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• W2004134377 abstract "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 tubulinin vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied β2-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. 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 tubulinin vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied β2-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. Agonist occupancy of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; β-AR, β2-adrenergic receptor; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Gβγ, the βγ 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).1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; β-AR, β2-adrenergic receptor; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Gβγ, the βγ 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). facilitates the exchange of bound GDP for GTP on heterotrimeric G proteins. The activated GTP bound G protein then dissociates into its constituent α- and βγ-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. 1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1086) Google Scholar, 2Inglese J. Freedman N.J. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 23735-23738Abstract Full Text PDF PubMed Google Scholar, 3Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (472) Google Scholar). 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 (β-arrestins) to the receptors (4Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (908) Google Scholar, 5Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). β-Arrestin binding has two functional consequences. First, the binding of β-arrestin sterically inhibits coupling of the receptor to its respective G protein (4Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (908) Google Scholar, 5Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). GRK-mediated receptor phosphorylation and β-arrestin binding thus lead to diminished receptor signaling, i.e. receptor desensitization (6Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar). Second, β-arrestin binding initiates the clathrin-mediated endocytosis (sequestration) of activated receptors (7Ferguson S.S. Downey W.E.J. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (844) Google Scholar). 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 (8Strasser R.H. Benovic J.L. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6362-6366Crossref PubMed Scopus (100) Google Scholar, 9Daaka Y. Pitcher J.A. Richardson M. Stoffel R.H. Robishaw J.D. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2180-2185Crossref PubMed Scopus (155) Google Scholar). The compartmentalization of GRK2 at the plasma membrane requires that its carboxyl-terminal pleckstrin homology domain binds both phosphatidylinositol 4,5-bisphosphate and the βγ-subunits of heterotrimeric G proteins (Gβγ) (10Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 11DebBurman S.K. Ptasienski J. Benovic J.L. Hosey M.M. J. Biol. Chem. 1996; 271: 22552-22562Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Since the membrane association of GRK2 requires free Gβγ and the release of Gβγ from the α-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 Gβγ regulate the rate of receptor phosphorylation by increasing the local GRK2 concentration. Additionally, allosteric activation of GRK2 occurs when it is complexed with Gβγand an activated receptor substrate (12Haga K. Haga T. J. Biol. Chem. 1992; 267: 2222-2227Abstract Full Text PDF PubMed Google Scholar, 13Kim C.M. Dion S.B. Benovic J.L. J. Biol. Chem. 1993; 268: 15412-15418Abstract Full Text PDF PubMed Google Scholar). This was demonstratedin vitro by measuring a potentiation of GRK-mediated phosphorylation of a peptide substrate in the presence of activated GPCR and Gβγ (13Kim C.M. Dion S.B. Benovic J.L. J. Biol. Chem. 1993; 268: 15412-15418Abstract Full Text PDF PubMed Google Scholar). 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. 14Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar). Protein kinase C-binding proteins (receptors for activated protein kinase C) (reviewed in Ref. 15Mochly-Rosen D. Smith B.L. Chen C.H. Disatnik M.H. Ron D. Biochem. Soc. Trans. 1995; 23: 596-600Crossref PubMed Scopus (91) Google Scholar) and protein kinase C substrates (16Chapline C. Ramsay K. Klauck T. Jaken S. J. Biol. Chem. 1993; 268: 6858-6861Abstract Full Text PDF PubMed Google Scholar, 17Hyatt S.L. Liao L. Aderem A. Nairn A.C. Jaken S. Cell Growth Differ. 1994; 5: 485-502PubMed Google Scholar, 18Liao L. Hyatt S.L. Chapline C. Jaken S. Biochemistry. 1994; 33: 1229-1233Crossref PubMed Scopus (70) Google Scholar) 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. 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 fromCLONTECH. 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. 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 (19Barak L.S. Ferguson S.S.G. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar). The (TAA) stop codon following the C-terminal leucine was replaced using site-directed mutagenesis (20Valette F. Mege E. Reiss A. Adesnik M. Nucleic Acids Res. 1989; 17: 723-733Crossref PubMed Scopus (175) Google Scholar) with an in frameBamHI restriction site. The proximalHindII/XhoI fragment was ligated with theXhoI/BamHI fragment into (pS65T-GFP) between theHindIII/BamHI polylinker restriction sites. 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. GRK2-binding proteins were identified using a modification of a procedure initially described by Leiser et al. (21Leiser M. Rubin C.S. Erlichman J. J. Biol. Chem. 1986; 261: 1904-1908Abstract Full Text PDF PubMed Google Scholar). Proteins in samples to be probed were separated by SDS-polyacrylamide gel electrophoresis (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar) and electrophoretically transferred to nitrocellulose membranes. The nitrocellulose filters were incubated in blotto (10 mm potassium phosphate buffer, pH 7.4, 0.15m 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. (23Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar), was autophosphorylated by incubation in 20 mm Tris, pH 7.5, 10 mmMgCl2, 2.0 mm EDTA, 1 mmdithiothreitol 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 [γ-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. Purified microtubules containing microtubule-associated proteins were prepared from homogenates of bovine brain using the antimitotic drug taxol as described by Vallee (24Vallee R.B. J. Cell Biol. 1982; 92: 435-442Crossref PubMed Scopus (391) Google Scholar). Purified tubulin was prepared from extracts of freshly isolated bovine brain as described by Simon et al. (25Simon J.R. Adam N.A. Salmon E.D. Micron Microsc. Acta. 1991; 22: 405-412Crossref Scopus (10) Google Scholar). 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 (26Voter W.A. Erickson H.P. J. Biol. Chem. 1984; 259: 10430-10438Abstract Full Text PDF PubMed Google Scholar). Purified tubulin was stored in aliquots at −80 °C until use. Western blots were performed by standard procedures using monoclonal antibodies against GRK2 (27Oppermann M. Diverse-Pierluissi M. Drazner M.H. Dyer S.L. Freedman N.J. Peppel K.C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7649-7654Crossref PubMed Scopus (82) Google Scholar) and polyclonal or monoclonal antibodies directed against β-tubulin (Sigma). Enhanced chemiluminescence detection of antigens (DuPont) was achieved with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). 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 (28Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). 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 (28Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and the human M2 Flag-tagged β2-adrenergic receptor in pcDNAs (28Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) were also used in this study. Serum-starved HEK-293 cells overexpressing GRK2 or GRK2 and β2-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-β-tubulin antibody (Sigma) (see Figs. 2 and 11) or a monoclonal anti-GRK2 antibody (27Oppermann M. Diverse-Pierluissi M. Drazner M.H. Dyer S.L. Freedman N.J. Peppel K.C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7649-7654Crossref PubMed Scopus (82) Google Scholar) (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.Figure 11Tubulin is phosphorylated following β-AR activation. HEK-293 cells transiently overexpressing GRK2 and β-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Activation 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 Large Image Figure ViewerDownload Hi-res image Download (PPT) 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[γ-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. 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) (23Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar) 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. Purified rod outer segment membranes (29Papermaster D.S. Dreyer W.J. Biochemistry. 1974; 13: 2438-2444Crossref PubMed Scopus (579) Google Scholar) or purified reconstituted β-AR (10Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 30Benovic J.L. Shorr R.G.L. Caron M.G. Lefkowitz R.J. Biochemistry. 1984; 23: 4510-4518Crossref PubMed Scopus (134) Google Scholar) 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.” β-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 β-AR phosphorylation. A rhodopsin concentration of ∼30 μmwas used in assays utilizing this 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 mmEDTA, 7.5 mm MgCl2, and 60 μm[γ-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 [γ-32P]ATP was subsequently removed by washing in 75 mm phosphoric acid as described previously (31Onorato J.J. Palczewski K. Regan J.W. Caron M.G. Lefkowitz R.J. Benovic J.L. Biochemistry. 1991; 30: 5118-5125Crossref PubMed Scopus (188) Google Scholar). 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. HEK-293 cells transiently overexpressing GRK2 and β-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 β-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.” 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. 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 β-AR (32Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). 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. 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 2.1m 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. Crude extracts (C) derived from various bovine tissues, together with a soluble (S) and a particulate (P) fraction derived from this extract (Fig.1 A) 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. 1 A, 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 pa" @default.
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• W2004134377 title "The G Protein-coupled Receptor Kinase 2 Is a Microtubule-associated Protein Kinase That Phosphorylates Tubulin" @default.
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