From the Departments of Biochemistry & Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Although the -adrenergic receptor kinase
(
ARK) mediates agonist-dependent phosphorylation and
desensitization of G protein-coupled receptors, recent studies suggest
additional cellular functions. During our attempts to identify novel
ARK interacting proteins, we found that the cytoskeletal protein
tubulin could specifically bind to a
ARK-coupled affinity column.
In vitro analysis demonstrated that
ARK and G
protein-coupled receptor kinase-5 (GRK5) were able to
stoichiometrically phosphorylate purified tubulin dimers with a
preference for
-tubulin and, under certain conditions, the
III-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of
ARK contains the primary tubulin binding determinants. In vivo interaction of GRK and
tubulin was suggested by the following: (i) co-purification of
ARK
with tubulin from brain tissue; (ii) co-immunoprecipitation of
ARK
and tubulin from COS-1 cells; and (iii) co-localization of
ARK and
GRK5 with microtubule structures in COS-1 cells. In addition,
GRK-phosphorylated tubulin was found preferentially associated with the
microtubule fraction during in vitro assembly assays
suggesting potential functional significance. These results suggest a
novel link between the cytoskeleton and GRKs that may be important for
regulating GRK and/or tubulin function.
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INTRODUCTION |
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Tubulin, along with a variety of associated proteins, constitute
microtubules, a major component of the cytoskeleton. Tubulin exists
principally in two forms, as either cytosolic soluble tubulin heterodimers consisting of various - and
-tubulin isotypes or as
insoluble assembled tubulin polymers (microtubules) (1). The ability of
tubulin to cycle between these two states is one of its most
fundamentally important features. In cells, microtubules are involved
in such diverse and dynamic functions as maintaining cell shape,
endocytosis, exocytosis, vesicle trafficking, cellular transport, and
mitosis (1). These dynamic functions require the coordinated regulation
of microtubule function and assembly, a process thought to reflect
integration of various extracellular signals (2). Indeed, many studies
have revealed functional relationships between tubulin and various
cellular signaling molecules. For example, tyrosine kinases such as the
insulin receptor and c-Src, as well as second messenger-responsive
kinases such as the cAMP-dependent and
Ca2+/calmodulin-dependent kinases, can regulate
microtubule function and/or assembly through phosphorylation (Ref. 3
and references therein). Moreover, a series of recent studies have
demonstrated direct binding interactions between G
and G
subunits and tubulin (4-6) suggesting new modes of regulation for
microtubule assembly. Other studies provide examples of tubulin or
microtubules regulating either the activity or the localization of
signaling molecules such as the A1 adenosine (7) and
-aminobutyric acid (8) receptors, phospholipase C-
1 (9), and
Ki-Ras (10). Taken together, these studies provide support for a novel
paradigm whereby information flow appears to be bi-directional between
signaling and cytoskeletal molecules.
-Adrenergic receptor kinase
(
ARK)1 is a member of a
family of G protein-coupled receptor kinases (GRKs) that includes
rhodopsin kinase (GRK1),
ARK (GRK2),
ARK2 (GRK3), GRK4, GRK5, and
GRK6. GRKs phosphorylate the agonist-activated form of G
protein-coupled receptors which in turn promotes the high affinity
binding of a second family of proteins, called arrestins (11). This
process functions to both uncouple the G protein from the receptor and to promote receptor endocytosis via clathrin-coated pits.
ARK is one
of the best characterized GRKs and has been shown to be important for
the phosphorylation and desensitization of a variety of receptors (11).
Moreover,
ARK activity appears to be regulated through interactions
with both free heterotrimeric G
subunits (12, 13) and negatively
charged membrane phospholipids (14-16). These interactions are thought
to be important largely for their ability to promote translocation of
ARK to the plasma membrane, facilitating interaction with receptor
substrates. Recent studies have provided novel information regarding
the function and cellular localization of
ARK. Specifically, it was
shown that
ARK can traffic along with
2-adrenergic
receptors to the endosome following receptor activation (17). Mayor and
co-workers (18) have also demonstrated an association of
ARK with
microsomes that appears to be mediated via an unidentified
ARK-binding protein. Furthermore,
ARK has been implicated to
function in developmental processes involved in cardiogenesis through
currently undefined mechanisms (19). Given these and other areas
of inquiry, it seems likely that additional cellular substrates and
regulators of
ARK exist. Thus, in this study a biochemical approach
was employed to explore this possibility. We describe here the
identification of tubulin as a novel GRK substrate and present
biochemical and cellular characterization of GRK/tubulin
interaction.
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EXPERIMENTAL PROCEDURES |
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Materials--
Dodecylmaltoside, urea, and protein A-agarose
were from Boehringer Mannheim, and [32P]ATP was from NEN
Life Science Products. Anti--tubulin monoclonal antibody and
enhanced chemiluminescence (ECL) reagents were from Amersham Pharmacia
Biotech. Polyvinylidene difluoride membrane was purchased from Applied
Biosystems. Nitrocellulose was from MSI, and
LipofectAMINETM and cell culture medium were from Life
Technologies Inc. Horseradish peroxidase-conjugated goat anti-mouse and
goat anti-rabbit antibodies and Affi-Prep 10 affinity chromatography
matrix were from Bio-Rad. Horseradish peroxidase-conjugated horse
anti-mouse antibody was from Vector Laboratories. Hemagglutinin
(HA)-specific polyclonal antibody was from Babco. GRK5-specific
polyclonal antibody was from Santa Cruz Biotechnology. Fluorescein
isothiocyanate-conjugated anti-mouse antibody,
tetramethylrhodamine-conjugated anti-rabbit antibody, and
SlowfadeTM mounting medium were from Molecular Probes Inc.
pGEX-2T GST gene fusion vector was from Amersham Pharmacia Biotech.
ATP, GTP, glutathione-agarose beads, paclitaxel (Taxol), crude soybean
phosphatidylcholine,
-mercaptoethanol, iodoacetic acid, and all
other chemicals were from Sigma.
Protein Expression and Purification--
ARK and GRK5 were
overexpressed in and purified from Sf9 insect cells (20, 21).
Purified rhodopsin kinase was generously provided by Drs. J. Pitcher
and R. Lefkowitz, and purified G
1
2 was
generously provided by Dr. S. P. Kennedy. Partially purified tubulin (PPT) containing microtubule-associated proteins was prepared from porcine brain by one to three cycles of
temperature-dependent microtubule assembly/disassembly as
described previously (6, 22). Purified tubulin, prepared from bovine
brain by assembly/disassembly followed by phosphocellulose
chromatography and 99% free of microtubule associated proteins (23),
was from Cytoskeleton and microSuppliers.
Gel Electrophoresis and Immunoblotting--
SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) was performed using standard methods
(24). Following electrophoresis, proteins were electroblotted onto
nitrocellulose. The membrane was then incubated in blocking buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Tween
20, and 5% non-fat dried milk) at 22 °C for 1 h followed by
probing with either an -tubulin-,
ARK-, GRK5-, or
arrestin-specific primary antibody diluted in blocking buffer.
Membranes were subsequently probed with a 1:2000 dilution of
horseradish peroxidase-conjugated secondary antibody in blocking buffer
for 20-60 min at 22 °C. All blots were developed by ECL following
the manufacturer's guidelines.
Synthesis of ARK-coupled Affinity Resin--
50 ml of
Affi-Prep 10 affinity chromatography matrix (20% v/v) was incubated
with or without
ARK at a final concentration of 0.3 mg/ml in 20 mM HEPES, pH 7.2, 5 mM EDTA, 0.02% Triton
X-100 for 5 h for 4 °C. The coupling reaction was stopped by
addition of 5 ml of 1 M Tris-HCl, pH 7.5, and incubation
overnight at 4 °C. A trace amount of
[35S]methionine-labeled
ARK was included in the
initial coupling reaction to enable a determination of coupling
efficiency (~1 mg
ARK/ml resin). The resin was then washed
extensively with 20 mM HEPES, pH 7.2, 200 mM
NaCl, 5 mM EDTA, 0.02% Triton X-100 and stored at
4 °C.
Identification of ARK-binding Proteins--
Fresh bovine calf
brain was stripped of connective tissue and minced in 200 ml of
homogenization buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 5 mM
benzamidine, 5 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 0.02% Triton X-100) using a Brinkman
Polytron (14,000 rpm, 30 s). The homogenate was centrifuged at
45,000 × g for 20 min and the resulting supernatant at
300,000 × g for 60 min. The final supernatant was
aliquoted and stored at
70 °C until use. One ml of
ARK-coupled
affinity resin (20% v/v) or control resin was incubated with 10 ml of
the soluble brain extract (or buffer) for 2 h at 4 °C. The
incubation mixture was centrifuged at 1000 × g for 1 min, and the pellet was washed four times with buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.02% Triton X-100). Bound proteins were released from the matrix by addition of 50 µl of SDS sample buffer and 100 µl of water to the final pellet followed by boiling for 10 min. The
samples were then subjected to 7.5% SDS-PAGE, and the separated
proteins were transferred to a polyvinylidene difluoride membrane. A
specific 55-kDa protein band was identified by Ponceau S staining,
excised, and then submitted to the Harvard Microchemistry Facility for
sequence analysis.
Phosphorylation Assay--
Phosphorylation reactions contained,
in a total reaction volume of 20-50 µl, 0.025-0.2 µM
ARK, GRK5, or rhodopsin kinase, 0.025-8 µM purified
tubulin dimers, 100 µM [
-32P]ATP
(2.0-7.5 cpm/fmol), 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 7.5 mM MgCl2 in the
absence or presence of 0.2 µM
G
1
2 and phosphatidylcholine (20 µg)-dodecylmaltoside (10 mM) mixed micelles (16).
Reactions were incubated at 30 °C for 5-90 min, stopped with SDS
sample buffer, and subjected to 10% SDS-PAGE. After autoradiography, the 32P-labeled tubulin bands were excised and counted to
determine the picomoles of phosphate transferred.
Identification of Tubulin Isotypes
Phosphorylated--
Phosphorylation reactions were performed as above
using either 0.025 or 2.5 µM ARK or GRK5 and 4.5 µM tubulin dimers in a total volume of 20 µl at
30 °C for 15 min. Phosphorylated tubulin was then reduced and
carboxymethylated essentially as described previously (25). Briefly,
0.5 M Tris-HCl, pH 8.6, 5 mM EDTA, 8 M urea, and 120 mM
-mercaptoethanol was
added to each phosphorylation reaction in a final volume of 100 µl
and incubated under nitrogen at 22 °C for 2 h. 250 mg/ml
iodoacetic acid, dissolved in 1 N NaOH, was then diluted
1:10 into the reduced tubulin solutions and incubated at 22 °C in
the dark for 30 min. Reactions were stopped with SDS sample buffer,
boiled, and subjected to 7.5% SDS-PAGE, Coomassie Blue staining, and
autoradiography.
GST-ARK Fusion Binding Assay--
Purified GST-
ARK fusion
proteins containing either the N-terminal domain (residues 1-184), the
central catalytic domain (residues 185-467), or the C-terminal domain
(residues 466-689) of
ARK were generated essentially as described
previously (26). Ten µg of GST or GST-
ARK fusion proteins
immobilized on glutathione-agarose beads were incubated with 0.4 µg
of soluble tubulin dimers in 100 µl of binding buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, and 0.02% Triton X-100) at 30 °C for 60 min.
Reactions were then chilled on ice for 5 min followed by precipitation
in a microcentrifuge for 10 s. The pellet was washed three times with 400 µl of binding buffer and then boiled with SDS sample buffer.
Samples were subjected to 10% SDS-PAGE and Western blotting using a
monoclonal
-tubulin-specific antibody.
Binding and Phosphorylation of Taxol-stable Microtubules by
GRKs--
Tubulin assembly reactions contained, in a total volume of
50 µl, 60 µg of tubulin (or control buffer), 1 mM GTP,
30% glycerol, and 10 µM Taxol in 100 mM
PIPES, pH 6.9, 1 mM EGTA, 1 mM
MgCl2. Reactions were incubated at 37 °C for 30 min
followed by pelleting at 150,000 × g for 10 min at
37 °C. The supernatant was removed and the Taxol-stable microtubule
pellets were gently resuspended in 50 µl of reaction buffer (100 mM PIPES, pH 6.9, 1 mM EGTA, 7.5 mM
MgCl2, 100 µM [-32P]ATP (5 cpm/fmol), 1 µM Taxol) in the absence or presence of 5 µg of
ARK or GRK5 and incubated for 30 min at 30 °C. Tubulin was then repelleted, and both the supernatant and pellet fractions were
boiled with SDS sample buffer and subjected to 10% SDS-PAGE, Coomassie
Blue staining, and densitometry. After autoradiography, the
32P-labeled tubulin bands were excised and counted to
determine the picomoles of phosphate transferred.
Cell Culture and Transfection--
COS-1 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin
sulfate at 37 °C in a humidified atmosphere containing 5%
CO2. Cells grown to 75-95% confluence were transfected
with 10 µg of either pcDNA3, pcDNA3-ARK, pBC12BI, or
pBC-GRK5 DNA using 65 µl of LipofectAMINETM per T75 flask
according to the manufacturer's instructions. For immunoprecipitation
experiments, cells were harvested 60 h after transfection. For
immunofluorescence experiments, cells were trypsinized and plated onto
12-mm glass coverslips in a 24-well dish 2 h after transfection
and analyzed at 24-36 h after transfection.
Immunoprecipitation--
COS-1 cells were transfected either
with pcDNA3 or pcDNA3-ARK as described above. At 60 h
after transfection, T75 flasks were rinsed with PBS, and the cells were
scraped into 1 ml of extraction buffer (10 mM Tris-HCl, pH
7.4, 1 mM EDTA, 5 mM dithiothreitol, 100 mM NaCl, 0.02% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
pepstatin A, and 10 µg/ml aprotinin) and lysed by freeze-thaw at
70 °C. Lysates were then centrifuged at 55,000 × g for 1 h at 4 °C, and the supernatant was stored at
70 °C. For immunoprecipitation, 100 µl of lysate was incubated with either a
ARK- or HA-specific polyclonal antibody or in the absence of antibody for 30 min at 4 °C followed by addition of 50 µl of 50% protein A-agarose pre-equilibrated in extraction buffer
and an additional 60 min incubation at 4 °C. Samples were then
centrifuged at 2000 rpm for 30 s in a microcentrifuge. The pellet
was washed three times with extraction buffer for 30 min at 4 °C and
then eluted in 50 µl of SDS sample buffer. Samples were subjected to
10% SDS-PAGE and Western blot analysis using either
ARK-specific or
-tubulin-specific monoclonal antibodies.
Immunofluoresence Microscopy--
Transfected COS-1 cells grown
on glass coverslips were rinsed twice with PBS at 37 °C and fixed
with methanol at 20 °C for 10 min. Alternatively, some cells were
preincubated with Dulbecco's modified Eagle's medium containing 10 µM (
)-isoproterenol for either 20 or 60 min at 37 °C
prior to fixing. Fixed cells were subsequently rinsed three times with
phosphate-buffered saline (PBS) and permeabilized with PBS containing
0.2% Triton X-100 for 10 min at 22 °C. These cells were blocked for
30 min at 37 °C with PBS containing 0.05% Triton X-100 (PBS/Triton)
and 5% non-fat dried milk and subsequently incubated with an
-tubulin-specific mouse monoclonal antibody and either a
ARK-,
GRK5-, or arrestin-3-specific rabbit polyclonal antibody diluted in the
same buffer for 1 h at 37 °C. Cells were rinsed three times
with PBS/Triton and then incubated at 37 °C for 30 min. The cells
were then incubated with a tetramethylrhodamine-conjugated goat
anti-mouse antibody and a fluorescein isothiocyanate-conjugated goat
anti-rabbit antibody both diluted 1:100 in PBS/Triton and 5% non-fat
dried milk for 1 h at 37 °C. Cells were rinsed five times with
PBS/Triton and then incubated at 37 °C for 30 min followed by fixing
with methanol at
20 °C for 5 min. Cells were rinsed with PBS and
mounted on a slide with SlowfadeTM mounting medium.
Fluorescence microscopy was performed on a Bio-Rad MRC600 laser
scanning confocal microscope (Hemmelholsteadt, UK) attached to a Zeiss
Axiovert 100 microscope, using a Zeiss Plan-Apo 63 × 1.40 NA oil
immersion objective at a zoom factor of 1.4.
Assembly of GRK-phosphorylated Tubulin Dimers--
Purified
tubulin dimers were subjected to a 10 min pre-spin at 150,000 × g at 4 °C to precipitate any existing tubulin aggregates. The supernatant was removed and utilized in the subsequent
phosphorylation assay. Phosphorylation reactions (40 µl total) were
incubated at 30 °C for 45 min and contained 60 µg of tubulin
dimers, 200 µM GTP, 100 mM PIPES, pH 6.9, 1 mM EGTA, and 3 mM MgCl2, in the absence or presence of 100 µM [-32P]ATP
(5 cpm/fmol) and either 4 µg of
ARK or GRK5. Assembly of phosphorylated or control tubulin was initiated by the addition of
glycerol (5-30% final concentration) and raising the temperature to
37 °C for 5-60 min. Assembly was quantitated by pelleting the microtubules at 150,000 × g for 10 min at 37 °C.
The supernatant and pellet fractions were then subjected to 10%
SDS-PAGE, Coomassie Blue staining, and densitometry. Distribution of
the 32P-labeled tubulin was assessed by autoradiography,
followed by excision and counting of the radiolabeled bands.
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RESULTS AND DISCUSSION |
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Identification of Tubulin as a ARK-interacting Protein--
In
an effort to identify novel GRK-interacting proteins, a crude bovine
brain cytosolic extract was chromatographed over a
ARK affinity
column. The column was washed extensively, and bound proteins were
eluted with boiling SDS sample buffer and subsequently subjected to
SDS-PAGE and Coomassie Blue staining. This strategy identified a total
of six proteins that specifically bound to the
ARK column (Fig.
1). The predominant interacting protein migrated at ~55 kDa, and additional proteins of ~50, ~46, ~44, ~38, and ~35 kDa were also observed. Since
ARK is known to bind G
subunits, we initially probed this blot using a
common antibody. This revealed that the ~35-kDa
protein was a G
subunit (data not shown).
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Phosphorylation of Tubulin by GRKs--
The ARK/tubulin
interaction was confirmed by the ability of purified
ARK to rapidly
phosphorylate highly purified bovine brain soluble tubulin dimers to a
stoichiometry of ~1.0 mol of Pi/mol of tubulin dimer
(Fig. 2). Addition of
ARK activators such as G
1
2 or
phosphatidylcholine-dodecylmaltoside mixed micelles increased the
stoichiometry to ~1.5 and ~1.6 mol/mol, respectively, or to ~2.0
mol/mol when added together. Addition of 10 mM
dodecylmaltoside alone, however, was without effect (data not
shown).
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In Vitro Interaction of GRKs with Soluble and Assembled
Tubulin--
Since the GRK/tubulin association was discovered via
their direct binding (Fig. 1), we were interested in further
characterizing this aspect of the interaction. Thus, we assessed the
binding of soluble tubulin dimers to GST-ARK fusion proteins
containing either the N-terminal, central catalytic, or C-terminal
domains of
ARK. When these GST-
ARK fusions were incubated with
purified soluble tubulin dimers and subsequently precipitated with
glutathione-agarose beads, it was found that the central catalytic
domain of
ARK (residues 185-467) contained the major binding
determinant for tubulin (Fig. 4). These
data are consistent with the ability of multiple GRKs to phosphorylate
tubulin as the catalytic domain is the most highly conserved region
among the GRKs (11). Whereas the N-terminal domain possessed a small
amount of specific binding, the C-terminal GST-
ARK fusion which
includes the PH domain and G
binding determinants demonstrated no
tubulin binding capacity. This raises the interesting possibility that
ARK, which appears to have distinct regions for interaction with
G
(the C-terminal domain) and tubulin (the catalytic domain), may
be able to concomitantly associate with both tubulin and G
to
form a
ARK·G
·tubulin ternary complex. This is consistent
with our observation that the apparent affinity of
ARK for tubulin
is increased ~2-fold by the presence of G
(Table I) and is
further supported by the recent demonstration that G
can directly
associate with microtubules (6). These observations suggest potentially
interesting implications for
ARK regulation.
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In Vivo Interaction of GRKs with Tubulin--
To address the
potential physiological relevance of the GRK/tubulin interaction, we
assessed the in vivo association of GRKs and tubulin using
three distinct approaches. First, a partially purified tubulin
preparation (PPT) was generated from porcine brain tissue by two
successive cycles of tubulin assembly (at 37 °C) and disassembly (at
4 °C) each followed by centrifugation. In this way tubulin, as well
as microtubule-associated proteins, are significantly enriched within
one or two cycles (22). This enrichment was quantitated by subjecting
varied amounts of PPT to SDS-PAGE, Coomassie Blue staining, and
densitometry revealing that tubulin represents ~93% of the protein
in this preparation, and the remaining ~7% is composed of
microtubule-associated proteins and other associated proteins (data not
shown). We then tested various amounts of PPT for the presence of
ARK by Western blotting using a
ARK-specific antibody. This
analysis revealed a significant amount of
ARK present in the PPT
preparation (0.05-0.1% of the total protein, Fig.
5A). A similar analysis of the
initial soluble porcine brain extract revealed that
ARK is present
at ~0.02% of the total protein (data not shown) suggesting a
2.5-5-fold enrichment of
ARK in PPT. Analysis of equal amounts of
the warm (37 °C) and cold (4 °C) supernatant and pellet fractions
revealed that
ARK is associated to a similar extent with the
"microtubule fractions" (i.e. the warm pellet and cold
supernatant) and the "non-microtubule fractions" (i.e.
warm supernatant and cold pellet). Given the above data that suggest
ARK may bind to both soluble and assembled forms of tubulin, it is
not surprising that
ARK does not strictly associate with the
microtubule fraction. Even so, a significant amount of
ARK is
present in the 2-cycle PPT (Fig. 5A) as well as in a 3-cycle
PPT (data not shown). Thus, the presence of
ARK in these tubulin
preparations is suggestive of an in vivo association between
ARK and tubulin that by necessity includes association with
microtubules, as
ARK does not precipitate on its own under these
conditions (data not shown). In order to demonstrate the specific
nature of the
ARK/tubulin association, the arrestins present in the
brain extract and PPT were also quantitated. While arrestins were found
to be expressed at ~10-fold higher levels than
ARK (~0.2% of
the protein in the pig brain extract), no arrestin was detected in 40 µg of PPT (data not shown).
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Analysis of the Functional Role of GRK/Tubulin Interaction-- While providing important in vitro and in vivo evidence for GRK/tubulin association, the above experiments fail to define any functional role for this interaction. Formally, three principal possibilities exist with respect to the functional significance of this interaction: (i) GRKs might regulate the assembly of tubulin; (ii) GRKs might regulate one or more of the many diverse functional properties of microtubules (i.e. independently of effects on tubulin assembly); or (iii) tubulin and/or microtubules may effect GRK function by regulating its cellular localization. Importantly, none of these possibilities are mutually exclusive, allowing the potential for complex functional relationships in cells.
Whereas the cellular functions of tubulin are diverse and complex, the ability of tubulin to dynamically cycle between soluble and assembled states is a fundamental property readily amenable to in vitro analysis (1, 2, 22, 23). Indeed, tubulin assembly is known to be subject to a wide variety of regulatory post-translational modifications including phosphorylation (3). Therefore, we initially focused on tubulin assembly as the most experimentally tractable and likely property of tubulin to be influenced by GRK-mediated phosphorylation. In this regard, we attempted to examine whether
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ACKNOWLEDGEMENTS |
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We thank M. Rasenick for partially purified
tubulin preparations; J. Pitcher and R. Lefkowitz for purified
rhodopsin kinase; J. Robishaw for the common antibody;
S. Kennedy for the purified G
1
2; and A. Pronin for purified GRK5 and valuable discussions.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grants GM44944, GM47419, and 5-T32-CA09662 (to C. V. 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.
Established investigator of the American Heart Association. To
whom correspondence should be addressed: Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4607; Fax: 215-923-1098; E-mail: benovic{at}lac.jci.tju.edu.
The abbreviations used are:
ARK,
-adrenergic receptor kinase; GRK5, G protein-coupled receptor
kinase-5; PAGE, polyacrylamide gel electrophoresis; GST, glutathione
S-transferaseHA, hemagglutininPPT, Partially purified
tubulinPIPES, 1,4-piperazinediethanesulfonic acidPBS, phosphate-buffered saline.
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REFERENCES |
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