(Received for publication, January 30, 1995; and in revised form, May 1, 1995)
From the
The aim of this study was to test the possible modification of
Physical interaction between
While Human mononuclear leukocytes (MNL) preparation and
treatments, cytosolic For immunoprecipitation and immunoblots, an
affinity-purified rabbit polyclonal anti- PKC was partially purified from bovine brain as
described(12) , except that PKC was eluted from the
DEAE-Sephacel column with a NaCl gradient of 0-200 mM to
improve the enzyme purity. The fractions containing peak PKC activity
for Histone IIIS ( Most of the in vitro experiments were performed using a
recombinant human For For desensitization
experiments MNL suspended in DMEM were untreated (control) or exposed
to 1 µM PMA for 15 min at 37 °C and then incubated in
the absence (undesensitized cells) or presence (desensitized cells) of
10 µM isoproterenol for 5 min at 37 °C to induce
desensitization, followed by extensive washing at 4 °C. For cAMP
accumulation experiments (16) , MNL were exposed to 10
µM isoproterenol for a further 5 min, and intracellular
cAMP accumulated was measured. For experiments with heparin, MNL were
permeabilized by streptolysin-O (0.8 unit/ml) for 5 min with or without
heparin (160 µg/ml; Sigma H-3125) after PMA pretreatment and prior
to induction of desensitization by isoproterenol, and membrane adenylyl
cyclase assay was carried out as in (17) . Titration with
heparin showed that 160 µg/ml heparin was the minimal concentration
for inhibition of homologous desensitization. Desensitization was
measured as the difference in isoproterenol-stimulated cAMP production
in cells or by membranes that were undesensitized and desensitized. DMEM and phosphate-free DMEM were obtained from Life Technologies,
Inc., RPMI 1640 medium from Bioproducts; glutamine and
penicillin/streptomycin from Seromed; Tris, SDS, bromphenol blue,
acrylamide, and bisacrylamide from Bio-Rad; EDTA, urea, and magnesium
chloride from Merck; DEAE-Sephacel from Pharmacia; Me MNL have been shown to express high levels of
Figure 1:
MNL cytosolic
Figure 2:
Characterization of MNL cytosolic
rhodopsin phosphorylating activity. A, rhodopsin
phosphorylation by cytosolic preparations obtained from MNL untreated (Control), treated with ionomycin (Iono, 1
µM) or PMA (1 µM) was carried out in the
presence and absence of light (agonist) or the
As
Figure 3:
In vitro phosphorylation of
Figure 4:
Identification of phosphorylated
For
kinetic and functional studies, PKC purified from bovine brain to
>90% homogeneity was used to avoid interfering proteins in the
phosphorylation assay (Fig. 5). Pure PKC was able to
phosphorylate
Figure 5:
Increased
The effect of PKC on kinetic
parameters of The
Figure 6:
Phosphorylation and activation of
To investigate
whether PKC can phosphorylate
Figure 7:
Phosphorylation of
To test the
physiological significance of PKC-mediated augmentation in cytosolic
Figure 8:
PMA pretreatment increased
In the present study we show that Direct phosphorylation of pure Phosphorylation
of Phosphorylation of To confirm the effect of PKC activation on
Four
subtypes of GRKs are expressed in MNL, namely The mechanism mediating Our results suggest a novel level of signal transduction cross-talk
by showing that the activity of
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptor kinase (
ARK) activity by second
messengers and/or their downstream components. Using human mononuclear
leukocytes (MNL), we found that calcium ionophores could elevate
ARK activity by about 80% in a protein kinase C (PKC)-dependent
manner. This was confirmed by the ability of the PKC activator phorbol
12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a
PKC-dependent modulation of
ARK activity. In vitro experiments with purified proteins showed that PKC could directly
phosphorylate
ARK1 with an apparent K
for
ARK1 of 6 nM. The ability of
ARK1 to
phosphorylate rhodopsin was 61% greater when it was phosphorylated by
PKC. The level of phosphorylation of
ARK1 immunoprecipitated from
MNL and Sf9 cells overexpressing this kinase was enhanced by about
2-3-fold after PMA treatment. Functional significance of
PKC-dependent increase in
ARK activity was demonstrated by
-adrenergic receptor (
AR) homologous desensitization
experiments in MNL.
AR desensitization, as induced by exposure to
10 µM isoproterenol (5 min at 37 °C), was increased
from 42 ± 10% in control to 68 ± 8% in PMA-pretreated
MNL.
ARK inhibitor heparin (160 µg/ml) prevented the
augmenting effect of PMA on
AR desensitization. These results show
that
ARK activity can be increased through phosphorylation by PKC,
thus indicating that
ARK can be preconditioned to modulate the
subsequent cellular responsiveness to receptor activation, providing
the cell with a mechanism by which specific homologous desensitization
can be regulated heterologously.
-Adrenergic receptor kinase (
ARK) (
)is a
serine-threonine kinase involved in the process of homologous
desensitization of G-protein-coupled receptors(1) , which bind
a large array of different molecules, ranging from photons,
neurotransmitters, and neuropeptides to autacoid substances, hormones,
and immunomodulators acting through different intracellular second
messengers. The mechanism of
ARK-mediated homologous
desensitization has been most extensively studied on the
-adrenergic receptors (
AR).
ARK
phosphorylates the agonist-occupied form of the receptor, enabling the
binding of its co-factor
-arrestin (2) to the receptor to
result in uncoupling of the receptor from G-proteins and hence effector
second messenger systems.
ARK is a member of a multigene family,
consisting of six known subtypes, which have also been named
G-protein-coupled receptor kinases (GRK 1-6) due to the
apparently unique functional association of such kinases with this
receptor family(1) . In this scheme rhodopsin kinase
corresponds to GRK1,
ARK1 to GRK2, and
ARK2 to
GRK3(1) . While the expression of rhodopsin kinase is
essentially confined to the retina, where it regulates
phototransduction, a wide tissue distribution has been reported for
many GRKs, the central nervous system as well as immune
cells(3, 4, 5, 6) being sites of
relevant expression of most subtypes.
ARK and receptor is favored by the binding of the kinase to
-subunits of heterotrimeric G proteins, and this results in
an enhanced receptor phosphorylation(7) . The binding of
ARK to
-subunits is shown to be mediated by a stretch of
amino acids near the C-terminal of the kinase, including sequences in
and extending beyond the most C-terminal region of the pleckstrin
homology (PH-) domain(8) .
ARK modulates
receptor-mediated production of second messengers, the likely existence
of a feedback loop by which second messengers and/or their downstream
components impinge on
ARK to modulate its activity remains an
intriguing open question. The present study was aimed to address this
point. We found that
ARK activity as well as
ARK-dependent
receptor homologous desensitization are enhanced in cells following
protein kinase C (PKC) activation. The molecular basis for these
effects is provided by in vitro experiments showing that
ARK can be directly phosphorylated by PKC and that this increases
the rhodopsin-phosphorylating activity of
ARK.
ARK preparation as well as bovine rod outer
segment (ROS) phosphorylation assay, and quantification of phosphate
incorporation were as described(4) . Phorbol 12-myristate
13-acetate (PMA) and ionomycin were dissolved in Me
SO
(0.01% Me
SO final concentration). Intracellular and
extracellular calcium was chelated by incubation with 1 mM EGTA and 10 nM ionomycin for 45 min, resulting in 80%
reduction in the subsequent ionomycin-induced calcium accumulation (not
shown). DEAE-Sephacel chromatography was carried out as described in (9) .
ARK1 antibody raised
against GST-human
ARK1 C terminus (222-amino acid) fusion protein
was used. For immunoprecipitation, 1 µg of antibody was used
according to (10) . The immune complexes were precipitated by
protein A/Sepharose CL-4B conjugate and washed three times with a
buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM
NaCl, and 0.5% Nonidet P-40, then SDS sample buffer added, boiled for 5
min at 100 °C, and analyzed by 10% polyacrylamide gel
electrophoresis. For immunoblots, 144 ng/ml antibody was used according
to (11) . Blots were developed with donkey anti-rabbit IgG
coupled to alkaline phosphatase according to the manufacturer's
instructions.
116 pmol phosphate/min/mg total eluted proteins)
were used. This gave an about 3-fold purification and the activity of
PKC, which in this preparation is estimated to represent
2% of
total protein(12, 13) , can also be expressed as 116
pmol phosphate/min/20 µg PKC. Histone IIIS phosphorylation activity
of 1 nmol of phosphate incorporation/min at 30 °C was defined as 1
unit of PKC. Pure PKC (13) was a gift of Dr. P. J. Parker.
ARK1 overexpressed in Sf9 insect cells infected
with a multiplicity of infection of 4 and purified as
described(14) .
ARK1 obtained using this procedure
migrated on the polyacrylamide gel as a doublet with apparent molecular
mass of about 80 kDa, as seen by Coomassie Blue staining and
immunoblot. More recently, using a different source of Sf9 cells
infected with a multiplicity of infection of 8 we obtained new
preparations in which purified
ARK1 migrated on polyacrylamide gel
as a single band (
80 kDa), as identified by immunoblotting. The
results obtained with the former preparation were confirmed using this
new preparation of purified
ARK1. In the PKC phosphorylation
assay, the phosphorylation mixture (48 µl) contained 30 mM Tris-HCl, pH 7.5, 7.5 mM MgCl
, 1 mM
CaCl
, 100 µg/ml phosphatidylserine, 1 µM PMA, 50 µM [
-
P]ATP
(2-8 µCi/reaction), with 200 microunits of PKC, and/or 10.4
nM of
ARK1 was usually incubated at 30 °C for 1 h.
The activity of
ARK1 was similar before and after this incubation
(4.3 ± 0.3 and 4.5 ± 0.9 nmol/min/mg respectively, n = 3). Since PKC undergoes autophosphorylation and migrates
on the gel near
ARK, at the end of the phosphorylation reaction
PKC was pelleted together with phosphatidylserine vesicles (70,000
g for 20 min at 4 °C), and hence separated from
ARK1. The resulting supernatant was electrophoresed on 8%
polyacrylamide gel and autoradiography of dried gels was for
12-18 h. Calphostin C was used as described(15) .
P labeling in living cells, MNL prepared as described
above were washed three times with phosphate-free Dulbecco's
modified Eagle's medium (DMEM) supplemented with L-arginine, L-cysteine, D-glucose, L-glutamine, L-inositol, L-leucine, and L-methionine according to the manufacturer's recipe, as
well as 25 mM of HEPES. MNL were then incubated in the same
medium. Sf9 cells were collected at 60 h after infection and washed
with phosphate-free Grace's medium (supplemented with 7.5 mM HEPES) three times before being incubated in the same medium. Both
cell types were incubated for 3 h in the presence of 1 mCi/ml
[
P]orthophosphate, at 37 °C for MNL, and at
27 °C for Sf9 cells. They were then treated with PMA (1
µM) or vehicle for 20 min. Cells were then washed three
times with ice-cold PBS before being lysed in cell lysis buffer and the
cytosol prepared as described(4) , and
ARK1 was
immunoprecipitated as described above.
SO
from Research Industries Corporations; cAMP assay kit,
[
-
P]ATP and
[
P]orthophosphate from Amersham; ionomycin and
H7 (1-5(isoquinoline sulfonyl)-2-methylpiperazine) from
Calbiochem; streptolysin-O from Wellcome; donkey anti-rabbit IgG
coupled to alkaline phosphatase from Pierce. All the other materials
were obtained from Sigma.
ARK mRNA
and activity(4, 5) , thereby providing an excellent
model for studying intracellular regulation of this kinase. We found
that exposure of MNL to calcium ionophores ionomycin and A23187 induced
a
70-80% increase in kinase activity (Fig. 1A) as measured by a ROS phosphorylation assay
which in these cells mostly measure the GRK subtype
ARK1(9) . The maximal effect was reached between 10 and 30
min of treatment with 1 µM ionomycin and was completely
prevented by calcium chelation (Fig. 1A). Since
calcium/calmodulin do not directly affect
ARK
activity(18) , the observed effect of calcium ionophores
appeared to be mediated by component(s) downstream of calcium. Possible
candidates include PKC and calcium/calmodulin-dependent
kinases(19, 20) . We found that coincubation of
ionomycin with two PKC inhibitors, H7 and staurosporin, reverted the
effect of ionomycin on
ARK activity (Fig. 1B),
suggesting that PKC mediates at least part of the effect of ionomycin.
This was confirmed by the finding that the PKC agonist PMA was able to
increase cytosolic
ARK activity to similar extents as by ionomycin (Fig. 1C). Characterization of the cytosolic rhodopsin
phosphorylating activity obtained from MNL treated with ionomycin or
PMA met the criteria for it being mediated by
ARK, i.e. the activity was completely inhibited by heparin and was agonist
(light)-dependent (Fig. 2A). Rhodopsin is also a
substrate of PKC (21) , but the contribution of PKC under our
assay conditions is excluded by the following pieces of evidence.
First, rhodopsin phosphorylation by PKC requires the addition of PKC
activators Ca
, diacylglycerol, and
phosphatidylserine(21) , which are absent in our
phosphorylation assay. Second, PKC phosphorylation of rhodopsin is
light independent (21) whereas light (agonist) is strictly
required to activate
ARK (see Fig. 2A). Third, the
augmented
ARK activity by ionomycin was still observed after the
samples had passed through DEAE-Sephacel gel, which retains PKC and
protein kinase A (18) thereby removing potentially interfering
kinases from the phosphorylation assay (not shown). Finally, the PKC
inhibitor calphostin C did not interfere with the phosphorylation of
rhodopsin (Fig. 2B). Activation of protein kinase A by
exposing MNL to 1 mM dibutyryl cAMP did not affect the level
of
ARK activity (not shown). Taken together these findings on
living MNL point to a direct or indirect modulation of
ARK
activity mediated by PKC.
ARK activity is
increased in a PKC-dependent manner. A, rhodopsin
phosphorylation by cytosolic
ARK obtained from freshly prepared
MNL untreated (control, C) or exposed for 20 min to calcium
ionophores A23187 1 µM, ionomycin 1 µM (I), and ionomycin after calcium chelation (I w/o
Ca
). The arrow indicates bands of
phosphorylated rhodopsin (opsin) as revealed by autoradiography after
polyacrylamide gel electrophoresis. B, the effect of ionomycin (I) was prevented by the presence of two PKC inhibitors H7 20
µM (I + H7), and staurosporin 0.5 µM (I + S). C, a similar increase of
ARK
activity was induced by ionomycin 1 µM (I) and
PMA 1 µM. The histogram on the right summarizes the
results from six independent experiments (means ± S.E.). *,
significantly different from control (p < 0.05; analysis of
variance). All experiments were repeated at least
twice.
ARK inhibitor
heparin (10 µg/ml). B, lack of effect of calphostin C on
rhodopsin phosphorylation by MNL cytosolic preparations. MNL were
untreated (Control) or treated for 20 min with 1 µM PMA and their respective cytosolic fractions prepared. Samples
containing 50 µg of cytosolic proteins were then tested for their
rhodopsin phosphorylating activities, and the ROS phosphorylation assay
was performed in the absence or presence of calphostin C (1
µM). All experiments were repeated at least
twice.
ARK contains in its amino acid
sequence a number of phosphorylation site motifs for PKC(4) ,
it represents a possible substrate for this kinase. Direct
phosphorylation by PKC may then lead to an increase of
ARK
activity. To investigate this hypothesis, a partially pure PKC was used
to phosphorylate
ARK1 purified from the baculovirus/Sf9 cell
expression system.
ARK1, which appeared on the polyacrylamide gel
as a doublet with apparent molecular mass of about 80 kDa, was indeed
phosphorylated by the PKC preparation, and this phosphorylation was
nearly completely inhibited by the selective PKC inhibitors
staurosporin and calphostin C (Fig. 3A), and was
prevented in the absence of the PKC activators PMA, calcium and
phosphatidylserine (not shown). Phosphate incorporation was increased
by
5-fold from the autophosphorylation level of 0.09 ±
0.01 to 0.49 ± 0.03 mol phosphate/mol of
ARK1 in the
presence of PKC (n = 4, p < 0.001), and the
time course of this reaction is depicted in Fig. 3, B and C. Immunoprecipitation using a specific
anti-
ARK1 antibody after the PKC phosphorylation reaction
specifically showed that
ARK1 was phosphorylated by PKC (Fig. 4A). By immunoblotting, it was shown that
ARK1 remained essentially in the supernatant after fractionation
by centrifugation (see ``Materials and Methods''). This also
shows that equal amounts of
ARK1 were present in the control and
PKC-phosphorylated samples (Fig. 4B). Autoradiography
of the same nitrocellulose membrane used for immunoblot confirmed the
phosphorylation of
ARK1 by PKC (Fig. 4B).
ARK1 by partially purified PKC. A, samples containing PKC
only (lane 1), PKC +
ARK1 (lane 2),
ARK1 only (lane 3), PKC +
ARK1 + 0.1
µM staurosporin (lane 4), and PKC +
ARK1 + 1 µM calphostin C (lane 5) were
incubated under phosphorylation conditions for 1 h. The arrow indicates the bands corresponding to
ARK1 as revealed by
autoradiography after polyacrylamide gel electrophoresis. B,
time course of
ARK1 phosphorylation by PKC. Samples containing PKC
only (lanes 1), PKC +
ARK1 (lanes 2), and
ARK1 only (lanes 3) were incubated for the indicated
times. C, graph showing average values of time-dependent
PKC-induced phosphate incorporation from two experiments, calculated
from radioactivity of excised bands of phosphorylated
ARK1 minus
autophosphorylated
ARK1 and PKC background. All experiments were
repeated at least twice.
ARK1. A, immunoprecipitation of
ARK1 after
phosphorylation by PKC. Samples containing PKC only (lane 1),
PKC +
ARK1 (lane 2),
ARK1 only (lane
3) were incubated under phosphorylation conditions for 1 h
followed by immunoprecipitation using an anti-
ARK1 antibody. The arrow indicates the bands corresponding to
ARK1 as
revealed by autoradiography after polyacrylamide gel electrophoresis of
the immunoprecipitated material. B, samples containing PKC
only (lanes 1), PKC +
ARK1 (lanes 2), and
ARK1 only (lanes 3) were incubated under phosphorylation
conditions for 1 h followed by centrifugation at 70,000
g for 20 min. The resulting supernatant and pellet were
electrophoresed on 8% polyacrylamide gel and transferred to
nitrocellulose paper.
ARK1 (arrows) was then detected by
immunoblotting (upper panel) and autoradiography (lower
panel) from the same blot. All experiments were repeated at least
twice.
ARK1 to a phosphate incorporation level of 0.74
± 0.07 mol/mol of
ARK1 (n = 3, p < 0.05 versus autophosphorylation, Fig. 5A). PKC showed a high affinity for
ARK1,
with an apparent K
of 6 nM, as
calculated from a double-reciprocal plot (Fig. 5B).
According to the experiments in intact cells (Fig. 1),
phosphorylation of
ARK1 by PKC should lead to increased rhodopsin
phosphorylating activity of the kinase. This was the case as the
ability of
ARK1 to phosphorylate rhodopsin was 61% greater when it
was phosphorylated by PKC compared to
ARK1 that was not exposed to
PKC (see Fig. 5C).
ARK1 activity upon
phosphorylation by PKC purified from bovine brain. 400 microunits of
PKC and 2.6 nM of
ARK1 (unless otherwise stated) were
used in PKC phosphorylation reactions. A, phosphorylation of
ARK1 by pure PKC. Samples containing PKC only (lane 1),
PKC +
ARK1 (lane 2), and
ARK1 only (lane
3) were incubated under phosphorylation conditions for 1 h. The arrow indicates the bands corresponding to
ARK1 as
revealed by autoradiography after polyacrylamide gel electrophoresis. Numbers on the left are molecular mass markers in kDa. B, assessment of the apparent K
of PKC for
ARK1 by double-reciprocal plot in which the
concentration of
ARK1 was varied. Each point is the average from
two experiments. C, assessment of rhodopsin phosphorylating
activity of
ARK1 after phosphorylation by PKC. Rhodopsin (250
nM) was phosphorylated in the presence of 21 µg/ml
purified brain
-subunits (35) by 20 µl of
supernatant from samples containing PKC only (lane 1), PKC
+
ARK1 (lane 2), and
ARK1 only (lane
3), which were previously incubated for 1 h at 30 °C to allow
phosphorylation of
ARK1 by PKC. The arrow indicates bands
of phosphorylated rhodopsin (opsin) as revealed by autoradiography
after polyacrylamide gel electrophoresis. Results from three
independent experiments (means ± S.E.) are shown in the
histogram on the right. *, significantly different from
ARK1
activity not exposed to PKC (p < 0.005, n =
3).
ARK phosphorylating activity were determined in
experiments in which different concentrations of rhodopsin were used as
substrate. Using a double-reciprocal plot, we found a 31% decrease in K
and a 10% increase in V
values (average of two experiments, data not
shown), and these changes can account for the increased phosphorylating
activity of
ARK induced by PKC.
ARK1 purified from
Sf9/baculovirus expression system used for the above experiments
migrated on the gel as a doublet, indicating that a fraction of protein
generated by this expression protocol was not properly processed. This
can explain the relatively low phosphorylation activity toward
rhodopsin of this
ARK1 preparation compared to those reported by
others(22, 23) . More recently, using slightly
different protocol of infection and a different batch of cells (see
``Materials and Methods''), we obtained new preparations in
which
ARK1 migrated on polyacrylamide gel as a single band
(
80 kDa) and had a much higher rhodopsin phosphorylating activity
(
4-6 nmol phosphate/min/mg in the absence of G/
)
compared to the previous batch. This activity is within the range of
the values previously reported(22, 23) . The
identification of this band was confirmed by immunoblotting.
Phosphorylation and activation of
ARK by PKC was confirmed in
experiments done with this new preparation of
ARK1. PKC was able
to phosphorylate this
ARK1 (Fig. 6A), and this
resulted in an increased ability of
ARK1 to phosphorylate
rhodopsin (from 3.6 to 7.0 nmol/min/mg without or with phosphorylation
by PKC, respectively, Fig. 6B).
ARK1, obtained by an alternative expression protocol, by PKC. A, a new
ARK1 preparation from Sf9 cells in which the
kinase appeared as a single band on the gel was used (see
``Materials and Methods'' for details). Samples containing
partially purified PKC only (lane 1), PKC +
ARK1 (lane 2), and
ARK1 only (lane 3) were incubated
under phosphorylation conditions for 1 h. The arrow indicates
the band corresponding to
ARK1 as revealed by autoradiography
after polyacrylamide gel electrophoresis. B, assessment of
rhodopsin phosphorylating activity of
ARK1 after phosphorylation
by PKC. Rhodopsin phosphorylation was carried out as described under
``Materials and Methods,'' using 20 µl of supernatant
from samples containing PKC only (lane 1), PKC +
ARK1 (lane 2), and
ARK1 only (lane 3),
which were previously incubated for 1 h at 30 °C to allow
phosphorylation of
ARK1 by PKC. The arrow indicates bands
of phosphorylated rhodopsin (opsin) as revealed by autoradiography
after polyacrylamide gel electrophoresis. These experiments are
representatives of two.
ARK in living cells, the
phosphorylation state of
ARK1 was examined in MNL and Sf9 cells
preloaded with labeled [
P]orthophosphate and
then treated with PMA. First, MNL were used since PKC activation in
these cells caused increases in
ARK activity (see Fig. 1).
We found that the level of phosphorylation of
ARK1
immunoprecipitated from MNL treated with PMA was enhanced by about
3-fold compared to that from control MNL (Fig. 7). Immunoblot
performed on parallel samples confirmed that equal amounts of
ARK1
were immunoprecipitated (Fig. 7). Sf9 cells overexpressing
ARK1 were also used for the same experiments to provide an
improved signal of phosphorylated
ARK1 and to obtain adequate
amount of immunoprecipitated
ARK1 for functional
assays(24) . We found that the PKC-induced
ARK1 phosphate
incorporation level was increased by approximately 2-fold compared to
the autophosphorylation level. The rhodopsin phosphorylating activity
of
ARK1 immunoprecipitated from PMA-treated Sf9 was
30%
higher than that from untreated cells (not shown).
ARK1 by PKC in
living cells. MNL and Sf9 cells overexpressing
ARK1, prelabeled
for 3 h in medium containing 1 mCi/ml
[
P]orthophosphate, were treated with PMA (1
µM) or vehicle for 20 min.
ARK1 was then
immunoprecipitated from their cytosolic preparations and analyzed by
polyacrylamide gel electrophoresis. On the left, immunoblot (imm) and autoradiogram (auto) of parallel samples of
immunoprecipitated
ARK1 from MNL are shown. On the right is the
autoradiogram of immunoprecipitated
ARK1 from Sf9 cells. The arrow indicates bands of
ARK1. The results shown are
representatives of two similar experiments.
ARK activity, we examined the effect of PMA pretreatment on
AR homologous desensitization in MNL. In control cells,
AR
homologous desensitization resulted in 42 ± 10% reduction in
receptor responsiveness as measured by isoproterenol-induced cAMP
accumulation, while in PMA-treated MNL, the level of desensitization
was increased up to 68 ± 8% (Fig. 8, left
panel). cAMP accumulation values (pmol/mg of proteins) were 375
± 32 and 220 ± 47 for undesensitized and desensitized
control MNL, respectively, and 464 ± 9 and 148 ± 39 for
undesensitized and desensitized PMA-treated MNL, respectively.
Forskolin-stimulated cAMP accumulation was unaffected by isoproterenol
pretreatment in both control and PMA-pretreated cells (not shown),
indicating that homologous (i.e. agonist-specific)
desensitization is selectively augmented by PMA. To confirm that the
PMA-induced increase in homologous desensitization was mediated by
ARK, experiments were carried out using the
ARK inhibitor
heparin on permeabilized cells(25) . In the presence of heparin (Fig. 8, right panel), homologous desensitization, as
assessed by receptor-stimulated adenylyl cyclase activity, was
decreased to almost the same level in cells with or without PMA
pretreatment. Isoproterenol-stimulated adenylyl cyclase activity values
(pmol cAMP/mg/min) were, in the absence of heparin, 48.8 ± 1.3
and 30.3 ± 0.8 for undesensitized and desensitized control MNL,
respectively, and 47.9 ± 0.4 and 23.5 ± 0.5 for
undesensitized and desensitized PMA-treated MNL, respectively; in the
presence of heparin, 36.2 ± 1.5 and 34.1 ± 0.3 for
undesensitized and desensitized control MNL, respectively, and 42.1
± 0.3 and 39.9 ± 1.5 for undesensitized and desensitized
PMA-treated MNL, respectively.
ARK-mediated
-adrenergic receptor homologous desensitization
in MNL. The level of desensitization (as induced by 10 µM
isoproterenol for 5 min at 37 °C) was compared in MNL untreated
(considered as 100%) or exposed to 1 µM PMA for 15 min. In
undesensitized and desensitized MNL, receptor responsiveness was
assessed by either agonist-stimulated cAMP accumulation (left
panel) or adenylyl cyclase activity in membrane preparation (right panel). Shown are means ± S.E.; *, significantly
different from relative control (for the left panel, p < 0.01, n = 5; for the right panel, p < 0.05, n = 4). The experiments on the right panel, in which heparin was used as
ARK inhibitor,
were performed on permeabilized MNL to allow heparin to enter the
cells.
ARK can be
phosphorylated by PKC and that this results in enhanced potency of
homologous desensitization. Different types of converging evidence
support this conclusion: (i) cytosolic
ARK activity was increased
after activation of PKC in MNL; (ii)
ARK1 was directly
phosphorylated by PKC; (iii) rhodopsin phosphorylating activity of
ARK1 was increased after phosphorylation by purified PKC; (iv)
ARK1 was phosphorylated in living cells after PMA treatment; (v)
the potency of
AR homologous desensitization was enhanced in
PMA-treated MNL, and this effect was prevented by the
ARK
inhibitor heparin.
ARK1 by PKC
was demonstrated using purified PKC in an in vitro assay.
Pharmacological proof that the phosphorylation of
ARK1 was due to
PKC was provided by its inhibition by staurosporin and, importantly, by
calphostin C, a highly specific PKC inhibitor which acts on the
regulatory domain of PKC, with negligible activity on other protein
kinases(15, 26) . Immunoprecipitation and
immunoblotting with anti-
ARK1 antibodies further identified
phosphorylated
ARK1. Finally, using a near homogeneous preparation
of PKC, phosphorylation of
ARK1 was confirmed.
ARK1 by PKC resulted in an increased
ARK activity toward
rhodopsin. The magnitude of this increase in
ARK activity was
similar to that observed on
ARK from living MNL in which PKC was
activated either by ionomycin or PMA (see Fig. 1C).
Significantly, PKC displays a high affinity toward
ARK1,
suggesting that the interaction between these two kinases is
biologically relevant.
ARK1 by PKC was
observed on living cells using two different cellular models. MNL
provided a physiological model since the quantitative and qualitative
parameters of
ARK1 and PKC were defined physiologically by the
cells. Sf9 cells with overexpressed
ARK1 provided a less
physiological model especially with regard to the PKC/
ARK1
stoichiometry. This may be the reason why the magnitude of increase in
phosphate incorporation into
ARK1 in PMA-treated cells was lower
in Sf9 cells than in MNL. It did nevertheless provide a convenient
means for visualization of
ARK1 phosphorylation(24) . In
addition, the high levels of
ARK1 in these cells allowed us to
examine specifically the activity of
ARK1 immunoprecipitated.
Activity of
ARK1 immunoprecipitated from Sf9 cells treated with
PMA was increased compared to that from control cells. These results
strongly indicate that the phosphorylation of
ARK1 by PKC is
physiologically relevant.
ARK with a functional approach on cells, the potency of
isoproterenol-induced rapid homologous desensitization following PMA
pretreatment was examined. In the present desensitization experiments,
5 min were allowed for 10 µM of isoproterenol to induce
homologous desensitization to ensure that the desensitization was
mediated mainly by
ARK(17, 27) . PMA pretreatment
potentiated the degree of desensitization from 42 to 68%, which is the
functional effect expected for the increased
ARK activity observed
in these cells (see Fig. 1). Similar results were obtained by
Chambaut-Guerin and Thomopoulos (28) in murine macrophage J774
cells. Additionally, as in their study, we also observed that PMA
increased
AR responsiveness, as measured by isoproterenol-induced
cAMP accumulation. This is not surprising as it is known that PKC may
affect the
AR-G
-adenylate cyclase axis to alter the
receptor-mediated response(29) . Using heparin to inhibit
ARK in permeabilized cells(25) , the increase in
homologous desensitization caused by PMA pretreatment was prevented,
thus confirming that
ARK was responsible for this effect.
ARK1,
ARK2,
GRK5, and GRK6(9) . The present results in vitro as
well as in living cells strongly suggest a major role of
ARK1 in
the PKC-induced increase in homologous desensitization. However, the
possible role of other subtype(s) in this process remains to be
investigated.
ARK-PKC interaction is
not known, although other studies suggest the involvement of
PH-domain(30, 31) . The PH-domain is composed of
approximately 100 loosely conserved amino acids, so far found to be
present in a large nubmer of intracellular signaling proteins,
including
ARK1 and 2(30) . Recently, two kinases have been
shown to bind directly to PKC through their respective
PH-domains(31, 32) . Although no consensus amino acid
sequence or structure within these two PH-domains critical for binding
PKC were identified, mutation of Arg-28 of the Bruton tyrosine kinase,
which causes the genetic disease agammaglobulinemia, reduced the
capacity of its PH-domain in binding PKC(31) . Arg-28 of Bruton
tyrosine kinase was therefore suggested to participate in PKC binding.
Interestingly, this amino acid is conserved in most of the PH-domains
identified so far, including that of
ARK1 (Arg-579)(30) .
This would support direct binding of PKC to
ARK1, as is suggested
by our observation of direct phosphorylation of
ARK1 by PKC.
ARK can be increased by direct PKC
phosphorylation. This indicates that the efficacy of homologous
desensitization, which regulates the responsiveness of many receptors,
can be modulated heterologously within the cell. Other members of the
GRK family may also be modified by PKC and/or other intracellular
enzymes and such a mechanism may underlie phenomena like
``receptor class desensitization''(33) . In view of
the fact that PKC is a key enzyme involved in signal transduction and
cell proliferation(19) , and that a number of G-protein-coupled
receptors have been shown to be protooncogenes(34) , the
present finding may have significantly wider implications.
ARK,
-adrenergic receptor kinase;
AR,
-adrenergic receptor;
DMEM, Dulbecco's modified Eagle's medium; GRK, G-protein
coupled receptor kinase; H7, (1-5(isoquinoline
sulfonyl)-2-methylpiperazine); MNL, mononuclear leukocytes; PKC,
protein kinase C; PMA, phorbol 12-myristate 13-acetate; ROS, rod outer
segment; PH-domain, pleckstrin homology domain; G/
,
-subunits of heterotrimeric G-protein.
We are grateful to Dr. P. J. Parker of the Imperial
Cancer Research Funds for the generous gift of pure PKC. We thank E.
Pompili for advice in immunoprecipitation, M. Sallese and S. Pirocchi
for Sf9 ARK1, M. Molino for calcium measurements, S. Sozzani for
advice in PKC experiments, D. Talone for excellent technical
assistance, and R. Bertazzi and S. Menna for expert assistance in
preparation of the figures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.