INTRODUCTION

Top Abstract Introduction Procedures Results & Discussion References

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.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results & Discussion References

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.

	RESULTS AND DISCUSSION

Top Abstract Introduction Procedures Results & Discussion References

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).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Detection of GRK2-binding proteins by nitrocellulose overlay assay. A and B, nitrocellulose overlay assays were performed, as described under "Experimental Procedures," using 32P-labeled GRK2 as a probe. Bound GRK2 was visualized following exposure of the nitrocellulose filters to film. Soluble (S), particulate (P), and crude extract (C) fractions from the indicated bovine tissues were probed for binding proteins. 15 µg of total protein was run in each lane. B, 15 µg of a purified microtubule preparation was run in lanes 1 and 5. The migration positions of molecular weight standards and purified tubulin, as determined by Ponceau S staining, are indicated. The blot shown is representative of at least three separate experiments.

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.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   GRK2 copurifies with microtubules. 25 µg of a crude bovine brain extract (C) or a purified microtubule preparation (MT) was subjected to Western blot analysis using a monoclonal anti-GRK2 (left panel) or anti-tubulin antibody (right panel). Purified GRK2 and tubulin were included on the blots as controls. The migration positions of molecular weight standards, GRK2, and tubulin are indicated. Similar results were obtained using three different preparations of taxol-precipitated microtubules.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   GRK2 coimmunoprecipitates with tubulin. Tubulin from HEK-293 lysates (approximately 1 mg of protein) was immunoprecipitated using a monoclonal anti- tubulin antibody (ip). Cells were transfected with GRK2 where indicated. Control immunoprecipitations (sham ip) in which the immunoprecipitating antibody was omitted were also included. Immunoprecipitates were subjected to Western blot analysis using an anti-GRK2 antibody (upper panel) or a polyclonal anti-tubulin antibody (lower panel). The migration positions of molecular weight standards and GRK2 are indicated. The Western blot shown is representative of three separate immunoprecipitations.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Expression and activity of GRK2, GRK2-Flag, and GRK2-Flag-GFP. A, GRK2, GRK2-Flag, and GRK2-Flag-GFP phosphorylate rhodopsin in a G-dependent fashion. Lysates (5 µg of protein) from cells transfected with empty vector, GRK2, GRK2-Flag, or GRK2-Flag-GFP were assayed for their ability to phosphorylate rhodopsin in the presence of the indicated concentrations of G. The results are expressed as pmol of Pi incorporated into rhodopsin/min/arbitrary unit of GRK2 protein. Cells expressing empty vector were used to determine the extent of rhodopsin phosphorylation in the absence of GRK2 overexpression, a value that was subsequently subtracted from that obtained using lysates overexpressing GRK2. The amount of GRK2 protein present in the assay was determined by Western blot analysis. The results presented represent the mean ± S.E. of three separate experiments. A representative Western blot showing the levels of GRK2 expressed in the four cell lines used is also shown. B, the sequestration of Y326A--AR in HEK-293 cells is rescued to the same degree by overexpression of either GRK2-Flag or GRK2-Flag-GFP. Results are presented from triplicate experiments (mean ± S.E.).

View larger version (95K): [in this window] [in a new window]   Fig. 5.   Association of GRK2-Flag-GFP and tubulin in HEK-293 cells. A-C, serial images of a mitotic HEK-293 cell by interference contrast (A), tubulin fluorescence (B), and GRK2-Flag-GFP fluorescence (C). Note the colocalization of the tubulin with the GFP-conjugated GRK2 in the paired images B and C. Paired images of tubulin and GRK2-Flag-GFP fluorescence in mitotic cells (D-G) and cells treated with 10 nM taxol (H-K) for 1.5 h at 37 °C. GFP fluorescence from the GRK2-Flag-GFP conjugate is enhanced in regions containing large concentrations of tubulin. This can be observed in the mitotic spindle in the center of the cell stained for tubulin in D and imaged for GRK2-Flag-GFP fluorescence in E or in the two daughter cells stained for tubulin (F) or viewed by GRK2-Flag-GFP fluorescence (G). Taxol-produced cytoplasmic aggregates yield qualitatively similar results when viewed for tubulin (H, J) and GRK2-Flag-GFP (I, K). Bars correspond to 50 µm for A-C, 25 µm for D-G and J-K, and 62 µm for H-I).

View larger version (129K): [in this window] [in a new window]   Fig. 6.   Immunoelectron micrographs of HEK-293 cells transfected with GRK2-Flag. A-C, HEK cells were labeled as described under "Experimental Procedures." The cells were labeled with no primary antibody (A), rabbit anti-tubulin (B), and mouse anti-Flag antibody, respectively (C), followed by either immunogold-conjugated goat anti-rabbit antibody or immunogold-conjugated goat anti-mouse antibody. A, treated with both secondary antibodies and exhibits essentially no labeling. B, treated with anti-rabbit 10-nm immunogold. Linear distributions of tubulin can be observed throughout the image. In C, GRK2-Flag can be observed using anti-mouse 10-nm gold to follow linear patterns and is localized at the surface of mitochondria. D, this cell field was stained for both GRK2-Flag and tubulin. Tubulin is labeled by the larger 10-nm gold particles, whereas GRK2-Flag is labeled by 5-nm gold, with the arrows highlighting sites of colocalization. Note the distribution of GRK2-Flag and tubulin on the right (arrowhead) that follows a filament back to the mitochondrial surface. Bars correspond to 720 nm in A-C and 360 nm in D. M, mitochondria; N, nucleus.

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.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   A tubulin kinase copurifies with microtubules. Taxol-precipitated microtubules, prepared as described under "Experimental Procedures," were incubated with [-32P]ATP for the times indicated. Reactions were quenched and subjected to electrophoresis, and gels were exposed to film (upper panel) and quantified using a PhosphorImager (lower panel). The results from a representative experiment are shown. Similar results were obtained with three different microtubule preparations. The migration position of tubulin is indicated on the autoradiograph.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   The endogenous tubulin kinase has biochemical properties similar to those of GRK2. A, the effect of the protein kinase A inhibitor, staurosporine, heparin, and GTP on tubulin phosphorylation mediated by the microtubule-associated protein kinase (light bars) or rhodopsin phosphorylation mediated by GRK2 (dark bars) is shown. Assays were performed as described under "Experimental Procedures" for 10 min. 100% activity is that activity measured in the absence of inhibitors. The results shown represent the mean values ± S.E. for three separate determinations. B, the effect of monoclonal antibodies on tubulin phosphorylation mediated by the microtubule-associated protein kinase (light bars) or rhodopsin phosphorylation mediated by GRK2 (dark bars). Phosphorylation reactions were performed for 10 min in the presence of 10 µg of the indicated antibodies as described under "Experimental Procedures." 100% kinase activity is that measured in the absence of antibody addition. The results represent the mean values ± S.E. from three separate determinations using two different microtubule preparations.

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 P_i/mol of tubulin, i.e. 2 mol P_i 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 K_m 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.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   GRK2 phosphorylates tubulin. Tubulin devoid of microtubule-associated proteins was incubated in the presence of ATP in either the absence (open symbols) or presence (closed symbols) of purified GRK2 (50 nM). Phosphorylation reactions were quenched at the indicated times. The results shown represent the mean values from two separate experiments.

View this table: [in this window] [in a new window]   Table I Kinetic parameters of the GRK2 for -AR, tubulin, and a soluble peptide substrate Reconstituted -AR (0.3-9.0 µM), purified tubulin (0.3-9.0 µM), or a soluble peptide substrate (10 µM to 1 mM) were phosphorylated as described under "Experimental Procedures." Phosphorylations were performed for 10 min at 30 °C, in either the presence or absence of the -subunits of heterotrimeric G proteins (, 200 nM). The results shown represent the mean values obtained from three separate determinations.

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).

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Activation of GPCRs facilitates the interaction of GRK2 with tubulin. GRK2 was immunoprecipitated from HEK-293 cell lysates transiently overexpressing this enzyme. Cells were either unstimulated (unstim.) or treated with isoproterenol (+ ISO), lysophosphatidic acid (+ LPA), or the thrombin agonist peptide, SFLLRN, (+ Thrombin) for 5 min prior to harvest. GRK2 immunoprecipitates were subsequently subjected to Western blot analysis using an anti-tubulin antibody. Purified tubulin (tubulin) and 10 µg of HEK-293 cell lysate (Cell lysate) were used as positive controls. A control in which the immunoprecipitating antibody was omitted (sham ip) is also shown. The migration position of molecular weight standards and tubulin is indicated. The result shown is representative of three separate experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 11.   Tubulin is phosphorylated following -AR activation. HEK-293 cells transiently overexpressing GRK2 and -AR were labeled with [32P]orthophosphate as described under "Experimental Procedures." Cells were subsequently left unstimulated () or treated with isoproterenol for 10 min (+). Following harvest, a membrane fraction was prepared from which tubulin was immunoprecipitated. Tubulin immunoprecipitates were fractionated on SDS-polyacrylamide gels and exposed to film. A control in which the immunoprecipitating antibody was omitted is also shown (sham ip). The results shown are representative of two separate experiments.

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.