From the Howard Hughes Medical Institute Laboratories and
Departments of Medicine and Biochemistry, Cell Biology,
and ¶ Microbiology, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT |
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The G protein-coupled receptor kinase 2 (GRK2) is
a serine/threonine kinase that phosphorylates and desensitizes
agonist-occupied G protein-coupled receptors (GPCRs). Here we
demonstrate that GRK2 is a microtubule-associated protein and
identify tubulin as a novel GRK2 substrate. GRK2 is associated with
microtubules purified from bovine brain, forms a complex with tubulin
in cell extracts, and colocalizes with tubulin in living cells.
Furthermore, an endogenous tubulin kinase activity that copurifies with
microtubules has properties similar to GRK2 and is inhibited by
anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin
in vitro with kinetic parameters very similar to those for
phosphorylation of the agonist-occupied 2-adrenergic
receptor, suggesting a functionally relevant role for this
phosphorylation event. In a cellular environment, agonist occupancy of
GPCRs, which leads to recruitment of GRK2 to the plasma membrane and
its subsequent activation, promotes GRK2-tubulin complex formation and
tubulin phosphorylation. These findings suggest a novel role for GRK2
as a GPCR signal transducer mediating the effects of GPCR activation on
the cytoskeleton.
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INTRODUCTION |
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Agonist occupancy of G protein-coupled receptors
(GPCRs)1 facilitates the
exchange of bound GDP for GTP on heterotrimeric G proteins. The
activated GTP bound G protein then dissociates into its constituent
- and
-subunits, both of which can activate a variety of
different effector systems. The G protein-coupled receptor kinases
(GRKs), a family of serine/threonine kinases, play an important role in
regulating this signal transduction process (reviewed in Refs. 1-3).
GRKs specifically phosphorylate agonist-occupied GPCRs, which are the
only known substrates for these enzymes.
GRK-mediated phosphorylation of agonist-activated GPCRs promotes the
high affinity binding of cytosolic arrestin proteins (-arrestins) to
the receptors (4, 5).
-Arrestin binding has two functional
consequences. First, the binding of
-arrestin sterically inhibits
coupling of the receptor to its respective G protein (4, 5).
GRK-mediated receptor phosphorylation and
-arrestin binding thus
lead to diminished receptor signaling, i.e. receptor
desensitization (6). Second,
-arrestin binding initiates the
clathrin-mediated endocytosis (sequestration) of activated receptors
(7). GRK-mediated phosphorylation of activated GPCRs thus plays a
critical role in regulating both the activity and number of plasma
membrane receptors.
GRK2 is predominantly a cytosolic enzyme that becomes
membrane-localized following GPCR activation (8, 9). The
compartmentalization of GRK2 at the plasma membrane requires that its
carboxyl-terminal pleckstrin homology domain binds both
phosphatidylinositol 4,5-bisphosphate and the -subunits of
heterotrimeric G proteins (G
) (10, 11). Since the
membrane association of GRK2 requires free G
and the
release of G
from the
-subunit is catalyzed by
receptor activation, the membrane association of GRK2 is
agonist-dependent. Thus GRK2 activity is regulated by
several interdependent mechanisms. Agonist occupancy of the receptor
and the targeting of GRK2 to different cellular compartments by
G
regulate the rate of receptor phosphorylation by increasing the local GRK2 concentration. Additionally, allosteric activation of GRK2 occurs when it is complexed with G
and an activated receptor substrate (12, 13). This was demonstrated in vitro by measuring a potentiation of GRK-mediated
phosphorylation of a peptide substrate in the presence of activated
GPCR and G
(13). Thus, in addition to serving as GRK2
substrates, agonist-occupied GPCRs bind to and directly activate
membraneassociated GRK2.
The activation of membrane-associated GRK2 by agonist-occupied GPCRs suggests, potentially, the existence of a signaling pathway in which GRK2 is the effector. To date, however, no substrates for these enzymes other than the receptors themselves have been found. Accordingly, we sought to identify GRK2-binding proteins and potential substrates by performing overlay assays and by examining the intracellular distribution of GRK2 using fluorescence and immunoelectron microscopy. Nitrocellulose overlay assays, in which protein extracts immobilized on nitrocellulose are incubated with a protein probe, have been successfully used to identify a number of proteins that interact with the regulatory subunits of protein kinase A, termed A kinase anchoring proteins (reviewed in Ref. 14). Protein kinase C-binding proteins (receptors for activated protein kinase C) (reviewed in Ref. 15) and protein kinase C substrates (16-18) have also been identified using similar procedures. In this study, we identify tubulin as a GRK2-binding protein and a novel GRK2 substrate. The potential implications of GRK-mediated tubulin phosphorylation on GPCR function are discussed.
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EXPERIMENTAL PROCEDURES |
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Materials
Propranolol and isoproterenol were from Sigma or RBI. Anti-mouse and anti-rabbit antibodies were obtained from Sigma and Molecular Probes, Inc. Mouse monoclonal antibodies against the 12CA5 (hemagglutinin) epitope were purchased from Boehringer Mannheim, and monoclonal M2 anti-Flag® antibody was purchased from Kodak IBI. Cell culture media were purchased from Medtech, and fetal bovine serum was purchased from Atlanta Biologicals. Physiological buffers were from Life Technologies, Inc. Restriction enzymes were obtained from Promega or New England Biolabs, T4 DNA ligase from Promega, and Hot Tub DNA polymerase from Amersham Pharmacia Biotech. Plasmids containing variants of green fluorescent protein were purchased from CLONTECH.
Plasmid Construction
GRK2-Flag-- The Flag peptide sequence (DYKDDDDK) was inserted by site-directed mutagenesis before the C-terminal leucine residue of the GRK2 backbone residing in the vector pcDNA1/Amp. A cDNA fragment coding for the insert was ligated between the XhoI restriction site of GRK2 and the SalI site of pcDNA1/Amp and verified by sequencing.
GRK2-Flag-GFP-- A mutant GFP (pS65T-GFP) with a red shifted excitation spectrum and enhanced fluorescence compared with wild type GFP was attached to the C terminus of the Flag® epitope tagged GRK2 (19). The (TAA) stop codon following the C-terminal leucine was replaced using site-directed mutagenesis (20) with an in frame BamHI restriction site. The proximal HindII/XhoI fragment was ligated with the XhoI/BamHI fragment into (pS65T-GFP) between the HindIII/BamHI polylinker restriction sites.
Fractionation of Bovine Tissue Extracts
To survey for the presence of GRK2-binding proteins, various bovine tissues (frozen in liquid nitrogen), were thawed and homogenized (5 ml/g, wet weight) in buffer A (20 mM Tris, pH 7.2, containing 0.25 M sucrose, 5 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 10 mM benzamidine-HCl). Tissue samples were homogenized using a Polytron homogenizer, and nuclei were pelleted by centrifugation at 700 × g for 15 min. The supernatant, termed crude homogenate, was further fractionated into particulate and soluble fractions by centrifugation at 150,000 × g for 1 h. The resulting particulate fractions were resuspended in buffer A (5 ml/g of tissue in the original homogenate). All operations were performed at 4 °C. Protein concentrations were determined with Bradford reagent (Bio-Rad) using bovine serum albumin as a standard.
Overlay Method for Detection of GRK2-binding Proteins
GRK2-binding proteins were identified using a modification of a
procedure initially described by Leiser et al. (21).
Proteins in samples to be probed were separated by SDS-polyacrylamide
gel electrophoresis (22) and electrophoretically transferred to nitrocellulose membranes. The nitrocellulose filters were incubated in
blotto (10 mM potassium phosphate buffer, pH 7.4, 0.15 M NaCl, 5% (w/v) nonfat dry milk, and 0.02%
NaN3) for 1 h at 4 °C and subsequently washed three
times with binding buffer (100 mM Tris, pH 7.4, 50 mM NaCl). GRK2-binding proteins were detected by incubating the nitrocellulose filters with purified autophosphorylated GRK2. GRK2
(3 µM) purified from baculovirus-infected Sf9
cells, described by Kim et al. (23), was autophosphorylated
by incubation in 20 mM Tris, pH 7.5, 10 mM
MgCl2, 2.0 mM EDTA, 1 mM
dithiothreitol containing 60 µM ATP (~6000 cpm/pmol) at
30 °C for 30 min. Prior to incubation with the nitrocellulose
filters, the GRK2 was desalted over G25 columns (1 ml) to remove excess
[-32P]ATP. The 32P-labeled GRK2 (0.2 µM) was incubated with the nitrocellulose filters in
binding buffer for 1 h at 4 °C. Blots were washed extensively with binding buffer to reduce nonspecific binding and were subsequently exposed to x-ray film.
Purification of Taxol-precipitated Microtubules and Tubulin
Purified microtubules containing microtubule-associated proteins were prepared from homogenates of bovine brain using the antimitotic drug taxol as described by Vallee (24).
Purified tubulin was prepared from extracts of freshly isolated bovine
brain as described by Simon et al. (25). Briefly, brain was
homogenized at a ratio of 0.5 ml of buffer/g of tissue in 100 mM Pipes, pH 6.9, containing 2 mM EGTA and 1 mM MgSO4 (PEM buffer), that also contained 1 mM ATP and protease inhibitors. The homogenate was
centrifuged at 100,000 × g for 1 h at 4 °C, and the supernatant was diluted 1:1 with PEM containing 60% glycerol and 0.2 mM GTP (PEMG buffer). After a 45-min incubation to
polymerize tubulin, microtubules were collected by centrifugation at
100,000 × g for 45 min at 29 °C. The microtubule
pellet was processed through a second depolymerization/polymerization
step by cycling between 4 °C and 37 °C. The two-cycle purified
tubulin was subsequently purified to >99% homogeneity using
phosphocellulose chromatography as described by Voter and Erickson
(26). Purified tubulin was stored in aliquots at 80 °C until
use.
Western Blots
Western blots were performed by standard procedures using
monoclonal antibodies against GRK2 (27) and polyclonal or monoclonal antibodies directed against -tubulin (Sigma). Enhanced
chemiluminescence detection of antigens (DuPont) was achieved with
horseradish peroxidase-conjugated secondary antibodies (Amersham
Pharmacia Biotech).
Cell Culture and Transfection
Human embryonic kidney (HEK) 293 cells were maintained in
minimal essential medium or Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin/streptomycin in a 5%
CO2 incubator at 37 °C. Cells were transfected with
2.0-5.0 µg of plasmid containing GRK2-Flag-GFP cDNA using
coprecipitation with calcium phosphate (28). Cells were maintained in
100-mm dishes or transferred to 22-mm square, ethanol-sterilized
coverslips in six-well plates as necessary. Cell lines permanently
expressing GRK2-Flag-GFP or the GRK2-Flag construct were made using
G418 (Geneticin) selection (0.5 mg/ml) of calcium phosphate-transfected HEK-293 cells. Plasmids encoding bovine GRK2 (28) and the human M2
Flag-tagged 2-adrenergic receptor in pcDNAs (28)
were also used in this study.
Immunoprecipitation of GRK2 and Tubulin
Serum-starved HEK-293 cells overexpressing GRK2 or GRK2 and
2-adrenergic receptor were treated with agonists as
described in the figure legends. Medium was subsequently removed, and
cell monolayers were washed twice with ice-cold phosphate-buffered saline (PBS). Cells were subsequently lysed by scraping into 1% CHAPS-HEDN buffer (HEDN contained 10 mM Hepes, pH 7.2, 1 mM EDTA, 1 mM dithiothreitol, and 100 mM NaCl), 1 ml of buffer per 150-mm plate of 80% confluent
cells. Lysates were cleared by centrifugation at 15,000 × g for 15 min at 4 °C, and the supernatants incubated with
15 µg of immunoprecipitating antibody. A monoclonal anti-
-tubulin antibody (Sigma) (see Figs. 2 and 11) or a monoclonal anti-GRK2 antibody (27) (see Fig. 10) was used. Incubations were performed at
4 °C for 1 h in the presence of 50 µl of a 50% slurry of
protein A/G-Sepharose (Calbiochem). Following this incubation period, protein A/G-Sepharose-bound immune complexes were recovered by centrifugation and washed three times in CHAPS-HEDN. Proteins were
removed from the Sepharose beads with SDS-polyacrylamide gel
electrophoresis sample buffer (8% SDS, 25 mM Tris, pH 6.5, 10% glycerol, 5% mercaptoethanol, 0.003% bromphenol blue), resolved by electrophoresis on 12% acrylamide gels, and subjected to Western blot analysis.
Phosphorylation Reactions
Phosphorylation of Tubulin by the Microtubule-associated Tubulin
Kinase--
Taxol-precipitated microtubules (200 nM) were
incubated in a volume of 25 µl in 20 mM Tris, pH 7.5, 2.0 mM EDTA, 10 mM MgCl2, 1 mM dithiothreitol containing 60 µM
[-32P]ATP (~6000 cpm/pmol) (buffer B). Incubations
were performed at 30 °C for the times indicated in the figure
legends. Reactions were stopped by the addition of an equal volume of
SDS sample loading buffer and electrophoresed on 10%
SDS-polyacrylamide gels. The dried gels were subjected to
autoradiography and PhosphorImager (Molecular Dynamics) analysis to
determine the number of pmol of phosphate transferred to tubulin.
Incubation with protein kinase A inhibitor (10 µg/ml), staurosporine
(10 nM), heparin (5 µM), GTP (10 mM), and monoclonal antibodies (10 µg) was used to
elucidate the biochemical characteristics of the microtubule associated tubulin kinase.
GRK2-mediated Phosphorylation of Tubulin-- Phosphorylation reactions were performed essentially as described above with two exceptions. First, tubulin purified by phosphocellulose chromatography and devoid of endogenous kinase activity was used as a substrate. Second, purified recombinant GRK2 (50 nM) (23) was included in the phosphorylation reactions. Tubulin concentrations ranging between 0.03 and 0.9 µM were incubated for 10 min at 30 °C to determine the kinetic parameters for GRK2-mediated tubulin phosphorylation.
GRK2-mediated Phosphorylation of Receptor
Substrates--
Purified rod outer segment membranes (29) or purified
reconstituted -AR (10, 30) were incubated in buffer B at 30 °C with 50 nM GRK2. Phosphorylation reactions were incubated
and analyzed as described under "Phosphorylation of Tubulin by the Microtubule-associated Tubulin Kinase."
-AR concentrations ranging between 0.03 and 0.9 µM were incubated at 30 °C for 10 min to determine the kinetic parameters of GRK2-mediated
-AR
phosphorylation. A rhodopsin concentration of ~30 µM
was used in assays utilizing this substrate.
GRK-mediated phosphorylation of a soluble synthetic peptide
substrate--
A stock solution of the purified peptide (RRREEEEESAAA)
was prepared, and the pH was adjusted to 7.2 by the addition of Tris base. GRK-mediated peptide phosphorylation was determined by incubating peptide (10 µM to 1 mM) and GRK2 (50 nM) in 20 mM Tris-HCl, pH 7.2, 2 mM
EDTA, 7.5 mM MgCl2, and 60 µM
[-32P]ATP (~2000 cpm/pmol). The final reaction
volume was 25 µl, and incubations were performed at 30 °C for 15 min. Phosphorylation reactions were linear over this time period.
Reactions were stopped by spotting onto P-81 phosphocellulose paper
(2 × 2-cm squares). Free [
-32P]ATP was
subsequently removed by washing in 75 mM phosphoric acid as
described previously (31). GRK2-mediated peptide phosphorylation was
determined by subtracting the counts incorporated in the absence of
peptide from the counts incorporated in the presence of this substrate.
Phosphorylation of Cellular Tubulin
HEK-293 cells transiently overexpressing GRK2 and -AR were
starved in phosphate-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) for 2 h. These cells were subsequently
incubated in the same medium containing
[32P]orthophosphate (0.2 mCi/ml) for 2 h to label
intracellular pools of ATP. Cells were treated with the
-adrenergic
agonist isoproterenol (10 µM for 10 min), washed three
times with ice-cold PBS, and harvested in 20 mM Tris, pH
7.4, 2 mM EDTA containing protease inhibitors. Following a
low speed spin to remove nuclei (600 × g for 15 min),
membranes were prepared by spinning the clarified cellular homogenate
at 150,000 × g for 20 min. Tubulin was subsequently immunoprecipitated from these cells as described under
"Immunoprecipitation of GRK2 and tubulin."
Immunofluorescence, Interference-Contrast, and Video Microscopy
GRK2-Flag-GFP or GRK2-Flag-expressing HEK-293 cells transfected as described were plated onto ethanol-sterilized glass coverslips in growth medium at least 24 h prior to observation. Coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Antibody labeling or washing of fixed cells was performed at room temperature in a solution of PBS containing 0.008% saponin (w/v) and 1% bovine serum albumin at pH 7.2. A primary rabbit, anti-tubulin antibody originally raised against sea urchin tubulin, a gift of Dr. K. Fujiwara, was kindly provided by Prof. Harold Erickson (Duke University) and was used at a 1:1000 dilution. The mouse monoclonal M2 anti-Flag® epitope antibody was used to localize GRK2-Flag. All antibody incubations were performed at room temperature for 40-60 min with three or four washes following each incubation. Either fluorescein or Texas Red-conjugated secondary antibody (anti-mouse or anti-rabbit) was used as required at 1:250 dilutions. Coverslips were inverted, mounted on glass slides over a drop of PBS, and sealed with clear nail polish prior to viewing. Samples were observed with a Leica model DM50 epifluorescence microscope with one port connected to an Optronics VI-470 CCD video camera system with 768 × 494 active pixels set in manual gain mode. GRK2-Flag-GFP fluorescence and fluorescein fluorescence were visualized using a fluorescein (GFP) excitation and emission filter cube, whereas Texas Red was observed using a broad band excitation rhodamine cube. The electronic cell images obtained from the camera were printed using a Sony model UP-5600 MD color video printer with a UPK-5502SC digital interface board, and imported into Adobe Photoshop (2.5) using the accompanying Sony import module.
Sequestration
Flow cytometry analysis was performed as follows. GRK2,
GRK2-Flag, or GRK2-Flag-GFP was coexpressed in HEK-293 cells with the
12CA5 epitope-tagged Y326A mutant -AR (32). Cells were grown in
six-well Falcon dishes at a density of 250,000-400,000 cells/well with
equal seeding per well. Following aspiration and washing of each well
with serum-free medium, serum-free media with or without isoproterenol
was added at 37 °C for 30 min. The incubations were stopped by
aspiration of medium and the addition of ice-cold PBS to each well.
Following washing in PBS, the cells were incubated for 30 min with a
1:400 dilution of anti-12CA5 antibody in Dulbecco's modified Eagle's
medium at 4 °C, washed three times in cold PBS, incubated with a
1:250 dilution of goat anti-mouse R-phycoerythrin-conjugated antibody,
and then fixed and stored in 3% formaldehyde for flow cytometry.
50,000 cells were analyzed for each condition using 520-nm
excitation.
Immunoelectron Microscopy of HEK-293 Cells for GRK2-Flag and Tubulin
Confluent 100-mm dishes of permanently transfected, GRK2-Flag-expressing HEK-293 cells or untransfected cells were fixed for 20 min with 4% paraformaldehyde/PBS, washed in PBS, and treated at room temperature for 60 min with a 0.008% saponin, 1% bovine serum albumin PBS solution containing a 1:500 dilution of M2 anti-Flag antibody or a 1:1000 dilution of rabbit anti-tubulin antibody. They were then washed three times with PBS to remove free antibody and prepared for electron microscopy as follows. Cells were pelleted, further fixed in paraformaldehyde in 200 mM Pipes, pH 7.0, coated with agar to hold them together, infiltrated with 2.1 M sucrose for cryoprotection, placed onto stubs, and then snap-frozen in liquid nitrogen. They were stored in a liquid nitrogen freezer until sectioned. Ultrathin cryosections were cut on a Reichert-Jung ultracut E, equipped with an FC4 cryochamber (Leica, Deerfield, IL). Sections were collected on Formvar and carbon-coated nickel grids, incubated on 5% fetal calf serum in PBS, and followed by 50 mM ammonium chloride in PBS. Grids not previously treated with anti-tubulin primary antibody were incubated over a 1:100 dilution of rabbit anti-tublulin antibody for 1 h at room temperature and washed. Grids were further labeled by incubation with goat anti-mouse and goat anti-rabbit IgG conjugated with either 5-nm or 10-nm colloidal gold (Aurion) at a 1:10 dilution. After thorough washing in PBS followed by washing in water, they were embedded in a 9:2 mixture of 2.1 M methyl cellulose and 2% aqueous uranyl acetate. Grids were viewed in a Philips EM300 electron microscope.
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RESULTS AND DISCUSSION |
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Detection of GRK2-interacting Proteins by Protein Overlay-- Crude extracts (C) derived from various bovine tissues, together with a soluble (S) and a particulate (P) fraction derived from this extract (Fig. 1A) were subjected to electrophoresis on SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose. The nitrocellulose filters were subsequently incubated with a purified preparation of autophosphorylated 32P-labeled GRK2. Following extensive washing, GRK2 retained on the filter was detected by autoradiography. As shown in Fig. 1A, very few GRK2-binding proteins were detected under these conditions. GRK2 was retained on the filter by proteins of 55-kDa present in the crude extracts and particulate fraction derived from bovine brain and retina. Additionally, a 42-kDa GRK2-binding protein was detected in the crude and particulate fraction derived from bovine heart. A similar pattern of GRK2 binding proteins was obtained when nitrocellulose filters were incubated with unphosphorylated GRK2, and bound GRK2 was detected immunologically (data not shown).
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GRK2 Is Associated with Tubulin in Intact Cells-- Tubulin is an extremely abundant protein representing approximately 5% of the total protein content of brain. Furthermore, in the nitrocellulose overlay assay, native GRK2 binds to denatured, immobilized tubulin. In light of these observations, is the interaction between GRK2 and tubulin of physiological significance? That GRK2 binds to native tubulin and that this interaction occurs in intact cells is shown in Figs. 2, 3, 5, and 6.
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Tubulin Kinase and GRK2 Have Similar Biochemical
Properties--
Microtubules consist of a core cylinder built from
heterodimers of - and
-tubulin monomers (36). As many as six
different genes encode for
- and
-tubulin, a heterogeneity that
is further increased by the postranslational modification of these
proteins (37). One such post-translational modification is
phosphorylation. Phosphorylation of tubulin on both
- and
-subunits has been reported, although to date only class
III
-tubulin from adult brain has been shown to be phosphorylated
in vivo (38, 39). The identity of the serine/threonine
kinase responsible for this phosphorylation event remains obscure.
Ca2+/calmodulin-dependent protein kinase (40),
casein kinase I (41), and casein kinase II (42, 43) can phosphorylate
tubulin in vitro, although it is currently unknown if these
kinases phosphorylate tubulin in vivo. A tubulin kinase
activity with biochemical properties similar to casein kinase II has,
however, been reported to copurify with microtubules (44). Indeed, we
find that the addition of Mg2+/ATP to a taxol-precipitated
microtubule preparation is sufficient to promote tubulin
phosphorylation (Fig. 7). Since GRK2 is
tightly associated with tubulin (Figs. 2, 3, 5, and 6), could GRK2 be a
tubulin kinase, possibly even the main microtubule-associated tubulin
kinase? To investigate this possibility, the biochemical characteristics of the microtubule-associated tubulin kinase were compared with those of purified GRK2. Protein kinase A inhibitor, staurosporine (a protein kinase C inhibitor), heparin (an inhibitor of
casein kinase II (45) and members of the GRK family (46)), and GTP were
used as potential inhibitors of either tubulin phosphorylation mediated
by the endogenous microtubule-associated tubulin kinase (Fig.
8A, light bars) or
rhodopsin phosphorylation mediated by purified GRK2 (Fig.
8A, dark bars). The addition of excess unlabeled GTP was used to determine the phosphoryl donor specificity of the
endogenous tubulin kinase. Since GRK2 utilizes exclusively ATP (46),
while casein kinase II can use both ATP and GTP as phosphate donors
(45), GTP was utilized in this study to distinguish the activities of
these two enzymes.
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GRK2 Phosphorylates Tubulin in Vitro--
The tubulin kinase
activity of GRK2 was examined in vitro using purified
proteins. Tubulin devoid of microtubule-associated proteins was
purified using reversible, temperature-dependent assembly
and phosphocellulose chromatography (25). As shown in Fig.
9, this highly purified tubulin
preparation is essentially free of endogenous tubulin kinase activity
(open symbols). The addition of purified GRK2 promotes
rapid, stoichiometric phosphorylation of tubulin (Fig. 9, closed
symbols). The maximal stoichiometry of phosphorylation approaches
1.0 mol of Pi/mol of tubulin, i.e. 2 mol
Pi incorporated per mol of /
heterodimer.
Furthermore, the kinetic parameters for GRK2-mediated tubulin
phosphorylation are similar to those for GRK2-mediated phosphorylation
of agonist-occupied GPCRs, the only previously identified physiological
substrates for GRK2. Table I lists the
kinetic parameters for GRK2-mediated phosphorylation of tubulin
together with those for GRK2-mediated, isoproterenol-stimulated,
-AR
phosphorylation. Notably, the Km for GRK2-mediated
tubulin phosphorylation is ~1.0 µM, while that for
GRK2-mediated phosphorylation of the best peptide substrate is ~1340
µM (Table I and Ref. 31). Tubulin thus represents an
approximately 1000-fold better substrate for GRK2 than peptide, suggesting that the tertiary structure of tubulin plays an important role in mediating the interaction with GRK2.
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Agonist Occupancy of GPCRs Promotes GRK2-tubulin Complex Formation
and Tubulin Phosphorylation--
Consistent with the model outlined
above, agonist occupancy of GPCRs promotes GRK2-tubulin complex
formation and tubulin phosphorylation in intact cells (Figs.
10 and
11). In HEK-293 cells transiently overexpressing GRK2, agonist occupancy of endogenously expressed GPCRs
dramatically enhances the amount of tubulin associated with GRK2 as
assessed by coimmunoprecipitation (Fig. 10). Activation of the -AR,
lysophosphatidic acid, and thrombin receptors increases the amount of
tubulin present in GRK2 immunoprecipitations by approximately 8-fold.
That this agonist-induced association of GRK2 and tubulin is
accompanied by increased tubulin phosphorylation is shown in Fig. 11.
Cells transiently overexpressing the
-AR were labeled with
orthophosphate and incubated for 10 min in the presence or absence of
isoproterenol (a
-AR agonist). Tubulin was subsequently
immunoprecipitated from either a whole cell lysate or a membrane
fraction derived from these cells and immunoprecipitates subjected to
autoradiography. Agonist occupancy of the
-AR promoted an
approximately 2-fold increase in the 32P content of total
cellular tubulin (data not shown). More dramatically, however, an
approximately 9-fold increase in the 32P content of
membrane-associated tubulin was observed upon GPCR activation (Fig.
11). That tubulin present in cellular membranes is specifically
phosphorylated following
-AR activation may potentially be explained
by the observations that (i) GRK2 associates with the plasma membrane
following GPCR activation (8, 9) and (ii) that phosphorylated tubulin
preferentially associates with lipid vesicles (47).
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GRK2-mediated Tubulin Phosphorylation: Potential Physiological
Significance--
GRK2-mediated -AR phosphorylation plays a
critical role in mediating rapid agonist-induced receptor
desensitization (reviewed in Refs. 1-3) and targets the
-AR for
internalization (7). Does this enzyme have additional cellular
functions? In this report we demonstrate that GRK2 associates with
microtubules and with soluble tubulin in a cellular extract and in
living cells and that tubulin represents an excellent substrate for
this enzyme in vitro. Notably, the kinetic parameters of
GRK2-mediated tubulin phosphorylation mirror those of GRK2-mediated
-AR phosphorylation, a physiological substrate of this enzyme, and
far surpass those of peptide substrates. Agonist occupancy of GPCRs
promotes GRK2-tubulin complex formation and tubulin phosphorylation.
Taken together, these observations suggest a potential role for GRK2 in
modulating the phosphorylation status of tubulin in intact cells.
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ACKNOWLEDGEMENTS |
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We thank Ron Uhing and W. Carl Stone for
purified 2-adrenergic receptor, Grace P. Irons and Linda
Czyzyk for virus and cell culture, and Donna Addison and Mary Holben
for excellent secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL16037 (to R. J. L), NS 19576 (to M. G. C.), and HL 03422 (to L. S. B.) and a Bristol Myers Squibb unrestricted grant award (to M. G. C.).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.
§ Recipient of a Michael Smith Postdoctoral Fellowship from the Medical Research Council Canada. Present address: The John P. Robarts Research Institute, P.O. Box 5015, 100 Perth Dr., London, Ontario N6A 5K8, Canada.
To whom correspondence should be addressed: Duke University
Medical Center, Box 3281, Durham, NC 27710. Tel.: 919-684-2974; Fax:
919-684-8875.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; GRK, G protein-coupled receptor kinase;
-AR,
2-adrenergic receptor; HEK, human embryonic
kidney; PBS, phosphate-buffered saline; G
, the
subunits of
heterotrimeric G proteins; GFP, green fluorescent protein; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes,
piperazine-N,N'-bis(2-ethanesulfonic acid).
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REFERENCES |
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