(Received for publication, August 14, 1996, and in revised form, November 12, 1996)
From the Department of Biochemistry and Molecular Pharmacology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
G protein-coupled receptor kinases (GRKs)
specifically recognize and phosphorylate the hormone-occupied form of
numerous G protein-coupled receptors, ultimately resulting in
termination of receptor signaling. While little is presently known
about the regulation of GRK function, recent studies suggest a role for protein kinase C (PKC) phosphorylation of the -adrenergic receptor kinase in membrane association and activation of the kinase. To assess
a potential general role for PKC in regulating GRK function, we
characterized the ability of PKC to phosphorylate GRK5, a recently identified member of the GRK family. We demonstrate that GRK5 can be
rapidly and stoichiometrically phosphorylated by PKC in vitro. Intact cell studies reveal that GRK5 is also
phosphorylated when transiently expressed in COS-1 cells following
treatment with the PKC activator, phorbol 12-myristate 13-acetate.
In vitro analysis reveals two major sites of PKC
phosphorylation within the C-terminal 26 amino acids of GRK5. GRK5
phosphorylation by PKC dramatically reduces its ability to
phosphorylate both receptor (light-activated rhodopsin) and
non-receptor (casein and phosvitin) substrates. Kinetic analysis
reveals an ~5-fold increased Km and ~3-fold
decreased Vmax for rhodopsin, with no change in
the Km for ATP. The reduced affinity of
PKC-phosphorylated GRK5 for rhodopsin was also evident in a decreased
ability to bind to rhodopsin-containing membranes, while direct binding
of GRK5 to phospholipids appeared unaltered. These results suggest that
PKC might play an important role in modulating the ability of GRK5 to
regulate receptor signaling and that GRK phosphorylation by PKC may
serve as a disparate mechanism for regulating GRK activity.
A basic feature of most cells is their ability to regulate their
responsiveness to extracellular stimuli. This phenomenon, often termed
desensitization, has been extensively studied for the
2-adrenergic receptor
(
2AR),1 which mediates
catecholamine stimulation of cAMP production (1, 2). Desensitization
appears to involve a multistep process that is initiated by receptor
activation and culminates in receptor phosphorylation and functional
uncoupling of receptor signaling. Phosphorylation of the
2AR can be mediated by multiple protein kinases,
including second messenger activated kinases such as protein kinase A
and protein kinase C (PKC), as well as a specific class of kinases
termed G protein-coupled receptor kinases (GRKs) (3, 4). GRKs are
unique in that they specifically recognize and phosphorylate the
agonist occupied or activated form of a receptor. To date, six members
of the GRK family have been identified.
While GRKs are involved in regulating G protein-coupled receptor
function, the activity of GRKs themselves also appears to be regulated.
Examples of such regulation include the activation of rhodopsin kinase
(5, 6), -adrenergic receptor kinase (
ARK) (7), and
GRK52 upon binding to an activated
receptor. This activation is manifested as an increased ability of the
kinase to phosphorylate exogenous peptide substrates and will likely be
a property shared by all of the GRKs. Another mechanism for regulating
GRK function appears to be via phospholipid interaction. All of the
GRKs can directly interact with phospholipids either via covalent
modifications such as farnesylation (rhodopsin kinase) (8) or
palmitoylation (GRK4 and 6) (9, 10), or via lipid binding domains such
as the pleckstrin homology domain in
ARK1 and 2 (11) or a poorly defined polybasic domain in GRK5 (12, 13). In addition,
ARK1 and 2 have the ability to interact directly with G protein
subunits,
an interaction that may be involved in kinase localization (14-16). At
least two of the GRKs also undergo rapid autophosphorylation (12, 13,
17). Autophosphorylation of rhodopsin kinase reduces its affinity for
phosphorylated rhodopsin, suggesting a potential role in dissociation
of the kinase from the receptor (18). In contrast, autophosphorylation
of GRK5 appears to significantly activate the kinase (12).
More recent studies have implicated calcium in the regulation of GRK
activity. In the visual system, rhodopsin kinase has been shown to be
inhibited by the Ca2+-binding protein recoverin (19).
Calcium binding to recoverin promotes its association with rhodopsin
kinase, inactivating the kinase and thereby reducing its ability to
phosphorylate rhodopsin. Since calcium levels are decreased upon light
activation of rod cells (20), recoverin binding to rhodopsin kinase
might provide a mechanism for adaptation of the system to ambient
light. Recent studies have also demonstrated that ARK can be
phosphorylated by PKC, a Ca2+ and
phospholipid-dependent kinase, leading to an ~2-3-fold
activation of the kinase possibly via an increased ability of the
kinase to bind to membranes (21, 22).
In the present study we evaluated whether GRK5 can be regulated by PKC.
We show that GRK5 is rapidly phosphorylated by PKC in vitro
as well as in transfected COS-1 cells. The major sites of PKC
phosphorylation were localized within the C-terminal ~26 amino acids
of GRK5. In contrast with ARK, PKC phosphorylation significantly
inhibits GRK5 activity. These findings suggest that PKC may play a
diverse role in the regulation of GRK function.
Restriction endonucleases, Vent DNA polymerase, and other molecular biology reagents were purchased from New England Biolabs or Boehringer Mannheim. SP-Sepharose was obtained from Pharmacia Biotech Inc. and protein A-agarose from Bio-Rad. Phosphatidylcholine (Type II-S from soybean) was from Sigma, while phosphatidylserine (bovine brain, 99% pure) was from Avanti Polar Lipids, Inc. Prestained Rainbow protein molecular weight markers were from Amersham. The GRK5 antibody SC-565 was purchased from Santa Cruz Biotechnology, Inc., while all other materials were from sources previously described (23).
Mutagenesis and ExpressionAll mutations were introduced
into the full-length clone pGRK5 (24) by PCR-mediated, site-directed
mutagenesis. To mutate Lys215 to arginine (GRK5-R), the
mutant sense primer 5-AATGTATGCCTGC
CGCTTGGAGAAGAAGA-3
(Arg codon is underlined) was used with the antisense primer
5
-TTCATGATGGTCAGGACCAAGC-3
to amplify a 171-base pair fragment of
pGRK5. The resulting PCR fragment was isolated and then used as the
antisense primer in a second PCR reaction with 5
-GTGCAAAGAACTCTTTTC-3
as the sense primer and pGRK5 as the template. The resulting PCR
product was digested with BstEII and XhoI and
used to replace a similar fragment in the baculovirus expression
construct pBacPAK-GRK5 (24). To replace Ser484 and
Thr485 with aspartate residues (GRK5-DD), the sense primer
5
-TGATCCGCGGCCGGGTGG-3
and the antisense primer
5
-GATTGACGCCCTTCAC
GAACTGCTCGATG-3
(Asp codons are
underlined) were used to amplify pGRK5. The PCR fragment was isolated,
digested with BglI and BsaHI, and used to replace
similar fragments in pBacPAK-GRK5 and pBacPAK-GRK5-R to generate the
mutants GRK5-DD and GRK5-RDD, respectively. The PCR-derived portions of
the constructs were sequenced in their entirety to confirm the presence
of the desired mutations.
Bovine and rat PKC, overexpressed and purified from Sf9 cells, were
a generous gift from Dr. C. Stubbs (25). Bovine
ARK and the mutant
GRK5s were overexpressed and purified from Sf9 cells as described (16,
23).
Autophosphorylation reactions
contained 30 pmol (2 µg) of either wild type GRK5 or GRK5-DD in 20 µl of 20 mM Tris-HCl, pH 8.0, 4 mM
MgCl2, 1 mM EDTA, 0.1 mM
[-32P]ATP (1000 cpm/pmol). Reactions were incubated at
30 °C for 0, 10, or 30 min and stopped with 5 µl of SDS sample
buffer. Samples were electrophoresed by the method of Laemmli (26), and
the gels were stained with Coomassie Blue, dried, autoradiographed, and
the 32P-labeled proteins were excised and counted to
determine the picomoles of phosphate transferred.
20 pmol (1.35 µg) of GRK5
(wild type or mutant) were phosphorylated in the presence or absence of
1 pmol (80 ng) of bovine PKC in 20 µl of 20 mM
Tris-HCl, pH 8.0, 6 mM MgCl2, 0.5 mM EDTA, 0.2 mM CaCl2, 0.5 µM phorbol 12-myristate 13-acetate (PMA), 0.9 mg/ml
phospholipid liposomes, 0.1 mM [
-32P]ATP
(1000 cpm/pmol). Phospholipid liposomes were prepared by sonicating 80 mg of phosphatidylcholine and 10 mg of phosphatidylserine in 5 ml of 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA on ice four times for 20 s. Reactions were
incubated for 6 min or as indicated at 30 °C and stopped with 5 µl
of SDS sample buffer. Samples were electrophoresed and processed as
described above. Kinetic studies used 1-60 pmol of GRK5 phosphorylated
with 0.3 pmol of PKC for 3 min at 30 °C. Km and
Vmax values were derived from double-reciprocal
plots of the data.
Expression plasmids for GRK5 were constructed by cloning the coding sequences of GRK5 and GRK5-R in the vector pBC12BI (27). COS-1 cells were grown to ~80-90% confluence in six-well dishes (35-mm wells) at 37 °C in a humidified atmosphere containing 5% CO2, 95% air in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected with 2 µg of DNA/well using LipofectAMINE following the manufacturer's instructions (Life Technologies, Inc.). Forty-eight hours after transfection, cells were either used for metabolic labeling or were harvested for immunoblotting analysis.
For metabolic labeling, cells were incubated for 1 h at 37 °C either in RPMI 1640 (lacking methionine) with 10% dialyzed fetal bovine serum for the [35S]Met labeling or in DMEM (lacking phosphate) with 1% fetal bovine serum for [32P]inorganic phosphate (32Pi) labeling. Cells were then incubated with either [35S]Met (0.1-0.2 mCi/well) or 32Pi (0.3-0.6 mCi/well) in 1 ml of the appropriate medium for 2.5 h. PMA (0.2 µM final concentration) or vehicle were added to the wells, and the cells were incubated for 30 min at 37 °C, washed with phosphate-buffered saline, harvested, and analyzed by immunoprecipitation.
Immunoblotting and ImmunoprecipitationGRK5 rabbit polyclonal antibodies were raised against glutathione S-transferase (GST) fusion proteins containing either amino acids 98-136 or 489-590 (12) of human GRK5. GST and the GST-GRK5 fusion proteins were expressed in Escherichia coli and purified on a glutathione-Sepharose column using standard procedures (28). A rabbit polyclonal antibody was also generated against a peptide corresponding to amino acids 556-571 of GRK5 that had been covalently coupled to hemagglutinin. The resulting GRK5-specific peptide antibodies were affinity-purified on a Sepharose column containing the covalently coupled peptide. The GRK5 rabbit polyclonal antibody SC-565, generated against amino acids 571-590 of GRK5, was from Santa Cruz Biotechnology, Inc.
Transfected COS-1 cells were harvested by scraping into 0.4 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 200 mM NaCl, 50 mM NaF, 0.8% Triton X-100 with protease inhibitors (5 µM aprotinin, 5 mM benzamidine, 20 µM leupeptin, 2 µM pepstatin A, 1 mM phenylmethylsulfonyl fluoride). Cells were lysed by freeze/thaw and supernatants prepared by centrifugation for 7 min at 100,000 × g. Fifteen µg of total protein were then electrophoresed and subjected to Western blot analysis with the indicated antibodies. Proteins were visualized by ECL (Amersham), while protein concentrations were determined by dye binding assay (Bio-Rad) using bovine serum albumin as a standard.
Metabolically labeled cells were harvested by scraping into 0.2 ml of ice cold 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 200 mM NaCl, 50 mM NaF, 0.1% Triton X-100 with protease inhibitors. Cells were lysed by freeze/thaw and supernatants prepared by centrifugation for 7 min at 100,000 × g. Supernatants were diluted 5-fold with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 20 mM NaF, 0.02% Triton X-100 with protease inhibitors, and enriched in GRK5 by batchwise SP-Sepharose chromatography. SP-Sepharose eluates (150 µl) were added to 300 µl of Tris-buffered saline (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.05% Tween 20) and 30 µl of GST-GRK5-(489-590) antiserum. Where indicated, 100 µg of GST-GRK5-(489-590) was added to block antibody binding to GRK5. Samples were incubated on ice for 15 min, and then 50 µl of a 50% protein A-agarose suspension were added and incubated an additional 30 min. The resin was washed twice with 1 ml of Tris-buffered saline, and labeled proteins were eluted with two 70-µl aliquots of SDS sample buffer heated to 70 °C. Supernatants were combined, and 25-µl aliquots of each sample were electrophoresed on a 10% SDS-polyacrylamide gel. The gel was fixed, soaked in 20% (w/v) PPO in acetic acid, washed with water, dried, and autoradiographed, and the 32P- or 35S-labeled proteins were excised and counted. PMA treatment of the cells did not significantly change the total amount of 35S-labeled GRK5 that was immunoprecipitated (92 ± 5% of untreated cells, n = 4).
SP-Sepharose Purification of Phosphorylated GRK5 and Determination of Phosphorylation SitesFollowing phosphorylation by PKC, GRK5 was purified by batchwise chromatography on SP-Sepharose. Briefly, phosphorylation reactions were stopped on ice, mixed with an equal volume of 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl, 0.4% Triton X-100, and then incubated for 10 min with 50 µl of a 50% suspension of SP-Sepharose in buffer A (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 50 mM NaCl, 0.02% Triton X-100). The resin was pelleted, washed two to three times with 1 ml of buffer A, and the bound kinase was eluted with two 75-µl aliquots of 20 mM Tris-HCl, 1 mM EDTA, 600 mM NaCl, 0.02% Triton X-100. The supernatants were combined, diluted with 150 µl of 20 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 1 mM EDTA, and then used for further analysis. Typically, 70-80% of the GRK was recovered by this procedure, while PKC did not bind to SP-Sepharose.
For phosphoamino acid analysis, 300 pmol of GRK5-RDD was phosphorylated with 7 pmol of PKC in a 120-µl reaction for 15 min and then purified on SP-Sepharose as described above. The stoichiometry of phosphorylation was ~2.1 mol of phosphate/mol of GRK5-RDD. Fifty µl of the SP-Sepharose eluant were lyophilized, dissolved in 200 µl of 5.7 M HCl, and subjected to acid hydrolysis for 2 h at 105 °C. Phosphoamino acids were separated on cellulose plates by two-dimensional TLC using isobutyric acid, 0.5 M ammonium hydroxide (5:3 v/v) in the first dimension, and 2-propanol, HCl, water (7:1.5:1.5 v/v) in the second dimension (29).
To determine the sites of phosphorylation, 3.6 nmol of purified GRK5-R was incubated with 60 pmol of rat PKC in a 1.2-ml reaction for 15 min and then purified on SP-Sepharose. The stoichiometry of phosphorylation was ~2.7 mol of phosphate/mol of GRK5-R. The eluate from SP-Sepharose was mixed with 400 µl of 10 mM Tris-HCl, pH 8.0, containing 6 µg of trypsin and then digested at 23 °C for 16 h. The resulting peptides were separated by reverse-phase HPLC on a C18 Vydac column eluted with a 15-ml linear gradient of 4-33% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. Peptides were detected by absorbance at 220 nm, while radioactivity was determined by Cerenkov counting. This yielded one major peak of radioactivity (90%) eluting at ~10% acetonitrile and one minor peak (10%) at ~23% acetonitrile. The fraction containing the major radioactive peak was diluted to ~5% acetonitrile, 0.1% trifluoroacetic acid, reapplied to the column, and eluted with a 15-ml linear gradient of 7.8-10.6% acetonitrile, 0.1% trifluoroacetic acid. This resulted in the resolution of two peaks of radioactivity, which were collected and then sequenced using a gas-phase amino acid sequencer (Applied Biosystems model 477A).
Effect of PKC Phosphorylation on GRK ActivitySixty pmol of
GRK5, GRK5-DD, or ARK were phosphorylated with 5 pmol of bovine PKC
in a 40-µl reaction for 10 min and then purified on SP-Sepharose.
Aliquots of the phosphorylated kinases before and after SP-Sepharose
purification were electrophoresed on an SDS-polyacrylamide gel to
enable assessment of autophosphorylation, phosphorylation by PKC, and
recovery from SP-Sepharose.
Four-µl aliquots (~0.6 pmol) of the SP-Sepharose-purified GRKs were
then assayed by incubating with either ROS membranes (60 pmol of
rhodopsin), casein (10 µg), or phosvitin (10 µg) in 20 µl of 20 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 1 mM EDTA, 0.1 mM [-32P]ATP
(1000 cpm/pmol) for 6 min at 30 °C in room light. The reactions were
stopped with 5 µl of SDS sample buffer, the samples were electrophoresed on a 10% SDS-polyacrylamide gel, gels were stained with Coomassie Blue, dried, and autoradiographed, and the
32P-labeled proteins were excised and counted. To assess
the kinetics of receptor phosphorylation, 25-660 pmol of rhodopsin
were phosphorylated with control or PKC-phosphorylated GRK5 in 20 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 1 mM EDTA, 0.1 mM [
-32P]ATP
(6000 cpm/pmol). To determine the kinetics for ATP, rhodopsin (60 pmol)
was phosphorylated with GRK5 in 20 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 1 mM EDTA, and 5-250
µM [
-32P]ATP (3000 cpm/pmol).
Km and Vmax values were
derived from double-reciprocal plots of the data. Urea-treated ROS
membranes containing rhodopsin were prepared from bovine retinas as
described previously (30).
The ability of GRK5 to associate with phospholipids and rhodopsin was analyzed by incubating 8-µl aliquots (~1.2 pmol) of SP-Sepharose-purified 32P-labeled GRK5 in the presence or absence of phospholipid liposomes (108 µg) or ROS membranes (250 pmol of rhodopsin) in 60 µl of 20 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 80 mM NaCl at 30 °C for 5 min in room light. The incubations were then pelleted at 100,000 × g for 6 min, the pellets were resuspended in 60 µl of reaction buffer, and equal aliquots of the supernatant and pellet fractions were electrophoresed on a 10% SDS-polyacrylamide gel. The gels were dried, autoradiographed, and the 32P-labeled proteins were excised and counted.
Previous studies have
demonstrated that GRK5 is rapidly autophosphorylated in a
phospholipid-dependent manner (12). GRK5 autophosphorylation results in the appearance of two slower moving forms of GRK5 when analyzed by SDS-PAGE (Fig.
1A, wt, 10 and 30 min). This transition of GRK5 into slower moving forms was not observed in the absence of ATP and paralleled the increased
incorporation of radioactivity into the kinase when incubated with
[-32P]ATP (Fig. 1B). Since the major sites
of GRK5 autophosphorylation were previously identified as
Ser484 and Thr485 (12, 13), phosphorylation of
one of these residues likely causes a shift in the electrophoretic
mobility of GRK5 while incorporation of a second phosphate creates an
even slower moving form of the kinase.
Since GRK5 autophosphorylation might make it difficult to assess the
ability of GRK5 to serve as a substrate for other protein kinases, we
generated several mutants, which would have a reduced ability to
autophosphorylate. These mutants were expressed in Sf9 cells using the
baculovirus system and purified to >95% homogeneity (Fig.
1A). One of these mutants targeted a lysine residue
(Lys215 in GRK5) that is conserved in all protein kinases
and appears to be involved in the phosphotransfer reaction (31).
Mutation of this conserved lysine into arginine generally results in a protein kinase with significantly reduced catalytic activity. The
corresponding K215R mutation in GRK5 yielded a protein (GRK5-R) that
had ~100-200-fold reduced ability to phosphorylate activated rhodopsin and 2-adrenergic receptor compared to wild
type GRK5 (data not shown). Similarly, GRK5-R was reduced
~100-200-fold in autophosphorylation (Fig. 2).
Replacement of Ser484 and Thr485 in the
autophosphorylation site with alanine leads to a significant reduction
in the ability of the protein to phosphorylate rhodopsin and
2-adrenergic receptor (12). Here we generated a mutant
with Ser484 and Thr485 replaced by negatively
charged aspartate residues, which we expected would mimic
autophosphorylation of GRK5. Indeed, the corresponding mutant protein
(GRK5-DD) showed a significant reduction in autophosphorylation (Fig.
1B) with no loss in its ability to phosphorylate rhodopsin compared to wild type GRK5. We also generated a mutant with both the
catalytic site (K215R) and the autophosphorylation site (S484D and T485D) mutated. Similar to GRK5-R, this mutant (GRK5-RDD) had
no significant catalytic activity (data not shown) and was not
autophosphorylated (Fig. 2A). Interestingly, the
introduction of negative charges into the autophosphorylation site also
results in a reduced electrophoretic mobility of both GRK5-DD and
GRK5-RDD compared to wild type GRK5 (Fig. 1A).
To test whether GRK5 serves as a substrate for PKC, we initially
examined the ability of purified PKC to phosphorylate wild type and
mutant GRK5. GRK5 phosphorylation by PKC was most evident for the
autophosphorylation-defective mutants, but an ~3-fold increase in the
phosphorylation of wild type GRK5 was also observed (Fig.
2A). Time-course studies revealed that GRK5-R was rapidly phosphorylated by PKC (t1/2 ~2 min) to a
maximal stoichiometry of 3.6 ± 0.3 mol of phosphate/mol of GRK5-R
(Fig. 2B). In contrast, GRK5-RDD was also rapidly
phosphorylated but only to a stoichiometry of ~2.1 ± 0.3 mol/mol. This suggests that Ser484 and/or
Thr485 at the autophosphorylation site might serve as
potential sites for phosphorylation by PKC. However, at low PKC
concentrations and short incubation times, PKC showed little
discrimination between GRK5-R and GRK5-RDD (Fig. 2C),
suggesting that Ser484 and/or Thr485 are most
likely not the primary phosphorylation targets of PKC. Kinetic studies
reveal a Km ~0.6 µM and
Vmax ~60 nmol phosphate/min/mg for both GRK5-R
and GRK5-RDD in the absence of PKC activators, while a
Km ~2 µM and
Vmax >900 nmol phosphate/min/mg were obtained
in the presence of PKC activators. These kinetics are comparable to
those of a number of well established PKC substrates (32) and
demonstrate that GRK5 is a good substrate for PKC in vitro.
Since GRK5 is a good
substrate for PKC in vitro, we next wanted to assess whether
GRK5 could also serve as a substrate for PKC in intact cells. COS-1
cells, either untransfected or transfected with the vector pBC12BI,
contain undetectable levels of GRK5 as assessed by immunoblotting (Fig.
3A, lane 2). When transfected with
the expression construct pBC-GRK5, GRK5 was expressed at a high level
(~3 µg/mg) and exhibited a broad immunostaining pattern suggestive
of a heterogeneous state of autophosphorylation (lane 3). In
contrast, GRK5-R was expressed at a much lower level (~0.5 µg/mg)
(lane 4), precluding us from further studying this construct in COS-1 cells.
To determine whether GRK5 is phosphorylated by PKC in COS-1 cells, cells transfected with vector alone or with wild type GRK5 were metabolically labeled with either [35S]methionine or 32Pi. The labeled cells were then treated with the PKC activator PMA for 30 min, and GRK5 was immunoprecipitated using an anti-GRK5 rabbit polyclonal antibody (12). No 35S- or 32P-labeled proteins were immunoprecipitated from COS-1 cells transfected with vector alone (Fig. 3B, lanes 1 and 5). However, in cells transfected with wild type GRK5, two or three 35S-labeled proteins were immunoprecipitated (lane 2). Immunoprecipitation of these proteins was completely blocked by the GRK5 fusion protein used to generate the antibodies (lane 4). The pattern of 35S-labeled bands observed in the absence of PMA treatment suggests that 60-70% of the GRK5 was autophosphorylated (slower moving form of the kinase), while the remainder has the same mobility as purified recombinant GRK5 and likely corresponds to GRK5 that is not autophosphorylated. This was confirmed with 32P labeling where only the slower moving band was radiolabeled (Fig. 3B, lane 6). Since autophosphorylation appears to activate GRK5, the high basal autophosphorylation level implies that most of the GRK5 expressed in COS-1 cells is in an activated state.
When the cells were treated with PMA, a significant change in the electrophoretic mobility of GRK5 was apparent with a noticeable decrease in the amount of slower moving and corresponding increase in the faster moving form (Fig. 3B, compare lanes 2 and 3). Since autophosphorylation decreases the electrophoretic mobility of GRK5, this suggests that PMA treatment may reduce the amount of autophosphorylated GRK5. Direct evidence for PKC phosphorylation of GRK5 in COS-1 cells was assessed by immunoprecipitation of the 32P-labeled proteins following treatment with PMA. These studies show that the total amount of immunoprecipitated 32P-labeled GRK5 was increased significantly (180 ± 20% compared to untreated cells, n = 3). Since phosphorylation of both the slower and faster moving forms of GRK5 was observed following PMA treatment (Fig. 3B, lane 7), this suggests that PKC phosphorylation does not alter the electrophoretic mobility of GRK5 and that Ser484 and Thr485 are not the primary sites of PKC phosphorylation. Thus, under basal conditions 60-70% of the GRK5 appears to be stoichiometrically autophosphorylated, whereas following PMA treatment ~30% of the GRK5 is autophosphorylated while ~70% is stoichiometrically phosphorylated, most likely by PKC.
Identification of the Sites of PKC PhosphorylationThe
in vitro and intact cell studies suggest that while PKC can
phosphorylate Ser484 and/or Thr485, the primary
sites of phosphorylation likely reside elsewhere in GRK5. Since
GRK5-RDD can be phosphorylated by PKC to a stoichiometry of ~2 mol of
phosphate/mol of GRK5-RDD (Fig. 2B), this suggests a minimum
of two primary sites of PKC phosphorylation. Because the C-terminal
tail of GRK5 is rich in serine, threonine, and basic residues, we
tested whether a GST fusion protein containing the C-terminal 102 amino
acids of GRK5 (GST-GRK5-(489-590)) could also serve as a substrate for
PKC. Indeed, GST-GRK5-(489-590) was phosphorylated by PKC to a
stoichiometry of ~1-1.5 mol of phosphate/mol of fusion protein,
while GST itself was not a substrate for PKC (Fig.
4A). Thus, one or two potential PKC
phosphorylation sites lie within the C-terminal 102 amino acids of
GRK5.
We next tested the ability of control and PKC-phosphorylated GRK5-R to be immunoblotted by antibodies targeted to different regions of GRK5. Both control and PKC-phosphorylated GRK5-R were comparably recognized by antibodies generated against either residues 98-136 or residues 489-590 of GRK5 (Fig. 4B, lanes 1-4). However, an antibody generated against residues 556-571 of GRK5 clearly immunoblotted the nonphosphorylated form of GRK5-R, but it did not recognize PKC-phosphorylated GRK5-R (lanes 5 and 6). This suggests that PKC likely phosphorylates GRK5 within the 16 amino acid antibody epitope KRPSQNNSKSSPSSK. The binding of an antibody generated against residues 571-590 of GRK5 was also reduced after phosphorylation by PKC, although it was still detectable (lanes 7 and 8). This domain is also serine- and threonine-rich, although there is only one basic residue nearby (Lys570).
Phosphoamino acid analysis of PKC-phosphorylated GRK5-RDD revealed that most of the radioactivity was incorporated into phosphoserine (83 ± 2%) with a small amount in phosphothreonine (17 ± 2%) (data not shown). To further define the PKC-phosphorylation sites on GRK5, PKC-phosphorylated 32P-labeled GRK5-R was digested with trypsin and chromatographed by reverse phase HPLC. This yielded one major (~90%) and one minor (~10%) peak of radioactivity. When the major peak was rechromatographed and eluted with a shallow gradient, two separate peaks of radioactivity were observed. The peaks were collected and subjected to gas phase sequencing. The first peak yielded the sequence of the extreme C terminus of GRK5 (Thr571-Ser590) (Fig. 4C). This peptide contains 7 serine and 2 threonine residues and represents the same region that was used to generate the antibodies whose recognition of GRK5 was reduced by PKC phosphorylation (Fig. 4B, lanes 7 and 8). The second radioactive peak yielded the sequence SSPSSK (residues 565-570) (Fig. 4C). This region is part of the peptide (Lys556-Thr571) used to generate the antibodies whose binding was completely blocked by PKC phosphorylation (Fig. 4B, lanes 5 and 6). These results demonstrate that at least two PKC phosphorylation sites lie within the C-terminal 26 residues of GRK5. Since both of these peptides contain multiple serine and/or threonine residues, we cannot determine the exact residues phosphorylated by PKC. However, since there is a requirement for basic residues to serve as a PKC substrate (33) and phosphoserine is the predominant amino acid in GRK5 phosphorylated by PKC, we envision the most likely sites to be Ser572 and either Ser566 or Ser568. Of note is the fact that Ser572 and Ser566 are conserved in human (24), bovine (13), and rat (34) GRK5. However, it is also possible that PKC has little preference for specific residues within this C-terminal domain, and that the phosphorylation of any residue would have a similar consequence. For example, PKC can phosphorylate three different serine residues within pleckstrin, but phosphorylation of any two of the three has the same effect on pleckstrin's ability to inhibit phosphoinositide hydrolysis (35).
Effect of PKC Phosphorylation on GRK5 ActivitySince GRK5 is
rapidly and stoichiometrically phosphorylated by PKC in
vitro, we next tested the effect of PKC phosphorylation on GRK5
activity. Since ARK has been demonstrated to be phosphorylated and
activated by PKC (21, 22), it was used as a control in this series of
studies.
ARK and GRK5 were phosphorylated by PKC, purified on
SP-Sepharose, and then assayed for their ability to phosphorylate light
activated rhodopsin. Under the conditions used,
ARK was only
phosphorylated by PKC to a stoichiometry of ~0.1 mol of phosphate/mol
of kinase. Thus, as might be expected, no significant change in the
ability of
ARK to phosphorylate rhodopsin was detected (Fig.
5A, lanes 2 and 3). In
contrast, under identical conditions GRK5 was phosphorylated by PKC to
a stoichiometry of ~2.4 mol of phosphate/mol of kinase, demonstrating that GRK5 is a much better substrate for PKC than is
ARK. Moreover, in striking contrast to
ARK, PKC-phosphorylated GRK5 had an ~90% reduced ability to phosphorylate rhodopsin (Fig. 5A, compare
lanes 5 and 6). This effect also required active
PKC since boiled PKC had no effect on GRK5 activity (compare
lanes 5 and 7). Similar results were obtained
with PKC-phosphorylated GRK5-DD (phosphorylated to a stoichiometry of
~1.9 mol/mol) (Fig. 5A, lanes 8-10),
demonstrating that the observed inhibition of GRK5 by PKC is not
mediated via reduced autophosphorylation of GRK5. Kinetic studies
revealed that phosphorylation of GRK5 by PKC caused an ~5-fold
increase in the Km (25.4 ± 3.1 µM for PKC-phosphorylated versus 5.5 ± 0.8 µM for control) and an ~2.8-fold reduction in
Vmax for rhodopsin (Fig. 5B). Thus,
phosphorylation by PKC reduces both the catalytic rate and affinity of
GRK5 for rhodopsin.
Since rhodopsin phosphorylation was dramatically reduced by PKC
phosphorylation of GRK5, we also tested the effect of PKC phosphorylation on non-receptor substrates. The ability of
PKC-phosphorylated GRK5 to phosphorylate soluble substrates such as
phosvitin and casein was also significantly inhibited (~75-85%)
compared to control GRK5 (data not shown). Moreover, similar results
were obtained with PKC-phosphorylated GRK5-DD, demonstrating again that
the reduced catalytic activity is not simply due to reduced autophosphorylation of GRK5. In contrast, there was no change in the
Km for ATP for PKC-phosphorylated and control GRK5
(data not shown). Thus, these results demonstrate that PKC phosphorylation reduces the catalytic activity of GRK5 toward both
receptor and non-receptor substrates. This is in contrast to ARK,
where PKC phosphorylation results in an ~1.6-fold reduction in the
ability of
ARK to phosphorylate a soluble peptide substrate (22) but
a 2-3-fold increase in rhodopsin phosphorylation (21, 22).
Previous studies have demonstrated that GRK5 can bind directly to
phospholipid membranes (12) as well as to ROS membranes in a
light-independent fashion (13). Here we tested the effect of PKC
phosphorylation on GRK5 binding to phospholipid vesicles and ROS
membranes. A small amount of GRK5 was pelleted when centrifuged alone,
while the addition of phospholipid vesicles significantly increased the
amount of pelleted GRK5 (from 16% to 36%) (Fig. 6A). Incubation of GRK5 with ROS membranes
resulted in an even larger increase in the amount of pelleted GRK5,
probably due to direct binding to rhodopsin (to 62%). PKC
phosphorylation of GRK5 did not change its binding to phospholipid
vesicles (33% versus 36%); however, a significant decrease
in binding to ROS membranes was observed (25% versus 62%)
(Fig. 6B). Thus, PKC phosphorylation of GRK5 does not affect
the ability of the kinase to bind to phospholipid vesicles, but it does
significantly reduce the direct binding of the kinase to receptor.
Again this is in contrast to ARK, where it has been suggested that
the enhancement in rhodopsin phosphorylation was the result of
increased membrane binding of
ARK (22), although it is not clear
whether this binding was to phospholipids or to the receptor
itself.
Conclusions
In summary, our findings suggest that PKC
phosphorylation may serve as a general mechanism for regulating GRK
function. PKC rapidly and stoichiometrically phosphorylates GRK5
resulting in a significant reduction in GRK5 activity. This is in
striking contrast with the ability of PKC to phosphorylate and activate ARK (20, 21). The differential regulation of
ARK and GRK5 by PKC
suggests that GRK diversity may play an important role in the
differential desensitization of various receptors. GRK expression
levels and subtype may also prove critical in determining how rapidly
receptor signaling is attenuated, in particular for those receptors
coupled to phospholipase C stimulation. For example, myocardial
ARK
and GRK5 appear to play an important role in regulating
2AR function, which is coupled to stimulation of cAMP
production (36, 37). In contrast, the myocardial type 1A angiotensin II
receptor, which stimulates phospholipase C and consequently PKC,
appears to be regulated by
ARK but not by GRK5. Similarly, recent
studies have demonstrated that coexpression of either the type 1A
angiotensin II or
1b-adrenergic receptors with
ARK
leads to increased agonist-induced receptor phosphorylation, while
coexpression of either receptor with GRK5 results in enhanced basal
phosphorylation but no significant agonist-induced phosphorylation of
these receptors (38, 39). Perhaps PKC activation by either angiotensin
II or
1b-adrenergic receptor stimulation may turn off
GRK5 activity.
ARK appears to be the predominant GRK in many cells (40, 41).
However, mRNA distribution reveals that GRK5 is particularly high
in lung, heart, skeletal muscle, placenta, colon, and retina (13, 24,
34). Moreover, Nagayama et al. (34) recently showed that
GRK5 is the predominant GRK in rat thyroid FRTL5 cells and is involved
in desensitization of the thyrotropin receptor. Interestingly,
overexpression of GRK6 in these cells has the same effect as GRK5, but
FRTL5 expresses little if any GRK6. Thus, the expression of GRK5 in
these cells is determined by reasons other than a simple requirement
for thyrotropin receptor desensitization. We speculate that the
expression of a particular GRK in a given cell is determined by a need
to integrate regulation of receptor desensitization in the cell with
other signals. In cells that express multiple GRKs, the relative
contribution of each GRK in the desensitization of a particular
receptor may well depend on the presence of other stimuli. Because PKC
activates
ARK and inhibits GRK5, stimulation of PKC would favor
signals regulated by GRK5 in such cells. Thus, phosphorylation by PKC
may play an important role in the heterologous regulation of GRK
activity.
We thank P. Kunapuli for valuable discussions and C. Stubbs for the purified protein kinase C preparations.