From the
The changing relationship between stimuli and responses after
prolonged receptor stimulation is a general feature of hormonal
signaling systems, termed desensitization. This phenomenon has been
best exemplified in the covalent modification of the G protein-linked
catecholamine receptors. However, other components within this
signaling pathway can be involved in desensitization. Here we present
evidence that desensitization occurs at the level of the effector
enzyme itself through phosphorylation. Type V adenylyl cyclase (AC) is
the major isoform expressed in the heart. Using purified enzymes, we
demonstrate that protein kinase A (PKA) directly phosphorylates and
thereby inhibits type V AC catalytic activity. This inhibition was
negated in the presence of PKA inhibitor. Analysis of enzyme kinetics
revealed that this inhibition was due to a decrease in the catalytic
rate, not to a decrease in the affinity for the substrate ATP. Our
results indicate that AC catalytic activity can be regulated through
PKA-mediated phosphorylation, suggesting another mechanism of
desensitization for receptor pathways which signal via increases in
intracellular cAMP.
Occupation of cell surface catecholamine receptors by
norepinephrine released from the synaptic terminal evokes signaling via
the stimulatory GTP regulatory protein, G
Besides initiating this downstream signaling cascade, PKA
also phosphorylates proteins located upstream of this signaling
pathway, i.e. catecholamine receptors, thereby mediating their
desensitization
(1) . Phosphorylation of the cytoplasmic domain
of the receptor makes the molecule less efficient in coupling to the G
protein. The receptor is also phosphorylated and uncoupled by another
kinase, termed
More
recently, we have demonstrated that AC itself can also be regulated by
phosphorylation
(2) . This occurs through protein kinase C (PKC)
in an isoenzyme-specific manner, and actually leads to an enhancement
of catalytic activity. In contrast, the exact role of AC modification
in the process of desensitization remains unknown. Amino acid sequence
analysis of AC reveals the presence of multiple putative PKA-sensitive
motifs within this molecule
(3) . However, PKA-mediated
phosphorylation of the AC molecule itself and consequent changes in
catalytic activity have not been clearly demonstrated; these were the
goals of the present study.
The present study demonstrates that PKA directly
phosphorylates and inactivates type V AC. Inactivation of type V AC
results from a decreased catalytic rate, not from a decreased affinity
for the substrate ATP. The sites of phosphorylation by PKA, as assessed
by phosphopeptide mapping, were different from those phosphorylated by
PKC. The degree of inhibition was more prominent when AC was stimulated
with forskolin or with G
Catecholamine-mediated desensitization
has been attributed to a variety of mechanisms, particularly,
uncoupling of the
While the vast majority of prior
studies of desensitization have focused at the level of the
receptor
(11, 12) , alteration of other components within
the
The recent cloning of multiple mammalian ACs has finally
allowed structure-function studies of this relatively poorly
characterized component of the cAMP signaling pathway to be carried
out
(17) . We had identified an AC isoform from a canine cardiac
cDNA library, designated as type V
(18) . The expression of this
isoform is restricted to the heart and brain. Interestingly, type V AC
is the major isoform in the adult heart where catecholamine-mediated
desensitization has been extensively investigated over the past decade
in various pathophysiological conditions
(19, 20) . We
have more recently reported that type V AC is potently regulated
through PKC-mediated phosphorylation
(2) . The two PKC
isoenzymes, PKC
PKA-mediated inactivation of AC creates a feedback system within the
cAMP signaling pathway, which is analogous to PKC-mediated inhibition
of the phospholipase C pathway
(23, 24) . Catecholamine
stimulation in the heart activates both the phospholipase C/PKC pathway
via
We thank Dr. R. Iyengar (Mount Sinai Medical School)
for helpful discussion.
Note Added in Proof-A similar
PKA-mediate inhibition of AC was found in type VI (R. Iyengar, personal
communication).
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, which in turn
activates adenylyl cyclase (AC).
(
)
AC is a
membrane-bound enzyme that catalyzes the conversion of ATP to cyclic
AMP. Cyclic AMP, an intracellular second messenger, activates protein
kinase A (PKA), which initiates an enzymatic cascade of phosphorylation
reactions within the cell. Examples are various enzymes involved in
myocyte contraction in the heart and those involved in glycolysis in
the liver.
adrenergic receptor kinase (BARK). A unique
feature of BARK is that it only phosphorylates the agonist-bound form
of the receptor. BARK presumably recognizes changes in the tertiary
structure of the receptor caused by ligand binding, thus
phosphorylating only the activated form of the receptor. Nevertheless,
in both cases receptor phosphorylation occurs only when the signaling
pathway is activated; it is triggered either by agonist occupation of
the receptor or by cyclic AMP formation as a result of receptor
activation, thus forming a closed loop of negative feedback.
Overexpression and Purification of Type V
AC
Plasmid construction and purification of the recombinant
hexa-histidine tagged type V AC were performed as described
previously
(2) . Briefly, High Five insect cells (1
10
cells) were infected with the recombinant baculovirus
and harvested 60-70 h after infection. Cells were lysed by
nitrogen cavitation at 800 p.s.i. for 30 min at 4 °C. Nuclei were
removed by centrifugation at 500
g for 10 min.
Membranes were harvested by centrifugation at 150,000
g at 4 °C for 40 min, followed by suspension in buffer A (20
mM Hepes (pH 8.0), 20% glycerol, 400 mM NaCl, 2
mM MgCl
, 1 mM EDTA, 2 mM
-mercaptoethanol) with protease inhibitors, and were recentrifuged
at 150,000
g at 4 °C for 30 min. The membranes
were then resuspended in buffer A with 0.8% dodecyl maltoside. After
incubation and centrifugation at 150,000
g for 30 min,
the supernatant was further incubated with forskolin-CH Sepharose 4B
(Pharmacia Biotech Inc.) for 16 h at 4 °C. After washing, AC was
eluted with 200 µM forskolin and 0.2% dodecyl maltoside in
buffer A. The eluate was further incubated with nickel-nitrilotriacetic
acid resin (Qiagen, CA) at 4 °C for 30 min. After washing, the
enzyme was eluted with buffer A containing 0.1% dodecyl maltoside and
100 mM imidazole. The eluate was buffer changed and
concentrated using Centricon-100 (Amicon, MA) and stored at -80
°C until use.
Phosphorylation of Type V AC by PKA
Type V AC was
incubated in the presence or absence of forskolin (100 µM)
or GTPS-G
in a buffer containing 50 mM
Hepes (pH 8.0), 5 mM MgSO
, 1 mM EDTA, and
1 mM dithiothreitol at 30 °C for 10 min. Various amounts
of PKA catalytic subunit (PKA-CS) (Sigma) were then added in a buffer
containing 20 mM Hepes (pH 8.0), 10 mM
MgCl
, 1 mM dithiothreitol, 0.2 mM ATP, 1
mM creatine phosphate, 8 units/ml phosphocreatine kinase in
the absence or presence of PKA regulatory subunit (PKA-RS) (Sigma) at
25 °C for 10 min. Cyclic AMP production, as a measurement of AC
catalytic activity, was measured at 30 °C for 10 min as described
previously
(4) . Briefly, the mixture was assayed in a solution
containing 1 mM creatine phosphate, 8 units/ml creatine
phosphokinase, 20 mM Hepes (pH 8.0), 5 mM
MgCl
, 0.1 mM cAMP, 0.2 mM ATP, and
[
-
P]ATP (0.2-5 mCi/assay tube)
followed by the addition of 100 µl of 2% sodium dodecyl sulfate.
[
H]cAMP was used as an internal standard to
measure overall recovery. Protein concentration was determined by
staining with Amido Black
(5) .
PKA Assay
Phosphorylation was monitored using a
PKA assay system (Life Technologies, Inc.) with Kemptide as substrate.
PKA-CS was incubated in 20 mM Hepes (pH 8.0), 10 mM
MgCl, 1 mM dithiothreitol, 0.2 mM ATP, 1
mM creatine phosphate, 8 units/ml phosphocreatine kinase, 0.25
mg/ml bovine serum albumin, [
-
P]ATP
(12-24 mCi/assay tube) with 50 mM Kemptide as substrate
at 30 °C for 10 min. The reaction was quenched by spotting the
sample mixture onto a phosphocellulose disc and the incorporation of
P into Kemptide from [
-
P]ATP
was measured using scintillation counting.
Phosphopeptide Mapping of Type V AC
Purified type
V AC was phosphorylated either with 6 units/ml PKC or with 200
units/ml PKA-CS in the presence of [
-
P]ATP
as described above and previously
(2) . After separation on 8%
SDS-PAGE, the phosphorylated type V AC was excised and rehydrated as
described
(6) . The phosphorylated protein was further digested
at 37 °C for 18 h in a buffer containing 50 mM
(NH
)
CO
and 0.3 mg/ml
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Sigma), followed by lyophilization. The trypsin digests were
resuspended in a loading buffer containing 1 M acetic acid,
0.05 M NH
OH, 6 M urea, 5% glycerol, and
applied to an acidic 45% polyacrylamide gel designed for separating
small peptides. The limit of resolution of this acidic gel
electrophoresis is approximately 100 daltons
(7) . After
electrophoresis, the gel was dried and autoradiography was carried out.
PKA Inactivates Type V AC Catalytic Activity
As
shown in Fig. 1, incubation of purified type V AC with PKA
resulted in a decrease in both basal (85 ± 3% of control, mean
± S.E., p < 0.05, n = 4) and
stimulated (with GTPS-G
, 72 ± 3%; with
forskolin, 57 ± 3%, p < 0.01, n = 4)
AC catalytic activity. The two different preparations of PKA-CS, one
partially purified from cardiac tissue (Sigma) and the other purified
from Escherichia coli overexpressing recombinant PKA-CS
(Upstate Biotechnology Inc., Lake Placid, NY), both produced a similar
degree of inhibition. When analyzed on SDS-PAGE, the partially purified
PKA showed a major band at 45 K
, while
that from E. coli showed a single band at the same molecular
weight (data not shown). We also examined whether PKA affects any of
the components in the cyclase assay buffer, such as creatine
phosphokinase. After phosphorylating AC with PKA in the absence of the
regeneration system, we started the cAMP production assay by adding the
hot mixtures and regeneration system in the presence of an excess
amount of PKA inhibitor. However, this did not alter the results (115
± 9 nmol/min/mg in the absence of PKA, 66 ± 9 nmol/min/mg
in the presence of PKA, n = 3, p < 0.05).
This indicates that the inhibition of AC by PKA is not through
affecting the regeneration system within the reaction mixture.
Figure 1:
Effect of protein
kinase A on the adenylyl cyclase catalytic activity. Purified type V AC
was incubated in the presence or absence of various stimulators at 30
°C for 10 min. Various amounts of PKA catalytic subunit
(0-200 units) were then added, followed by incubation at 25
°C for 10 min. Cyclic AMP production, as a measurement of AC
catalytic activity, was measured at 30 °C for 10 min. Open
circles, without any stimulators; closed circles, with
100 µM forskolin; triangles, with
GTPS-G
. The data are means ± S.E. from
four independent experiments. Specific activities in the absence of
PKA-CS were approximately 50 nmol/min
mg (basal), 200
nmol/min
mg (GTP
S-G
), and 250
nmol/min
mg (forskolin). *, p < 0.05;**, p < 0.01 from that in the absence of
PKA-CS.
In
order to examine the specificity of this inactivation, we examined the
effect of PKA-RS, a specific inhibitor of PKA-CS, on PKA-mediated
inhibition of type V AC catalytic activity (Fig. 2). Addition of
PKA-RS negated the inhibition by PKA in a concentration dependent
manner (0-300 units/ml). We also performed a time course study on
this inhibition. Type V AC was incubated in the presence or absence of
PKA. The degree of phosphorylation was time-dependent and paralleled
that of inhibition as assessed by the incorporation of P
into type V AC (Fig. 3).
Figure 2:
Effect of the protein kinase A regulatory
subunits (PKA-RS) on protein kinase A-mediated inhibition of type V
adenylyl cyclase catalytic activity. Forskolin-stimulated (100
µM), purified type V AC was incubated with 100 units/ml
PKA-CS (Sigma) in the presence of increasing concentrations of PKA-RS
(0-300 units/ml) (Sigma). AC catalytic activity was measured as
described under ``Materials and Methods.'' Each point was
determined in duplicate. The data are means ± S.E. of four
independent experiments.
Figure 3:
Time course study on adenylyl cyclase
inhibition by protein kinase A. AC catalytic activity was plotted as a
function of time. Type V AC was incubated in a buffer containing 20
mM Hepes (pH 8.0), 10 mM MgCl, 1
mM dithiothreitol, 1 mM creatine phosphate, 8
units/ml phosphocreatine kinase, 0.1 mM cAMP, 0.2 mM
ATP, and [
-
P]ATP and 100 µM
forskolin in the presence or absence of PKA (200 units/ml) (Sigma) or
PKI (10 units/ml) (New England Biolabs) for 30 min. Small aliquots of
the reaction mixture were removed at 2, 5, 10, 20, and 30 min after the
initiation of the reaction, and were added to 2% SDS to terminate the
reaction. The
P-phosphorylation of AC was similarly
performed except that [
-
P]ATP was used
instead of [
-
P]ATP, followed by SDS-PAGE
and autoradiography. Circles, without PKA and PKI;
squares, with PKA without PKI; triangles, with PKA
and PKI.**, p < 0.01, n =
4.
Enzyme Kinetic Analysis
We then examined whether
this PKA-mediated inactivation of type V AC results from either a
decreased AC catalytic rate (V) or decreased
affinity for the substrate ATP (K
).
Enzyme kinetic analysis using a Lineweaver-Burk plot revealed that this
inhibition was due to a decrease in the catalytic rate
(V
= 410 ± 32 nmol/min
mg
without PKA-mediated phosphorylation, 231 ± 30 nmol/min
mg
with PKA-mediated phosphorylation, p < 0.01, n = 3) (Fig. 4). The K
for ATP was unaltered (38 ± 9 µM without
PKA-mediated phosphorylation, 39 ± 3 µM with
PKA-mediated phosphorylation, p = not significant,
n = 3).
Figure 4:
Kinetic
analysis of type V adenylyl cyclase catalytic activity in the presence
or absence of protein kinase A-mediated phosphorylation. The AC
catalytic activities were measured with 100 µM forskolin
in the presence or absence of 200 units/ml PKA-CS at 30 °C for 30
min in the cAMP assay buffer described under ``Materials and
Methods'' containing different concentrations of ATP.
V, 410 ± 32 nmol/min
mg without
PKA-mediated phosphorylation (open circles); 231 ± 30
nmol/min
mg with PKA-mediated phosphorylation (closed
circles) (p < 0.01). K, 38 ± 9
µM without PKA-mediated phosphorylation; 39 ± 3
µM with PKA-mediated phosphorylation (p =
NS). Means ± S.E. from three independent experiments are
shown.
Phosphopeptide Mapping
We also compared the
pattern of phosphorylation catalyzed by PKA to that of PKC. We had
previously shown that PKC-mediated phosphorylation of type V AC leads
to an enhancement of its catalytic activity
(2) . Type V AC was
phosphorylated in the presence of [-
P]ATP
either with PKA-CS or with PKC
, followed by trypsin digestion. The
digests were analyzed on SDS-PAGE. The phosphopeptide mapping pattern
was then determined by SDS-PAGE and autoradiography. As shown in
Fig. 5
, the sites phosphorylated by the two kinases were
different.
Figure 5:
Phosphopeptide mapping of type V adenylyl
cyclase. Purified type V AC was phosphorylated either with 200 units/ml
PKA-CS, or with 6 units/ml PKC, followed by trypsin digestion and
separation on acidic SDS-PAGE as described under ``Materials and
Methods.'' Similar results were obtained in three independent
experiments.
. However, the basal form of
AC was more heat-unstable, which has made it difficult to interpret
these data. We did not see, on the other hand, gross difference in the
degree and sites of phosphorylation in the phosphopeptide mapping
studies between basal and stimulated AC. PKA-mediated phosphorylation
and inactivation of AC may represent an alternative mechanism for
heterologous desensitization of the G protein-coupled receptor pathways
that lead to cAMP production.
-adrenergic receptor from G
by
phosphorylation
(1) . Indeed, regulation of the efficiency of
receptor coupling to G proteins by phosphorylation is a well accepted
mechanism to explain both homologous (by BARK) and heterologous (by
PKA) desensitization. Desensitization may also result from
sequestration and down-regulation of receptors. Multiple approaches
have been utilized to study the role of kinases involved in
desensitization of the
-adrenoreceptor/cAMP signaling pathway. It
was first demonstrated that in vitro phosphorylation of the
purified receptor by the two kinases, PKA and BARK, diminishes receptor
function as measured by GTPase activity
(8) . Thereafter,
utilizing a series of receptor mutants in which the phosphorylation
sites had been removed, the same group demonstrated that these mutant
receptors bound ligand and activated AC normally, but underwent
markedly less agonist promoted desensitization upon
stimulation
(9, 10) .
-adrenoreceptor/cAMP production pathway has been suggested to
result in heterologous desensitization. Earlier studies had suggested
PKA-dependent modulation of G
activity as a mechanism for
regulation
(13, 14, 15) . More recent studies
have suggested that modulation of the catalyst AC may play a role in
heterologous desensitization
(16) . Treatment of chick
hepatocytes with glucagon or 8-bromo-cAMP results in desensitization of
receptor-stimulated AC activity. The addition of excess purified
G
to desensitized hepatocyte membranes, however, did not
fully restore G
-stimulated AC activity, pointing to
hormone-induced desensitization at the level of catalyst as the
mechanism.
and PKC
, directly phosphorylate type V AC at
unique residues, leading to a 10-20-fold increase in catalytic
activity. The degree of this activation is greater than that achieved
by forskolin, the most potent AC agonist. Furthermore, the two PKC
isoenzymes are additive in their capacity to activate AC. These data
indicate that phosphorylation is a potent mechanism to activate type V
AC, which, unlike other AC isoforms, is insensitive to other
modulators, such as G protein
subunits or
calcium/calmodulin. In contrast, the present study demonstrates that
PKA-mediated phosphorylation inhibits type V AC. Thus, type V AC is
subject to dual regulation by phosphorylation: activation by PKC and
inhibition by PKA, mediated via phosphorylation at unique residues
within the type V molecule as demonstrated by phosphopeptide mapping. A
similar dual regulation by these two kinases has been shown in the case
of potassium channels
(21, 22) . Within type V AC, there
are 14 serine/threonine residues encompassed by a consensus sequence
for PKA-mediated phosphorylation and 11 residues for PKC-mediated
phosphorylation; however, we do not know the exact residues responsible
for mediating the functional alteration in catalytic activity.
-adrenoreceptors and the AC/PKA pathway via
-adrenergic
receptors. Thus, dual regulation of AC by PKC and PKA may play a major
role in integrating these two principal signal transduction pathways
and thereby modulate neuronal and hormonal input to the heart. We do
not know, however, whether other AC isoforms are similarly regulated
through PKA- and PKC-mediated phosphorylation.
adrenergic receptor kinase;
PKC, protein kinase C; GPT
S, guanosine
5`-O-(3-thiotriphosphate); PKA-RS, PKA-regulatory subunit;
PKA-CS, PKA-catalytic subunit; PAGE, polyacrylamide gel
electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.