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• W2059636645 abstract "Tuning of γ-aminobutyric acid type A (GABAA) receptor function via phosphorylation of the receptor potentially allows neurons to modulate their inhibitory input. Several kinases, both of the serine-threonine kinase and the tyrosine kinase families, have been proposed as candidates for such a modulatory role in vivo. However, no GABAAreceptor-phosphorylating kinase physically associated with the receptor has been identified so far on a molecular level. In this study, we demonstrate a GABAA receptor-associated protein serine kinase phosphorylating specifically β3-subunits of native GABAA receptors. The characteristics of this novel kinase clearly distinguish it from enzymatic activities that have been shown so far to phosphorylate the GABAA receptor. We putatively identify this protein kinase as the previously described GTAP34 (GABAA receptor-tubulin complex-associated protein of molecular mass 34 kDa). Using expressed recombinant fusion proteins, we identify serine 408 as a major target of the phosphorylation reaction, whereas serine 407 is not phosphorylated. This demonstrates the high specificity of the kinase. Phosphorylation of serine 408 is known to result in a decreased receptor function. The direct association of this kinase with the receptor indicates an important physiological role. Tuning of γ-aminobutyric acid type A (GABAA) receptor function via phosphorylation of the receptor potentially allows neurons to modulate their inhibitory input. Several kinases, both of the serine-threonine kinase and the tyrosine kinase families, have been proposed as candidates for such a modulatory role in vivo. However, no GABAAreceptor-phosphorylating kinase physically associated with the receptor has been identified so far on a molecular level. In this study, we demonstrate a GABAA receptor-associated protein serine kinase phosphorylating specifically β3-subunits of native GABAA receptors. The characteristics of this novel kinase clearly distinguish it from enzymatic activities that have been shown so far to phosphorylate the GABAA receptor. We putatively identify this protein kinase as the previously described GTAP34 (GABAA receptor-tubulin complex-associated protein of molecular mass 34 kDa). Using expressed recombinant fusion proteins, we identify serine 408 as a major target of the phosphorylation reaction, whereas serine 407 is not phosphorylated. This demonstrates the high specificity of the kinase. Phosphorylation of serine 408 is known to result in a decreased receptor function. The direct association of this kinase with the receptor indicates an important physiological role. The ionotropic GABAA 1The abbreviations used are: GABA and GABAA, γ-aminobutyric acid and γ-aminobutyric acid type A, respectively; PAGE, polyacrylamide gel electrophoresis; bd24-beads, antibody bd24 covalently coupled to protein A beads; MBP, maltose-binding protein; GST, glutathione S-transferase; GTAP, GABAA receptor-tubulin complex-associated protein receptor is abundantly expressed in the mammalian brain, and its major function is to mediate fast synaptic inhibition (for reviews, see Refs. 1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1799) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (458) Google Scholar, 3Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar). Binding of GABA to the extracellular domain of the receptor leads to the opening of an intrinsic ion channel followed by chloride influx, counteracting depolarization of the neuronal resting potential. Also, excitatory actions of the receptor depending on an altered chloride equilibrium potential have been reported (4Michelson H.B. Wong R.K.S. Science. 1991; 253: 1420-1423Crossref PubMed Scopus (228) Google Scholar, 5McLean H.A. Caillard O. Ben-Ari Y. Gaiarsa J.-L. J. Physiol. 1996; 496: 471-477Crossref PubMed Scopus (102) Google Scholar). A hallmark of the GABAA receptor is the many clinically important ways of modulation (3Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar, 6Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar). Biochemical purification of the GABAA receptor (7Sigel E. Stephenson F.A. Mamalaki C. Barnard E.A. J. Biol. Chem. 1983; 258: 6965-6971Abstract Full Text PDF PubMed Google Scholar) and cloning of the first two subunits (8Schofield P.R. Darlison M.G. Fujita N. Burt D.R. Stephenson F.A. Rodriguez H. Rhee L.M. Ramachandran J. Reale V. Glencorse T.A. Seeburg P.H. Barnard E.A. Nature. 1987; 328: 221-227Crossref PubMed Scopus (1275) Google Scholar) were followed by the description of so far 14 different mammalian subunits named α1–6, β1–3, γ1–3, δ, and ε. From these subunits, diverse heterooligomeric receptor subtypes are generated neuronally (9McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1085) Google Scholar), bearing most likely a pentameric structure (10Nayeem N. Green T.P. Martin I.L. Barnard E.A. J. Neurochem. 1994; 62: 815-818Crossref PubMed Scopus (378) Google Scholar, 11Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar) equivalent to that of the related nicotinic acetylcholine receptor (12Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (717) Google Scholar). As also demonstrated for other ion channels (13Smart T.G. Curr. Opin. Neurobiol. 1997; 7: 358-367Crossref PubMed Scopus (164) Google Scholar), GABAAreceptor function can be regulated by phosphorylation. This provides a mean for fine tuning inhibitory neuronal inputs. A variety of studies has given evidence for the phosphorylation of the GABAAreceptor by serine/threonine kinases such as protein kinase C (14Sigel E. Baur R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6192-6196Crossref PubMed Scopus (176) Google Scholar, 15Sigel E. Baur R. Malherbe P. FEBS Lett. 1991; 291: 150-152Crossref PubMed Scopus (47) Google Scholar, 16Kellenberger S. Malherbe P. Sigel E. J. Biol. Chem. 1992; 267: 25660-25663Abstract Full Text PDF PubMed Google Scholar, 17Moss S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (246) Google Scholar, 18Krishek B.J. Xie X. Blackstone C. Huganir R.L. Moss S.J. Smart T.G. Neuron. 1994; 12: 1081-1095Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 19Lin Y.-F. Angelotti T.P. Dudek E.M. Browning M.D. Macdonald R.L. Mol. Pharmacol. 1996; 50: 185-195PubMed Google Scholar,23Moss S.J. Doherty C.A. Huganir R.L. J. Biol. Chem. 1992; 267: 14470-14476Abstract Full Text PDF PubMed Google Scholar), cAMP-dependent protein kinase (17Moss S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (246) Google Scholar, 20Heuschneider G. Schwarz R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2938-2942Crossref PubMed Scopus (95) Google Scholar, 21Tehrani M.H.J. Hablitz J.J. Barnes E.M. Synapse. 1989; 4: 126-131Crossref PubMed Scopus (61) Google Scholar, 22Porter N.M. Twyman R.E. Uhler M.D. Macdonald R.L. Neuron. 1990; 5: 769-789Abstract Full Text PDF Scopus (160) Google Scholar, 23Moss S.J. Doherty C.A. Huganir R.L. J. Biol. Chem. 1992; 267: 14470-14476Abstract Full Text PDF PubMed Google Scholar, 24Kapur J. Macdonald R.L. J. Neurophys. 1996; 76: 2626-2634Crossref PubMed Scopus (44) Google Scholar, 25McDonald B.J. Amato A. Connolly C.N. Benke D. Moss S.J. Smart T.G. Nat. Neurosci. 1998; 1: 23-28Crossref PubMed Scopus (204) Google Scholar), cGMP-dependent protein kinase (26Leidenheimer N.J. Mol. Brain Res. 1996; 42: 131-134Crossref PubMed Scopus (23) Google Scholar, 27McDonald B.J. Moss S.J. J. Biol. Chem. 1994; 269: 18111-18117Abstract Full Text PDF PubMed Google Scholar), Ca2+/calmodulindependent kinase II (27McDonald B.J. Moss S.J. J. Biol. Chem. 1994; 269: 18111-18117Abstract Full Text PDF PubMed Google Scholar), and also by the tyrosine kinase Src (28Moss S.J. Gorrie G.H. Amato A. Smart T.G. Nature. 1995; 377: 344-348Crossref PubMed Scopus (199) Google Scholar, 29Valenzuela C.F. Machu T.K. McKernan R.M. Whiting P. VanRenterghem B.B. McManaman J.L. Brozowski S.J. Smith G.B. Olsen R.W. Harris R.A. Mol. Brain Res. 1995; 31: 165-172Crossref PubMed Scopus (72) Google Scholar). These reports point to the intracellular loops of GABAA receptor β- and γ-subunits as major phosphorylation targets. The functional consequences of phosphorylation are either inhibition or enhancement of GABAA receptor-mediated chloride currents. The results obtained for the effect of a given kinase are sometimes conflicting, probably reflecting the complex nature of GABAA receptor regulation by phosphorylation in vivo. Kinase activities copurifying with native GABAA receptors have previously been reported (30Sweetnam P.M. Lloyd J. Gallombardo P. Malison R.T. Gallager D.W. Tallman J.F. Nestler E.J. J. Neurochem. 1988; 51: 1274-1284Crossref PubMed Scopus (68) Google Scholar, 31Bureau M.H. Laschet J.J. J. Biol. Chem. 1995; 270: 26482-26487Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). A kinase physically associated with native GABAA receptors can be expected to be of major functional relevance in vivo, but the molecular identity of such a protein has not been described so far. This study was conducted to answer the question whether one of the recently identified GABAA receptor-associated proteins named GTAPs (32Kannenberg K. Baur R. Sigel E. J. Neurochem. 1997; 68: 1352-1360Crossref PubMed Scopus (39) Google Scholar) is a receptor-associated kinase. Our results indeed imply the identity of GTAP34 with a serine kinase that phosphorylates GABAAreceptor β3-subunits. GABAA receptors were immunoprecipitated from Triton X-100- or Zwittergent 3-14-solubilized calf brain membranes as described previously (32Kannenberg K. Baur R. Sigel E. J. Neurochem. 1997; 68: 1352-1360Crossref PubMed Scopus (39) Google Scholar). For precipitation, the monoclonal antibody bd24 (33Häring P. Stähli C. Schoch P. Takacs B. Staehelin T. Möhler H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4837-4841Crossref PubMed Scopus (158) Google Scholar, 34Ewert M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Cell Biol. 1990; 110: 2043-2048Crossref PubMed Scopus (137) Google Scholar) covalently coupled to protein A-Sepharose (bd24-beads) was used. With 10 μl of packed bd24-beads, GABAA receptor containing approximately 1 μg of α1-subunit was precipitated. This value was determined by comparing the staining intensity of the α1-subunit, migrating in SDS-PAGE (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) as a sharp 50-kDa band and identified by Western blotting, with known amounts of marker proteins in silver-stained gels. Since around 55 kDa several GABAA receptor β-subunits and also receptor-associated tubulin comigrated, the amount of β-subunits present could not be determined. For standard assays, 5 μl of bd24-beads obtained after immunoprecipitation were washed five times with 1 ml of ice-cold buffer 1 (25 mm Tris, pH 7.2, 10 mm MgCl2, 0.1 mm Na-EGTA, 1% (w/w) β-octyl glucoside). 35 μl of buffer 1 were added, and the reaction was started by adding radiolabeled ATP to concentrations ranging from 50 to 400 μm. The specific activity was between 1000 and 10,000 cpm/pmol using [γ-32P]ATP (3000 Ci/mmol; Hartmann Analytic, Braunschweig, Germany). Incubation was done under gentle agitation at 24 °C in a water bath for 30 min unless indicated otherwise. The reaction was stopped by adding SDS-PAGE sample buffer and heating to 95 °C for 10 min. After 8 or 10% SDS-PAGE,1 silver staining (36Morrissey J.H. Anal. Biochem. 1981; 117: 307-310Crossref PubMed Scopus (2942) Google Scholar), and drying of the gel, phosphorylated proteins were detected by autoradiography using a Kodak BioMax HE enhancer screen. To quantify32P incorporation, phosphorylated protein bands were excised from gels and homogenized, and the radioactivity was determined by scintillation counting. To test for the presence of associated kinases already known to phosphorylate GABAA receptor subunits in vitro, the following assay conditions were employed: (a) protein kinase C: 20 mm HEPES-NaOH, pH 7.5, 10 mmMgCl2, 0.5 mm CaCl2, 50 mg/ml phosphatidylserine; (b) cAMP-dependent protein kinase: 40 mm HEPES-NaOH, pH 7.0, 10 mmMgCl2, 0.1 mm EGTA, 10 μm cAMP; (c) calcium/calmodulin-dependent kinase: 35 mm HEPES-NaOH, pH 8.0, 10 mmMgCl2, 1 mm CaCl2, 0.1 mm dithiothreitol, 1 μm calmodulin; (d) cGMP-dependent protein kinase: 40 mm Tris-HCl, pH 7.4, 20 mm MgCl2, 2 mm cGMP. All buffers additionally contained 1% (w/w) β-octyl glucoside, and all assays were done with an ATP concentration of 100 μm. To deglycosylate phosphorylated GABAA receptor probes, 5 μl of bd24-beads obtained from immunoprecipitation were first subjected to a phosphorylation assay (as described above, 50 μm ATP, 60 min). The assay was terminated by washing the bd24-bead precipitate two times with 400 μl of buffer 2 (25 mm Tris-HCl, pH 7.2, 10 mmEDTA, 1% (w/w) β-octyl glucoside, pepstatin, antipain, and leupeptin each at a concentration of 5 μg/ml, 1 mmphenylmethanesulfonyl fluoride). 25 μl of buffer 2 and 5 μl (1 unit) of PNGase F (N-glycosidase F; Roche Molecular Biochemicals) were added, followed by 45 min of incubation at 37 °C. For controls, the enzyme was omitted. Deglycosylation was terminated by adding SDS-PAGE sample buffer and heating the probe. After 8% SDS-PAGE, proteins were transferred to nitrocellulose sheets (Hybond-ECL; Amersham Pharmacia Biotech) and immunodecorated in a manner similar to that described previously (32Kannenberg K. Baur R. Sigel E. J. Neurochem. 1997; 68: 1352-1360Crossref PubMed Scopus (39) Google Scholar). The following primary antibodies were used: bd24 (2 μg/ml), GABAA receptor β2/3-subunit-specific monoclonal antibody bd17 (33Häring P. Stähli C. Schoch P. Takacs B. Staehelin T. Möhler H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4837-4841Crossref PubMed Scopus (158) Google Scholar, 34Ewert M. Shivers B.D. Lüddens H. Möhler H. Seeburg P.H. J. Cell Biol. 1990; 110: 2043-2048Crossref PubMed Scopus (137) Google Scholar), GABAA receptor β1-subunit-specific antibody β1-(350–404) (5 μg/ml; Refs. 37Sperk G. Schwarzer C. Tsunashima K. Fuchs K. Sieghart W. Neuroscience. 1997; 80: 987-1000Crossref PubMed Scopus (276) Google Scholar and 38Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar), GABAAreceptor β2-subunit-specific antibody β2-(351–405) (2 μg/ml; Refs. 37Sperk G. Schwarzer C. Tsunashima K. Fuchs K. Sieghart W. Neuroscience. 1997; 80: 987-1000Crossref PubMed Scopus (276) Google Scholar and 38Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar), GABAA receptor β3-subunit-specific antibody β3-(345–408) (5 μg/ml; Refs. 37Sperk G. Schwarzer C. Tsunashima K. Fuchs K. Sieghart W. Neuroscience. 1997; 80: 987-1000Crossref PubMed Scopus (276) Google Scholar, 38Jechlinger M. Pelz R. Tretter V. Klausberger T. Sieghart W. J. Neurosci. 1998; 18: 2449-2457Crossref PubMed Google Scholar, 39Slany A. Zezula J. Tretter V. Sieghart W. Mol. Pharmacol. 1995; 48: 385-391PubMed Google Scholar, 40Todd A.J. Watt C. Spike R.C. Sieghart W. J. Neurosci. 1996; 16: 974-982Crossref PubMed Google Scholar), GABAAreceptor γ2-subunit-specific antibody γ2-(1–33) (5 μg/ml), and α-tubulin-specific monoclonal antibody B-5-1-2 (Sigma; 1:5000). After incubation with appropriate secondary antibodies, positive bands were detected with the ECL system (Amersham Pharmacia Biotech). For consecutive decoration of the same blot strip with different antibodies, bound antibodies were stripped off from the blot strip according to the protocol provided by the manufacturer (Amersham Pharmacia Biotech). The nomenclature of the fusion proteins was based on the published sequences for the GABAA receptor α1-subunit (8Schofield P.R. Darlison M.G. Fujita N. Burt D.R. Stephenson F.A. Rodriguez H. Rhee L.M. Ramachandran J. Reale V. Glencorse T.A. Seeburg P.H. Barnard E.A. Nature. 1987; 328: 221-227Crossref PubMed Scopus (1275) Google Scholar), β1–3-subunits (41Ymer S. Schofield P.R. Draguhn A. Werner P. Kohler M. Seeburg P.H. EMBO J. 1989; 8: 1665-1670Crossref PubMed Scopus (353) Google Scholar), and γ2-subunit (42Shivers B.D. Killisch I. Sprengel R. Sontheimer H. Koehler M. Schofield P.R. Seeburg P.H. Neuron. 1989; 3: 327-337Abstract Full Text PDF PubMed Scopus (432) Google Scholar), beginning with the first amino acid (position 1) of the mature protein after removal of the signal peptide. The cDNAs coding for cytoplasmic loop regions of β1 (amino acid residues 350–417), β2 (amino acid residues 351–417), β3 (amino acid residues 345–408 or 345–415), or γ2 (amino acid residues 319–366) subunits were amplified from rat brain cDNA and cloned into a modified pMALc vector that additionally encoded a His7 tag on the C terminus of the fusion proteins (43Mossier B. Togel M. Fuchs K. Sieghart W. J. Biol. Chem. 1994; 269: 25777-25782Abstract Full Text PDF PubMed Google Scholar). The mutant β3 constructs β3-(345–415; S381A), β3-(345–415; S395A), β3-(345–415; S407A), and β3-(345–415; S408A) were prepared from the β3-(345–415) construct by using the QuickChangeTM mutagenesis kit (Stratagene). The resulting MBP-β-loop fusion proteins were expressed and purified on a nickel-nitrilotriacetic acid column under denaturing conditions according to the recommendations of Qiagen Inc. α1 (amino acid residues 328–382) was prepared in a similar way as a GST fusion protein. After immunoprecipitation of GABAA receptors, washing with buffer 1, and adding 35 μl of buffer 1 to 5 μl of washed bd24-beads as described above, 2 μg of intracellular loop fusion proteins or an equimolar amount of histone H1 (fraction III-S; Sigma) was added. Since most of the fusion proteins were stored in 8m urea stock solutions, their addition led to urea concentrations during the phosphorylation assay of 0.13–0.3m. No effect of these concentrations of urea on the kinase activity was apparent (data not shown). Reactions were started by adding radiolabeled [γ-32P]ATP to a concentration of 50 μm, followed by incubation unter gentle agitation in a 24 °C water bath for 60 min. For determining the stoichiometry of phosphorylation in the course of a time dependence (see Fig. 2), modified conditions were employed, i.e. 5 μl of washed bd24-beads in a final volume of 20 μl with 1 μg of MBP-β3-(345–408) fusion protein and 400 μm ATP. Assays were terminated by pelleting the bd24-beads with the bound kinase activity by a brief centrifugation and removing the supernatants containing the intracellular loop fusion proteins. The supernatants were either analyzed by SDS-PAGE and autoradiography or by spotting aliquots onto Whatman P82 filters, extensive washing of the filters with 0.85% ortho-phosphoric acid, and scintillation counting. Immunoprecipitates bound to bd24-beads were extracted with buffer 1 containing 0.6m NaCl (high salt extraction) essentially as described previously (32Kannenberg K. Baur R. Sigel E. J. Neurochem. 1997; 68: 1352-1360Crossref PubMed Scopus (39) Google Scholar). To maximally maintain kinase activity for phosphorylation assays, the primary incubation of the immunoprecipitate in high salt buffer was shortened to 1 min, and the extracts were diluted immediately after extraction 3-fold with buffer 1, resulting in a NaCl concentration of 0.2 m. Buffer 1 containing 0.2m NaCl was also used for the phosphorylation assays of the other probes to be compared with the high salt extract. The assay volume was 120 μl for each probe: the nonextracted bd24-beads, the extracted beads (both 5 μl of beads) and the high salt extract (extracted from 5 μl of beads). 5 μg of MBP-β3-(345–408) fusion protein or 0.78 μg of histone were used as kinase substrate. The probes were incubated for 120 min at 24 °C in the presence of 50 μm radiolabeled ATP. Analysis of the assay supernatants was performed as described above. Gel filtration of high salt-extracted proteins was done in buffer 1 containing 0.6 m NaCl with a fast protein liquid chromatography system on a Superose 12 HR10/30 column (Amersham Pharmacia Biotech). Undiluted extract obtained from 140 μl of bd24-beads was separated at a flow rate of 0.1 ml/min. Starting at the void volume, 0.3 ml of eluate fractions were collected. For phosphorylation assays, 25 μl of each fraction were diluted with 50 μl of buffer 1 containing 4 μg of the MBP-β3-(345–408) fusion protein. To monitor autophosphorylation of separated proteins, identical probes lacking the fusion protein were prepared. Assays were performed for 120 min in the presence of 50 μm ATP, followed by 8% SDS-PAGE and autoradiography. Calf brain membranes were solubilized in 10 mm HEPES, 5 mm EDTA, 1% SDS at 95 °C for 10 min. The solution was diluted 10-fold with 10 mm HEPES, 5 mm EDTA, 50 mm NaCl, and 1% Triton X-100, resulting in a SDS concentration of 0.1%. After centrifugation at 165,000 × g for 30 min, the supernatant was precleared with protein A-beads for 90 min at 4 °C. 6 ml of the precleared solution containing 6 mg of protein were incubated with a 5 μg/ml concentration of either the rabbit antibody β2-(351–405) or antibody β3-(1–13) that recognizes β2- and β3-subunits (11Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar) overnight at 4 °C. The antibodies were recaptured with 15 μl of packed protein A-beads and washed five times with buffer 1. 30 μl of 1:1 slurry protein A-beads were mixed with 90 μl of high salt-extracted kinase (prepared in Zwittergent 3-14, extracted and diluted as described above) and incubated for 120 min at 24 °C in the presence of 50 μm radiolabeled ATP. Phosphorylated β2- and β3-subunits were analyzed by SDS-PAGE and autoradiography. One- and two-dimensional analysis of radiolabeled phosphoamino acids were performed essentially as described (44Hunter T. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1311-1315Crossref PubMed Scopus (1560) Google Scholar). In brief, GABAA receptor immunoprecipitate was incubated in buffer 1 with 50 μmATP for 60 min, either alone or in the presence of MBP-β3-(345–408) fusion protein. After SDS-PAGE of the whole immunoprecipitate or only the probe supernatant, silver staining, and autoradiography, protein was eluted from gel pieces containing labeled GABAAreceptor subunits or labeled MBP-β3-(345–408) fusion protein. Following acidic hydrolysis, amino acids were separated by two-dimensional thin layer chromatography and compared with phosphoamino acid standards. Phosphorylation of immunoprecipitated GABAA receptors and MBP-β3-(345–415) fusion proteins was done as described above. bd24-beads containing the bound phosphorylated receptor were washed with Lys-C digestion buffer (25 mm Tris HCl, pH 8.5, 1 mm EDTA). The supernatant of the phosphorylation assays containing the loop fusion proteins was subjected to a chloroform-methanol precipitation (45Wessel D. Flügge U.I. Anal. Biochem. 1983; 138: 141-143Crossref Scopus (3191) Google Scholar). Both the phosphorylated receptors and the loop constructs were digested in 200 μl of Lys-C digestion buffer containing 0.1 μg of Lys-C (Roche Molecular Biochemicals) for 19 h at 37 °C. Peptides were lyophilized twice in H2O and separated on cellulose thin layer plates in formic acid, glacial acetic acid, and H2O at a ratio of 50:156:1794, pH 1.9, 1.8 kV, for 15 min. and by ascending chromatography in isobutyric acid, pyridine, glacial acetic acid, 1-butanol, and H2O at a ratio of 65:5:3:2:29 in the second dimension (46Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). GABAA receptors were immunoprecipitated from Triton X-100-solubilized calf brain membranes with bd24-beads. The immunoprecipitate was assessed for the presence of an associated kinase activity by incubation with [γ-32P]ATP and subsequent SDS-PAGE analysis. In order to allow different kinases to exert optimal activity during the assay, various buffer compositions were used. In the presence of Mg2+, a strong phosphorylation of a 55-kDa band was obtained (Fig. 1, lanes 1 and 3–5). Replacement of Mg2+ with Mn2+ largely abolished this activity (Fig. 1,lane 2). Under conditions designed to stimulate protein kinase C (Fig. 1, lane 3), cAMP-dependent protein kinase (Fig. 1, lane 4), calcium/calmodulin-dependent kinase (Fig. 1,lane 5), or cGMP-dependent protein kinase (not shown), phosphorylation of the 55-kDa band was not stimulated, and no other phosphorylated band of comparable intensity was detected, although some additional bands became evident. This study focused on the phosphorylation of the 55-kDa band under conditions similar to those given for Fig. 1, lane 1. Analysis of the time dependence of specific incorporation of radioactivity under conditions promoting high enzymatic turnover showed that the phosphorylation was substantial with regard to the total amount of isolated receptors (Fig. 2). The addition of 100 μm sodium vanadate to the assay buffer had no effect, indicating the absence of a relevant phosphatase activity (not shown). No qualitative differences of the phosphorylation patterns were observed with concentrations of ATP ranging from 50 μm to 400 μm (results not shown). Following phosphorylation, GABAA receptor immunoprecipitates were enzymatically deglycosylated, subjected to SDS-PAGE, and blotted to nitrocellulose membranes, parallel to nondeglycosylated probes. Deglycosylation was done in order to unequivocally differentiate between receptor subunits and subunit isoforms and tubulin. It was indeed found that some proteins comigrating around 55 kDa (i.e. the GABAA receptor β2-subunit (Fig.3, lane 7), the GABAA receptor β3-subunit (Fig. 3, lane 11), and α-tubulin (Fig. 3, lane 27)) showed a differential migration after deglycosylation (Fig. 3, lanes 8, 12, and28). Radioactivity on the blot was detected by autoradiography, and the resulting pattern of labeled bands was compared with the signals obtained after immunodecoration with specific antibodies recognizing GABAA receptor α1-, β1-, β2-, β3-, β2/3-, and γ2-subunits and α-tubulin (Fig. 3). After deglycosylation, the number of radioactive bands increased. This might be due to incomplete deglycosylation or to a partial protein degradation during deglycosylation. The predominant radioactive signals at 55 kDa in the nontreated probe (Fig. 3, lanes 1, 5, 9, 13, 17,21, and 25, upper band) and at 52 kDa in the deglycosylated probe (Fig. 3, lanes 2, 6, 10, 14,18, 22, and 26, upper band) colocalized best with the signals obtained with an antibody specific for GABAA receptor β3-subunits (Fig. 3,lanes 9–12). Also, a weak radioactive band at 36 kDa, possibly representing a degradation product of β3-subunits generated during deglycosylation by contaminating proteases, comigrated with a β3-subunit positive signal (Fig. 3, lanes 14 and 16). Only one other antibody, recognizing GABAA receptor β2- and β3-subunits, also reacted with the dominant radioactive bands of glycosylated and deglycosylated probes (Fig. 3, lanes 13–16), confirming the result with the β3-subunit-specific antibody. The additional bands recognized by the β2/3-subunit-specific antibody were due to the detection of β2-subunits, as evident from the comparison with a β2-subunit-specific antibody (Fig. 3, lanes 7–8). With this antibody, as well as with the antibodies directed against GABAA receptor α1-, β1-, and γ2-subunits and for α-tubulin, a match with each of the two predominant radioactive bands of the receptor probes (55 kDa for the glycosylated receptor, 52 kDa for the deglycosylated probe) was not obtained. These results indicated a preferential phosphorylation of GABAA receptor β3-subunits by the GABAAreceptor-associated kinase activity. If it is assumed that β3-subunits are present in the receptor in a stoichiometry of 1:1 with α1-subunits, phosphorylation of β3-subunits almost occurs stoichiometrically (see Fig. 2). However, it should be pointed out that this probably represents an underestimate of the stoichiometry, since α1-subunits presumably assemble preferentially with β2- and not with β3-subunits (9McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1085) Google Scholar). In 8 of 19 experiments, in addition to the dominant 55-kDa band also a minor 50-kDa band was observed to be phosphorylated (see Fig. 3,lane 1). The minor radioactive bands at 50 kDa in the nondeglycosylated and at 45 kDa in the deglycosylated probe could be matched with immunopositive signals for GABAA receptor α1-subunits (Fig. 3, lanes 17–20), β1-subunits (Fig. 3, lanes 1–4), and also β3-subunits (Fig. 3, lanes 9–12). The immunostaining for the β1-subunit revealed a molecular mass of roughly 50 kDa for the glycosylated protein. This value is clearly lower than expected from the sequence similarity between the β1-subunit and the β2- and β3-subunits, both migrating at 55 kDa. Most likely, this discrepancy can be explained by partial degradation of the β1-subunit during receptor purification, resulting in the loss of a terminal portion of the protein. The GABAA receptor immunoprecipitate was incubated, under conditions promoting high enzymatic turnover, with a fusion protein consisting of amino acid residues 345–408 of the intracellular loop of the GABAA receptor β3-subunit fused to MBP (MBP-β3-(345–408)). A stoichiometry of phosphorylation of 0.79 mol of bound phosphate/mol of MBP-β3-(345–408) was obtained (Fig. 4). This result indicated the usefulness of fusion proteins of intracellular loops of GABAA receptor subunits for further experiments. In another experiment under comparable conditions, a similar enzymatic" @default.
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• W2059636645 title "A Novel Serine Kinase with Specificity for β3-Subunits Is Tightly Associated with GABAA Receptors" @default.
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