± S.E. unless otherwise noted.
p values of <0.05 were taken as statistically significant.
To determine whether AC7 protein could be phosphorylated by PKC,
an N-terminal tagged T7 and His epitope AC7 expression vector was
constructed that would allow for efficient purification and identification of AC7 by immunoprecipitation. There were no PKC consensus sites within or created by the addition of the T7 tag to the
AC7 protein. As seen in Fig.
1A, the catalytic subunits of
rat brain PKC phosphorylated a protein (that corresponded in size to
AC7) from the membranes of Sf9 cells that were infected with the
T7-AC7 vector but not the T7 control vector. This protein was also
enriched bys immunoprecipitation (IP) using the T7 monoclonal antibody
(Fig. 1A). Identification of this protein from infected Sf9 cells, as AC7, was accomplished by Western blot analysis
using the T7 monoclonal antibody to the N-terminal T7 tag and a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Fig.
1A). The protein identified as AC7 by Western blot analysis corresponded in size to the protein that was phosphorylated by the rat
brain PKC mixture (Fig. 1A).
-stimulated adenylyl cyclase (AC) activity. Of
the nine isoforms of AC, type 7 (AC7) is the most sensitive to ethanol.
The potentiation of AC7 by ethanol is dependent on protein kinase C
(PKC). We designed studies to determine which PKC isotype(s) are
involved in the potentiation of G
s-activated AC7
activity by ethanol and to investigate the direct phosphorylation of
AC7 by PKC. AC7 was phosphorylated in vitro by the
catalytic subunits of PKCs. The addition of ethanol to AC7-transfected
HEK 293 cells increased the endogenous phosphorylation of AC7, as indicated by a decreased "back-phosphorylation" of AC7 by PKC in vitro. The potentiation of G
s-stimulated
AC7 activity by either phorbol 12,13-dibutyrate or ethanol, in HEL
cells endogenously expressing AC7, was not through the
Ca2+-sensitive conventional PKCs. However, the potentiation
of AC7 activity by ethanol or phorbol 12,13-dibutyrate was found to be reduced by the selective inhibitor of PKC
(rottlerin), a
PKC
-specific inhibitory peptide (
V1-1), and the expression of the
dominant negative form of PKC
. Immunoprecipitation data indicated
that PKC
could bind and directly phosphorylate AC7. The results
indicate that the potentiation of AC7 activity by ethanol involves
phosphorylation of AC7 that is mediated by PKC
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of G-proteins, intracellular Ca2+
acting independently or in concert with calmodulin, and phosphorylation events that are postulated to involve protein kinase A and protein kinase C (PKC)1 (2, 3). The
current evidence indicates that each of the nine adenylyl cyclase (AC)
isoforms differs in its repertoire of regulatory controls (2, 3), and
all of the isoforms can be regulated coincidentally by multiple signals
to modulate the production of cAMP.
s-activated AC activity (5, 6), and
studies with HEK 293 cells transfected with the various isoforms of AC
have demonstrated a broad range of AC sensitivity to ethanol. Some
isoforms were found to be insensitive to ethanol (types 1, 3, 8a, and
8b), others were moderately sensitive (types 2, 5, 6, 8c, and 9), and
Type 7 AC was at least two to three times more sensitive to the
stimulatory actions of ethanol in comparison with all other tested ACs
(4). Agonist-stimulated AC7 activity in transfected HEK 293 cells can
be significantly potentiated by 10-20 mM ethanol (which
corresponds to tissue concentrations of 46-92 mg/100 ml ethanol), and
the potentiation by ethanol of AC7 activity is not mediated through the
inhibition of phosphodiesterase activity or an adenosine
receptor-mediated event (7). Ethanol has been shown to further
potentiate AC activity when AC is activated through a variety of
different membrane receptor systems, including dopamine D1a,
-adrenergic, and prostaglandin receptors (4, 5, 8). These data
indicate that the ability of ethanol to potentiate cAMP accumulation is
independent of transmitter receptors and is dependent primarily on the
AC isoform present in the cell.
s-activated AC7 activity by ethanol, and
we investigate the phosphorylation of AC7 by PKC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
V1-1
(
PKC8-16, AFNSYELGS),
V1-2
(
PKC14-21, EAVSLKPT), and a control nonsense
octapeptide (LSETKPAV). These peptides were conjugated to a
Drosophila antennapedia peptide (RQIKIWFQNRRMKWKK) to make
them more cell permeant. Catalytic subunits of PKC purified from rat
brain were prepared by trypsin digestion by Dr. Michael D. Browning
(University of Colorado Health Sciences Center, Denver, CO). Dr. Trevor
Biden (Garvan Institute of Medical Research, Sydney, Australia)
kindly provided the replication deficient adenovirus (Ad5 DL312)
vectors carrying either the wild-type or dominant negative PKC
.
Full-length recombinant human PKC
and PKC
were purchased from
Calbiochem (La Jolla, CA).
-glycerophosphate, 2 mM NaF, and
Calbiochem Protease Inhibitor Mixture Set III (l mM
AEBSF, 0.8 µM aprotinin, 50 µM
bestatin, 15 µM E-64, 20 µM leupeptin, and
10 µM pepstatin A). The cell suspension was sonicated
briefly on ice and centrifuged at 500 × g for 10 min
at 4 °C. The resulting supernatant was then centrifuged at
90,000 × g for 45 min at 4 °C, and the pellet was
solubilized as described below.
-glycerophosphate,
1 mM benzamidine, 10 mM dithiothreitol, and
Calbiochem Protease Inhibitor Mixture Set III. The cell suspension was
drawn through a 22-gauge syringe 10 times, while the preparation was
kept on ice and then centrifuged at 500 × g for 3 min
at 4 °C. The resulting supernatant was then centrifuged at
90,000 × g for 60 min at 4 °C. Both the pellet (membrane protein) and supernatant (cytosolic protein) were saved. For
immunoprecipitation (phosphorylation) experiments and Western blot
analysis, the membrane protein was solubilized in 1.0% SDS. The
samples were then incubated for 20 min at 80 °C and sonicated briefly. The protein concentration was determined using the BCA method (Pierce).
80 °C.
-glycerophosphate, and protease inhibitors as
described above, and 1.2% Nonidet P-40) to a final SDS concentration of 0.08-0.12% (a minimum 10-fold Nonidet P-40/SDS ratio). The samples
were incubated for 5 min with washed (IP buffer) protein A-agarose
(PAA) beads (ImmunoPure immobilized protein A; Pierce) at a ratio of
10-20%:80-90% beads to solubilized protein in IP buffer,
respectively, to remove material that bound nonspecifically to the
beads. The beads were pelleted by centrifugation (10,000 × g), and the supernatants were collected and shaken with 6 µg of monoclonal anti-T7 antibody (Novagen) for 2 h at room
temperature and then incubated with washed (IP buffer) PAA beads for
1 h. The antigen/antibody/PAA conjugates were pelleted
(10,000 × g) and washed once with IP buffer. The beads
were then washed twice at 22 °C with phosphorylation buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM EGTA, 0.1% Triton X-100) and incubated for 2-5 min at
37 °C in 40 µl of phosphorylation buffer including 0.3 µM [
-32P]ATP (6000 Ci/mmol; PerkinElmer
Life Sciences) and 250 nM of the constitutively active PKC
catalytic subunit purified from rat brain (20). The reaction was
terminated with 10 µl of 200 mM Na2EDTA, pH
8.0. The beads were pelleted and washed two times with IP buffer. The
samples were then reduced and alkylated to produce a tight band of AC
during the PAGE. The sample was reduced by incubation in 40 mM dithiothreitol at 80 °C for 15 min. After a 15-min
incubation at room temperature with 65 mM
N-ethylmaleimide, loading dye was added, and the sample was
briefly boiled before the PAGE. The precast 8% Tris-glycine
polyacrylamide gels were from Novex/Invitrogen (Carlsbad, CA). After
the electrophoresis, the gel was dried, and Kodak X-Omat Blue film was
used for the autoradiography.
(0.8 specific activity units) or PKC
(1.3 specific activity units) and 10 µCi of [
-32P]ATP
(3000 Ci/mmol from PerkinElmer Life Sciences; final ATP concentration
equaled 40 µM) in phosphorylation buffer (40 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
0.2 mM CaCl2, 1 mM dithiothreitol, 25 mM
-glycerol phosphate, 1 mM
Na3VO4, 2 µg/ml phosphatidylserine, 0.2 µg/ml diolein, 10 µM PDBu, and 0.02% Triton X-100).
The reaction mixture was pelleted to separate the T7-AC7/anti-T7
antibody/PAA immunocomplex from the supernatant containing PKC and
[
-32P]ATP. The pellet was washed with 40 mM Tris-HCl, pH 8.0, and the supernatant was precipitated
with trichloroacetic acid. Aliquots from both the pelleted fraction and
the supernatant were dithiothreitol- and
N-ethylmaleimide-treated, boiled in gel loading buffer, and loaded onto an 8% Tris-glycine gel for electrophoretic separation. The
gel was dried and then exposed to x-ray autoradiography film.
or PKC
(2 µg) was 32P-labeled through autophosphorylation for 30 min at 30 °C in the presence of 10 µCi of
[
-32P]ATP (3000 Ci/mmol from PerkinElmer Life
Sciences; final ATP concentration equaled 40 µM) in
phosphorylation buffer. After this reaction, the
32P-labeled PKC was trichloroacetic acid-precipitated
(10%) on ice, and the free 32P was removed with three
washes in 95% ice-cold ethanol. 32P-Labeled PKC was
resuspended at 100 ng/µl in 50% glycerol buffer (100 mM
NaCl, 2 mM EGTA, 2 mM EDTA, 0.05% Triton
X-100, and 5 mM TCEP adjusted to pH 7.8 with HEPES).
-glycerophosphate, 1.2% Nonidet P-40, 0.5 µg/ml
-globulin free BSA, and protease inhibitors as described above). The
membrane protein mixture was then added to washed PAA beads for 5 min
at 4 °C to remove the nonspecific binding proteins. The beads were
discarded, and the supernatant was incubated overnight at 4 °C with
4 µg of anti-T7 antibody. The solution containing the
T7-AC7·anti-T7 immunocomplex was added to washed PAA beads and
incubated for 120 min at 4 °C. The beads containing the
immunocomplex were pelleted and washed once in IP binding buffer A to
remove residual SDS. The beads were then resuspended in IP binding
buffer A, and 100 ng of 32P-PKC
or
was added and
incubated at 4 °C for 120 min. The beads containing the
immunocomplex and bound 32P-PKC were pelleted and washed
twice in IP binding buffer A. The supernatant and washes were combined
and counted along with the pellets. 32P-PKC binding to PAA
beads in the absence of T7 antibody or membrane protein was used for
determination of nonspecific binding of 32P-PKC and was
subtracted as background from the pelleted samples.
PKC antibody, 1:5000 dilution of anti-
-actin antibody, or the other appropriately diluted isoform specific PKC antibodies (PKC
, 1:750; PKC
, 1:5000; PKC
, 1:750; PKC
, 1:2500; PKC
, 1:750; PKC
,
1:750; PKC
, 1:1000; and PKC
, 1:750). The blot was then quickly
rinsed with fresh 3% NFDM-TBST and washed three times with 0.1%
liquid fish gelatin-TBS, 0.1% Tween 20, and 0.1% Triton X-100 at
22 °C. The blot was then incubated with 1:10,000 dilution of goat
anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody in
3% NFDM-TBST for 1 h at 22 °C and then washed three times with
0.1% fish gelatin in TBS, 0.1% Tween 20 at 22 °C. The blots
were rinsed in TBS prior to immunoreactive band detection using
enhanced chemiluminescence (Renaissance, PerkinElmer Life Sciences).
Rmin)/(Rmax
r) × (380 min/380 max) using 224 nM as the
Kd for the calcium complex of Fura-2 at 37 °C
(23). The Rmax was determined in the presence of
0.1% Triton X-100, and the Rmin was determined
in the presence of Ca2+ free buffer containing 3 mM EGTA. All of the traces represent individual
experiments, but all of the determinations were repeated at least on
two separate occasions. The reagents were added in volumes of 2-20
µl to give the final concentrations noted in the text and figure legends.
20 °C) chloroform was added to the virus suspension
followed by sonication for 10 min on ice at the interphase to form a
viscous emulsion. The emulsion was separated by a 15-min centrifugation
(setting #6 on IEC clinical centrifuge), and the upper aqueous phase
was isolated from the lower chloroform phase. The recovered aqueous
phase was added to an equal volume of prechilled (
20 °C)
chloroform. The mixture was sonicated (~5 min on ice) at the
interphase until viscous. The upper aqueous phase was again separated
by centrifugation and then overlaid on a two-step CsCl gradient
consisting of 1.25 and 1.4 g/ml CsCl. The virus was banded by
centrifugation at 95,000 × g for 2 h in a Beckman
L-90K Ultrafuge. The white virion band was collected at the gradient
interface (lower band; the top band is empty capsids) by sterile side
puncture. The virion band was dialyzed twice for periods of 1 h
each against 500 ml of virion dialysis buffer (10 mM HEPES,
pH 8.0, 1 mM EDTA, in phosphate-buffered saline) in a
10-kDa molecular weight cut-off slide-A-lyzer (Pierce) and then
dialyzed against virion dialysis buffer in 50% glycerol for 1 h
and stored at
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
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Fig. 1.
Phosphorylation of AC7 by PKC.
A, T7-tagged AC7 or T7 control transfected Sf9 cells
were solubilized, immunoprecipitated using the T7 monoclonal antibody,
and then phosphorylated in vitro by the catalytic subunit of
PKC. A, left panel, the conjugates were washed
(sup), and the immunoprecipitated proteins (IP)
were then separated by SDS-PAGE and subjected to autoradiography.
Right panel, identification of the immunoprecipitated AC7
protein from infected Sf9 cells was accomplished by Western blot
analysis using the T7 monoclonal antibody to the N-terminal T7 tag.
B, to assess cellular phosphorylation of AC7 by PKC,
back-phosphorylation was performed. T7-tagged AC7 or T7 control
transfected HEK 293 cells were pretreated for 5 min with
PGE1 (10 µM) or PGE1 in
combination with 100 mM EtOH or 200 mM EtOH
prior to solubilization and immunoprecipitation. B,
left panel, shown is an autoradiograph of AC7 from
T7-AC7-transfected HEK 293 cells back-phosphorylated in
vitro by the catalytic subunit of PKC. The extent of
back-phosphorylation of AC7 from T7-AC7-transfected HEK 293 cells was
assessed in three separate experiments by densitometry. B,
right panel, PKC-specific intracellular phosphorylation of
AC7 with different treatments was determined by calculating a ratio of
the densitometric measurements of in vitro
back-phosphorylation of AC7 by PKC (mean ± S.E.,
n = 3). The densitometric measurement for the in
vitro back-phosphorylation of control (EtOH untreated) AC7 extract
was 2.62 ± 0.36 units. This was set to a value of 1.0 on the
graph, and the EtOH-treated cell values were expressed as ratios to the
untreated control. The AC7 protein between lanes was normalized to the
heavy chain of the T7 primary antibody.
To determine whether AC7 could be phosphorylated by PKC within live cells, the back-phosphorylation of AC7 after various treatments of transfected cells was assessed. The back-phosphorylation method allows for the quantitation of the remaining phosphorylation sites that can be phosphorylated by a particular kinase in vitro after endogenous phosphorylation within live cells has occurred (24). If a treatment of live cells increases the PKC-mediated phosphorylation of AC7, then the subsequent in vitro incorporation of 32P, catalyzed by exogenous PKC, will be diminished because a number of PKC-sensitive sites will have already been phosphorylated within the live cell. As shown in the autoradiogram in Fig. 1B, less 32P-labeled phosphate was incorporated into AC7 (identified by immunoblotting) during the in vitro PKC phosphorylation under conditions where live cells were pretreated with PGE1 (10 µM) and ethanol (100 or 200 mM) compared with assays in which cells were treated only with PGE1. The extent of back-phosphorylation of AC7 harvested from T7-AC7-transfected HEK 293 cells was assessed in three separate experiments (including the representative autoradiograph shown in Fig. 1B). The densitometric measurements of these experiments were combined to calculate the intracellular PKC-sensitive phosphorylation of AC7 after exposure to ethanol. Using the ratio of back-phosphorylation units (densitometric analysis of 32P-incorporation) for AC7 from untreated HEK 293 cells divided by the back-phosphorylation units for AC7 from ethanol-treated cells, we determined that the addition of 100 mM ethanol produced a back-phosphorylation ratio of 1.44 ± 0.31 and that the presence of 200 mM ethanol increased this ratio to 2.24 ± 0.47 (Fig. 1B).
Because we also wished to investigate the actions of PKC and ethanol on
AC7 in nontransfected cell systems, we chose to use the HEL cells from
which we have previously isolated AC7 mRNA (17). The
Ca2+-sensitive adenylyl cyclases (cyclases 1, 5, 6, and 8)
have been shown to be regulated by store-operated Ca2+
influx (25). To induce store-operated Ca2+ influx, HEL
cells were depleted of intracellular Ca2+ with 100 nM thapsigargin (15 min of pretreatment) in the absence of
external Ca2+. External Ca2+ (5 mM)
was then added simultaneously with 10 µM
PGE1, and the cAMP accumulation was measured for 5 min.
Fig. 2A shows that the cAMP
accumulation in the presence of 10 µM PGE1 in
normal calcium containing media (0.9 mM Ca2+)
did not differ from the cAMP accumulation under conditions where store-operated Ca2+ influx was induced, indicating that HEL
cells have little or no Ca2+-regulated AC activity. In
contrast, a 30-min pretreatment with the non-isoform-selective PKC
inhibitor chelerythrine, at 20 µM, significantly reduced
the PGE1-induced cAMP accumulation (Fig. 2A).
When HEL cells were pretreated for 30 min with increasing concentrations of chelerythrine prior to the addition of 10 µM PGE1, both the ethanol (200 mM) and PDBu (100 nM) enhancement of
PGE1-stimulated AC activity in HEL cells was inhibited in a dose-dependent fashion (Fig. 2B). The dose
response of inhibition by chelerythrine did not demonstrate any
preferential sensitivity between ethanol and PDBu effects, and, at 20 µM, chelerythrine almost completely inhibited the
potentiation by either ethanol or PDBu. This AC activity data, along
with our previous results based on the measurements of AC mRNA in
HEL cells (17) suggests that the predominant AC activity in HEL cells
is an AC that is nonresponsive to changes in intracellular
Ca2+ but is sensitive to ethanol in a
PKC-dependent manner.
|
Using the C6C anti-AC antibody, an immunoreactive band in the range of 110 kDa was identified in the membrane fraction of HEL cells that corresponded in size to an immunoreactive band from T7-tagged AC7-infected Sf9 cells (data not shown) and the T7-tagged AC7-transfected HEK 293 cells. In addition, both the anti-T7 antibody and the anti-AC antibody recognized the same band on immunoblots for both the Sf9 (data not shown) and HEK 293 cell-derived T7-tagged AC7 (see Fig. 8A). The C6C type II family anti-AC monoclonal antibody was produced to recognize a region in the C2 domain of the type II family of ACs: AC2, AC4, and AC7 (21). Because we have previously demonstrated that HEL cells lack mRNA for AC2 and 4 (17), one can conjecture that the major AC in HEL cells is AC7.
To differentiate which PKC isotypes might be present in HEL cells and
thus better focus our search for the particular PKC involved in the
potentiation by ethanol, we used PKC isotype-specific antibodies. The
results from the Western blot analysis shown in Fig.
3 reveal that HEL cells express ,
,
, µ,
, and
(
) PKCs. The
and
isotypes were not
expressed at detectable levels in HEL cells, and only after very long
exposures did a faint band in the molecular mass range for PKC
appear (data not shown). We did not test for the presence of the PKC
isotype.
|
PKC inhibitors and biomolecular reagents were next employed to
differentiate which PKC isotype(s) might be involved in the potentiation of the endogenous AC7 in HEL cells by PDBu or ethanol. The
Ca2+ sensitivity of the conventional PKCs (,
and
) is well documented, and their activation and catalytic activity
can be greatly attenuated by preventing increases in Ca2+
influx or by preventing Ca2+ release from intracellular
stores. Preincubation with 1 mM EGTA (to reduce free
extracellular Ca2+ to ~50 nM) had no
significant effect on the potentiation of AC activity by either 200 mM ethanol or 100 nM PDBu in the presence of 5 µM PGE1 (Fig.
4A). Ethanol enhanced AC
activity by 68 ± 10 and 75 ± 16% in the absence and
presence, respectively, of 1 mM EGTA in the medium.
Chelating extracellular Ca2+ prevents Ca2+
influx but does not exclude the possibility that PGE1, in
addition to activating Gs, could also be activating a
Gq-coupled receptor (26) leading to subsequent
phosphatidylinositol 4,5-bisphosphate turnover and release of
internally stored Ca2+. To eliminate the possibility that
internally released Ca2+ might be activating conventional
PKCs, HEL cells were pretreated with 100 nM thapsigargin, a
sarcoendoplasmic reticulum Ca2+-ATPase inhibitor that
depletes inositol 1,4,5-triphosphate-sensitive Ca2+ stores.
In the presence of 1 mM external EGTA alone, 5 µM PGE1 transiently raised intracellular
Ca2+ to 309 ± 22 nM from a basal level of
106 ± 15 nM (Fig. 4B). However, the
pretreatment of HEL cells with 100 nM thapsigargin and 1 mM EGTA was sufficient to prevent any
PGE1-induced rise in [Ca2+]i (Fig.
4B). As shown in Fig. 4A, depleting the
intracellular Ca2+ stores prior to PGE1
stimulation had no effect on the potentiation of AC activity by either
200 mM ethanol or 100 nM PDBu. Ethanol enhanced
AC activity by 68 ± 10 and 60 ± 22% in the absence and presence of thapsigargin and EGTA, respectively.
|
Gö 6976 has been shown to selectively inhibit the conventional
PKCs and PKCµ (27). Rottlerin, on the other hand, has been shown to
selectively inhibit PKC and
(28, 29). Pretreating HEL cells for
30 min with increasing concentrations of Gö 6976 had little or no
effect on the potentiation of PGE1-stimulated AC activity by either PDBu or ethanol (Fig.
5A). However, when HEL cells
were pretreated for 30 min with rottlerin, the potentiation of AC
activity by both ethanol and PDBu was potently inhibited in a
dose-dependent fashion (Fig. 5B). At 2.5 µM rottlerin, the potentiation by ethanol was reduced
from 83 ± 10 to 28 ± 7%, and the potentiation by PDBu was
reduced from 112 ± 11 to 59 ± 12%. In the absence of
ethanol or PDBu, concentrations of rottlerin above 2.5 µM
significantly inhibited the PGE1-stimulated AC activity in
HEL cells.
|
Peptides containing specific sequences within the N-terminal V1 regions
of PKCs, which are unique and not conserved across the PKC family, have
been recently used to competitively inhibit the binding of PKCs to
scaffolding proteins called receptors for activated C kinases (RACK)
(30). These PKC-derived peptides have been shown to be PKC
isotype-specific (31). Dr. Daria Mochly-Rosen provided us with an
octapeptide (V1-1) from the V1 region of PKC
that has been shown
to be a selective inhibitor of PKC
localization and does not
cross-react with other PKCs (18, 32, 33). The PKC-derived peptides have
been conjugated via a disulfide linkage to a 16-amino acid antennapedia
signal peptide from Drosophila that enables the chimeric
peptides to translocate across the plasma membrane and after reduction
of the disulfide bond be trapped within the cell (34). When HEL cells
were pretreated with either PKC-derived inhibitory peptide or a control
peptide at 2 µM for 2 h prior to the stimulation by
PGE1, the potentiation of AC activity by 100 mM
ethanol was selectively attenuated by ~50% by the PKC-derived inhibitory peptide,
V1-1, which is selective for PKC
(18, 32,
33). In contrast neither the inhibitory peptide (
V1-2) selective
for PKC
, which is another member of the novel PKC family, nor the
control nonsense octapeptide had any effect (Fig.
6). The Student's t test was
used for pairwise comparisons and indicated that the PKC
peptide
significantly reduced the ethanol potentiation of
PGE1-stimulated AC activity, from 68 ± 16 to 36 ± 3% (*, p < 0.05), whereas the PKC
peptide, and
control peptide had no significant effect on the potentiation by
ethanol, 62 ± 11 and 68 ± 16%, respectively. The PKC
peptide also reduced the PDBu potentiation compared with control
(p < 0.05), whereas the PKC
peptide and the control
nonsense octapeptide were without effect.
|
Overexpression of the dominant negative, catalytically inactive forms
of PKC have previously been shown to be selective inhibitors of a
targeted PKC isotype by competing with the endogenous PKC for
cofactors, substrates, and cellular binding sites (35, 36). The
dominant negative construct for PKC has a lysine mutated to a
methionine within the ATP-binding site, making it catalytically inactive (36). HEL cells were infected with a replication deficient adenovirus (Ad5 DL312) carrying either the wild-type (WT) or dominant negative (DN) form of PKC
at two different adenovirus titers, 10 × 103 or 50 × 103
particles/cell. When normalized to
-actin levels, HEL cells infected
with the DN-PKC
adenovirus demonstrated a 174% increase in PKC
immunoreactive protein, representing the expression of the dominant
negative mutant PKC
in addition to the endogenous expression of
wild-type PKC
(Fig. 7A).
When HEL cells were assayed for AC activity 20 h after viral
infection, the potentiation of PGE1-stimulated AC activity
by 100 mM ethanol was no longer statistically significant
in cells infected with the DN-PKC
adenovirus, at either virus titer.
At the highest virus titer of 50 × 103
particles/cell, the potentiation of ethanol was reduced to 16 ± 7% in cells pretreated with DN-PKC
as compared with 52 ± 18% in cells infected with the WT-PKC
adenovirus (Fig. 7B).
The potentiation of AC activity by ethanol in cells infected with the
WT-PKC
was comparable with that of either HEL cells infected with a
control adenovirus carrying lacZ or uninfected HEL cells
under the same conditions, 52 ± 18% versus 44 ± 4% versus 41 ± 6%, respectively. Two-way analysis of
variance indicated a significant difference between DN and WT
expressing cells (F(1,28) = 5.748, p < 0.023) and between the treatments (ethanol versus no
ethanol; F(2,28) = 8.222, p < 0.002).
There was no statistically significant interaction between virus
exposure and treatments (F(2,28) = 1.507, p > 0.239). The post-hoc Tukey-test also revealed a
significant enhancement of PGE1-stimulated cAMP
accumulation in the presence of ethanol for only WT-treated cells (*,
p < 0.05 compared with PGE1 stimulation alone).
|
Because the above described data demonstrated that the ethanol
potentiation of AC activity in HEL cells was dependent on PKC, we
next determined whether AC7 was a substrate for PKC
in
vitro. Western blot analysis of the immunoprecipitated fractions
of solubilized, AC7-transfected HEK 293 cell membranes confirmed that
the T7-tagged AC7 was successfully expressed in these cells and that
T7-tagged AC7 protein bands could be detected at ~110 kDa (Fig.
8A). No bands were detected in
HEK 293 cells that were transfected with the control
pcDNA3.1-T7.His in this molecular mass range (Fig. 8A,
lanes 1 and 4). Cell membranes from
PGE1-stimulated AC7-transfected HEK 293 cells were then
prepared under conditions that included phosphatase inhibitors. The
extracted T7-tagged AC7 was then subjected to immunoprecipitation and
to phosphorylation by exogenously added full-length recombinant PKC
or PKC
. Shown in Fig. 8B is an autoradiogram of
immunoprecipitated T7-tagged AC7 from the transfected HEK 293 cells.
Fig. 8 demonstrates a preferential phosphorylation of AC7 by PKC
.
The arrow indicates 32P incorporation within a
protein band running at ~110 kDa that is unique to the T7-AC7
immunoprecipitate (lanes 2p and 5p). Longer exposures revealed a faint 110-kDa band in lane 5p. The
darker bands that appear below AC7 are residual amounts of the
autophosphorylated forms of PKC
(molecular mass = 77 kDa) or
(molecular mass = 88 kDa). No bands were evident, in the
110-kDa molecular mass range, at any length of exposure in the control
lanes where either the T7-tagged AC7 (lanes 1p and
4p) or the anti-T7 antibody (lanes 3p and
6p) was not present. Parallel Western blots using
the anti-T7 monoclonal antibody gave further evidence that the
phosphorylated band at 110 kDa corresponded to AC7.
|
Prior sequence analysis of AC7 suggested that a possible PKC-binding
site (see "Discussion") exists within the AC7 structure. Using
32P-labeled PKC
or
as a probe, we were able to show
an increased immunoprecipitation of 32P-PKC
together
with the T7-tagged AC7 when the anti-T7 antibody was used to
immunoprecipitate the protein complex. In contrast, we could not detect
an interaction between 32P-PKC
and the T7-tagged AC7
under our experimental conditions (Table
I).
|
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DISCUSSION |
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To date only AC2, AC5, and Ac6 have been shown to be
directly phosphorylated by PKC (37-40). The potentiation of
Gs-stimulated AC2 activity and the enhancement of the
basal activity of this enzyme by phorbol esters and PKC itself has been
the most extensively investigated (37, 38, 41). PKC
was shown to
markedly enhance the in vitro phosphorylation and the
sensitivity of AC2 to G
s stimulation, whereas PKC
reduced the sensitivity of AC4 to G
s (38). Although AC2
has been shown to be phosphorylated on Thr1047 after
phorbol ester treatment (37) and this post-translational modification
has been indicated to alter AC2 responsiveness to Gs
(38), little evidence currently exists 1) demonstrating that another
member of the type II AC family (i.e. AC7) is covalently modified by PKC-mediated phosphorylation, 2) demonstrating the resultant effects of such a phosphorylation on
Gs
-stimulated AC7 activity, and 3) as to whether a
particular isoform(s) of PKC may dominate the phosphorylation of AC7
and mediate the stimulatory effects of ethanol or PDBu on AC7 activity
in cells.
Our data show that immunoprecipitated AC7, overexpressed in Sf9 cells, is phosphorylated in vitro by a mixture of PKCs isolated from rat brain. In addition, the back-phosphorylation experiments demonstrated an increase in a PKC-specific phosphorylation of AC7 within HEK 293 cells after exposure of the intact cells to ethanol. To elucidate which PKC isozyme(s) were mediating the effects of ethanol, we utilized pharmacologic and intracellularly expressed inhibitors of PKC.
A role for the involvement of the atypical PKCs, which are not
sensitive to either Ca2+ or diacylglycerol/phorbol
esters, in the actions of ethanol was previously eliminated, because
down-regulation of conventional and novel PKCs by prolonged treatment
of HEL cells with phorbol esters blocked the effect of ethanol on HEL
cell AC activity (8). The involvement of Ca2+-sensitive
PKCs (conventional PKCs) was eliminated in the current work by showing
that depletion of intracellular Ca2+ and prevention of
Ca2+ influx into the cells did not alter the ability of
ethanol to increase agonist-stimulated AC7 activity (Fig. 4). This
conclusion was supported by the ineffectiveness of a selective
inhibitor of the conventional PKCs, Gö 6976 (27), to modulate the
potentiation of AC activity in HEL cells produced by ethanol or PDBu
(Fig. 5A). On the other hand, rottlerin, which is a
selective inhibitor of the novel PKC isoforms and
(28, 29),
significantly reduced the potentiation of PGE1-stimulated
AC activity in HEL cells by ethanol (Fig. 5B). Examination
of the dose-response curves for rottlerin for the inhibition of the
effects of PDBu and ethanol on AC7 activity revealed that higher
concentrations of rottlerin were necessary to produce the initial
inhibition of the actions of ethanol (Fig. 5B). The
differential effects of rottlerin on PDBu and the actions of ethanol
may well be due to the location of the pools of PKC
engaged by PDBU
and by ethanol to carry out the post-translational modification of AC7.
Ethanol may be acting on PKC
, which is already in the vicinity of
(bound to) AC7 (see below), whereas PDBu is activating and
translocating an intracellular pool of PKC
to accomplish its effect
on AC7 activity.
To further examine the involvement of PKC, additional molecular
reagents were used. The dominant negative form of PKC
is catalytically inactive but can compete with the endogenous PKC
for
substrates or cofactors (35, 36). On the other hand, the N-terminal V1
fragment of PKC
and the related
V1-1 octapeptide can selectively
block the proper localization and phorbol ester-induced translocation
of PKC
(18, 31-33, 42). We demonstrated that the effect of ethanol
on agonist-stimulated AC7 activity was blocked by either the dominant
negative form of PKC
(Fig. 7) or a PKC
-specific peptide (
V1-1)
derived from the N-terminal V1 region of PKC
(Fig. 6). In contrast,
the corresponding PKC
-specific peptide (
V1-2) (18, 31) and the
control nonsense octapeptide were without effect (Fig. 6).
Although it is has been thought that the PKC family shows little
sequence specificity toward its substrates, recent studies have
suggested that PKC tends to favor acidic residues downstream of the
S/T phosphorylation site, whereas PKC
tends to favor hydrophobic residues (43). The suggestive substrate selectivity of PKC
over that
of PKC
for AC7 in Fig. 8 is not without precedence. Both the high
affinity receptor for IgE and the elongation factor eEF-1
proteins
have been shown to be selectively phosphorylated by PKC
compared
with PKC
(44, 45).
Our experimental data in Table I also suggest a selective association
between AC7 and PKC under our experimental conditions. Examples of
selective binding of PKC
to other proteins have been demonstrated
(46-48). Many of these PKC-protein associations do not fit the defined
interaction of a PKC with a RACK protein. In cases where a RACK protein
is involved, PKC binds only after PKC is activated and does not
phosphorylate the RACK. Nevertheless, an interaction between the
substrate protein, GAP-43, and the N-terminal V1 region of PKC
has
been well demonstrated (47), as has the binding of the SRBC protein to
PKC
(46).
An examination of the AC7 sequence illustrates a possible binding site
for PKC that is not present in the sequences of the other ACs. When
putative PKC binding sites on AKAP 79, AKAP 250 (gravin), and other
PKC-binding proteins were aligned with full-length AC7, using the
ClustalW 1.60 multiple sequence alignment algorithm set with gap
penalties, in all cases, a contiguous sequence was best fit to the
C1b region of AC7. Furthermore, a region of the SRBC
protein, which has been shown to bind to and be phosphorylated by
PKC
(46), also aligned to the C1b domain of AC7. The following sequence from the C1b region of human AC7
491GAARPFAHLNHRESVSSGETHVPNGRRPKSVPQRHRRTPDRSMSPKGRSE541
contained the greatest degree of alignment with the putative PKC
-binding region of the SRBC protein. Two putative PKC
phosphorylation sites unique to AC7 and conserved within AC7 across
different species (mouse, cow, and human) are indicated by the residues italicized and underlined. Three additional putative PKC
phosphorylation sites are located just outside this region.
Our demonstration of the involvement of PKC in the phosphorylation
of AC7 and modulation of ethanol-potentiated AC7 activity coincides
with other studies that have implicated the novel PKCs in a number of
both acute and chronic effects of ethanol. Chronic ethanol exposure was
found to up-regulate the density and function of L-type
Ca2+ channels via a PKC
-dependent mechanism
(42), whereas short term ethanol exposure of NG108-15 cells was found
to alter the subcellular localization and presumably the function of
PKC
and
(49).
Whereas the exact mechanism or mechanisms by which ethanol utilizes
PKC to alter AC7 activity remains unknown, one possibility would be
that ethanol is modulating the interaction between and colocalization
of AC7 and PKC
. Ethanol could promote a conformational change in AC7
that provides or enhances availability of a site(s) for PKC-mediated
phosphorylation. This phosphorylation event could then act as an
electrostatic switch for regulating enzymatic activity and/or
regulating protein-protein (AC/G
s) or protein-lipid
interactions. The work presented here demonstrates a role for
PKC-induced phosphorylation in mediating the effects of ethanol on AC7,
and quite possibly this observation will be expanded to include other
neuronal signaling proteins in the future.
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ACKNOWLEDGEMENTS |
---|
We thank Sabine Lazis and Josphine Tsao for technical assistance and Dr. Jerry Schaak (University of Colorado Health Sciences Center, Denver, CO) for assistance in the use and propagation of the adenovirus.
![]() |
FOOTNOTES |
---|
* This work was supported by the Banbury Fund and by National Institute on Alcohol Abuse and Alcoholism Grant AA 9014.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: Dept. of Pharmacology,
UCHSC School of Medicine, 4200 East 9th Ave., C236, Denver,
CO 80262. Tel.: 303-315-3125; Fax: 303-315-0708; E-mail:
boris.tabakoff@uchsc.edu.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M210386200
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; BSA, bovine serum albumin; AC, adenylyl cyclase; AC7, adenylyl cyclase type 7; HEL, human erythroleukemia cells; HEK 293, human embryonic kidney cells; NFDM, nonfat dairy milk; RACK, receptor for activated C-kinase; PDBu, phorbol 12,13-dibutyrate; PGE1, prostaglandin E1; Sf9, Spodoptera frugiperda ovarian cells; EtOH, ethanol; IP, immunoprecipitation; PAA, protein A-agarose; TBS, Tris-buffered saline; WT, wild-type; DN, dominant negative.
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