Phosphorylation and Regulation of Antidepressant-sensitive Serotonin Transporters
1998; Elsevier BV; Volume: 273; Issue: 4 Linguagem: Inglês
10.1074/jbc.273.4.2458
ISSN1083-351X
AutoresSammanda Ramamoorthy, Elena Giovanetti, Yan Qian, Randy Blakely,
Tópico(s)Receptor Mechanisms and Signaling
ResumoAntidepressant-sensitive serotonin (5-hydroxytrypta-mine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45–47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5–6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by β-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking. Antidepressant-sensitive serotonin (5-hydroxytrypta-mine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45–47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5–6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by β-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking. The biogenic amine, serotonin (5-hydroxytryptamine, 5HT), 1The abbreviations used are: 5HT, 5-hydroxytryptamine (serotonin); SERT, serotonin transporter; DAT, dopamine transporter; NE, norepinephrine; NET, norepinephrine transporter; PKC, protein kinase C; PKA, protein kinase A; PKG, protein kinase G; CaM kinase, Ca2+/calmodulin-dependent kinase; PP1/2A, protein phosphatase 1/2A; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; BAPTA-AM, [1,2-bis(o-aminophenoxy)ethane-N,N,N′-tetraacetic acid tetra(acetoxymethyl) ester; KN-62, [1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine]; KN-93, [2-[N-(2hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamul)-N-methylbenzylamine]; h, human; PMA, phorbol 12-myristate 13-acetate; PDBu, phorbol 12,13-dibutyrate; DMEM, Dulbecco's modified Eagle's medium.1The abbreviations used are: 5HT, 5-hydroxytryptamine (serotonin); SERT, serotonin transporter; DAT, dopamine transporter; NE, norepinephrine; NET, norepinephrine transporter; PKC, protein kinase C; PKA, protein kinase A; PKG, protein kinase G; CaM kinase, Ca2+/calmodulin-dependent kinase; PP1/2A, protein phosphatase 1/2A; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; BAPTA-AM, [1,2-bis(o-aminophenoxy)ethane-N,N,N′-tetraacetic acid tetra(acetoxymethyl) ester; KN-62, [1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine]; KN-93, [2-[N-(2hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamul)-N-methylbenzylamine]; h, human; PMA, phorbol 12-myristate 13-acetate; PDBu, phorbol 12,13-dibutyrate; DMEM, Dulbecco's modified Eagle's medium. is a neurotransmitter in the central nervous system and peripheral nervous system (2$$Google Scholar, 3Jacobs B. 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Hamer D.H. Murphy D.L. Science. 1996; 274: 1527-1531Crossref PubMed Scopus (4325) Google Scholar), major depression (25Oglivie A.D. Battersby S. Bubb V.J. Fink G. Harmar A.J. Goodwin G.M. Smith C.A.D. Lancet. 1996; 347: 731-733Abstract Full Text PDF PubMed Scopus (498) Google Scholar), and autism (26Cook Jr., E.H. Courchesne R. Lord C. Cox N.J. Yan S. Lincoln A. Haas R. Courchesne E. Leventhal B.L. Mol. Psychiatry. 1997; 2: 247-250Crossref PubMed Scopus (385) Google Scholar). hSERT gene expression is significantly regulatable by activation of both PKA and PKC pathways (17Blakely R.D. Ramamoorthy S. Qian Y. Schroeter S. Bradley C. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 29-72Crossref Google Scholar), and potential target sites for second messenger-mediated regulation of gene expression have been identified at or near transcription initiation and mRNA splicing sites (27Lesch K.P. Balling U. Gross J. Strass K. Wolozin B.L. Murphy D.L. Riederer P. J. Neural Transm. 1994; 95: 157-162Crossref PubMed Scopus (579) Google Scholar, 28Heils A. Teufel A. Petri S. Stober G. Riederer P. Bengel D. Lesch K.P. J. Neurochem. 1996; 66: 2621-2624Crossref PubMed Scopus (1950) Google Scholar, 29Bradley C.C. Blakely R.D. J. Neurochem. 1997; 69: 1356-1367Crossref PubMed Scopus (97) Google Scholar). Recent findings suggest that the more delayed and long term changes in 5HT uptake activity mediated by altered SERT gene expression are mirrored by rapid, posttranscriptional events that alter 5HT uptake capacity at sites of expression (17Blakely R.D. Ramamoorthy S. Qian Y. Schroeter S. Bradley C. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 29-72Crossref Google Scholar). For example, platelet, endothelial, and brain SERTs are down-regulated within minutes by PKC activation (30Myers C.L. Lazo J.S. Pitt B.R. Am. J. Physiol. 1989; 257: L253-L258Crossref PubMed Google Scholar, 31Anderson G.M. Horne W.C. Biochim. Biophys. Acta. 1992; 1137: 331-337Crossref PubMed Scopus (88) Google Scholar, 32Anderson G.M. Vaccadino F.M. Hall L.M. Soc. Neurosci. Abstr. 1995; 21: 344.16Google Scholar), phenomena recapitulated in transfected COS (33Sakai N. Sasaki K. Nakashita M. Honda S. Ikegaki N. Saito N. J. Neurochem. 1997; 68: 2618-2624Crossref PubMed Scopus (55) Google Scholar), HEK-293 cells (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar), and LLC-PK1 cells. 2S. Ramamoorthy, unpublished observations.2S. Ramamoorthy, unpublished observations. We have recently shown that PKC-mediated down-regulation of 5HT uptake in stably transfected HEK-293 cells (HEK-hSERT) occurs via a specific reduction in cell-surface transporter protein (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar). The presence of multiple, canonical serine and threonine phosphorylation sites on SERT cytoplasmic domains (18Blakely R.D. Berson H.E. Fremeau Jr., R.T. Caron M.G. Peek M.M. Prince H.K. Bradley C.C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (683) Google Scholar, 19Hoffman B.J. Mezey E. Brownstein M.J. Science. 1991; 254: 579-580Crossref PubMed Scopus (504) Google Scholar, 20Ramamoorthy S. Bauman A.L. Moore K.R. Han H. Yang-Feng T. Chang A.S. Ganapathy V. Blakely R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2542-2546Crossref PubMed Scopus (778) Google Scholar, 34Miller K.J. Hoffman B.J. J. Biol. Chem. 1994; 269: 27351-27356Abstract Full Text PDF PubMed Google Scholar) and the ability of NH2 and COOH termini, where most of these sites lie, to serve as substrates for purified protein kinases (e.g. PKC, PKA (17Blakely R.D. Ramamoorthy S. Qian Y. Schroeter S. Bradley C. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 29-72Crossref Google Scholar, 35Qian Y. Melikian H.E. Moore K.R. Duke B.J. Blakely R.D. Soc. Neurosci. Abstr. 1995; 21: 344.7Google Scholar)) suggests that rapid kinase-mediated regulation of 5HT uptake may occur, in part, as a consequence of transporter phosphorylation. Direct protein phosphorylation is known to regulate the activity and/or surface distribution of many ion channels, receptors, and transporters; however, to date, SERT phosphorylation has not been described. To explore regulatory posttranslational processing of SERT proteins, we have generated and characterized SERT-specific antibodies that immunoprecipitate and immunoblot SERT polypeptides in vitroand in vivo (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar, 36Qian Y. Melikian H.E. Rye D.B. Levey A.I. Blakely R.D. J. Neurosci. 1995; 15: 1261-1274Crossref PubMed Google Scholar). We now report the use of these antibodies to establish the direct phosphorylation of hSERT proteins using 293-hSERT cells. hSERT proteins in this system are phosphorylated under basal conditions, and phosphorylation can be significantly elevated by both PKC and cyclic nucleotide (cAMP and cGMP)-activated protein kinases. In addition, studies with phosphatase inhibitors reveal endogenous pathways leading to SERT phosphorylation independent of PKC, PKA, and PKG. These findings reveal a highly dynamic, and potentially complex, process of SERT phosphorylation and dephosphorylation whose regulation coincides, in part, with altered trafficking of SERT proteins. We propose that direct SERT phosphorylation may be a determinant of receptor and second messenger-mediated changes in 5HT transport capacity and discuss the potential roles of SERT phosphorylation in altered plasma membrane expression. 293-hSERT cells were previously generated and characterized in this laboratory (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar). Trypsin, glutamine, penicillin, streptomycin, G418, and phosphate-free Dulbecco's modified Eagle's medium (DMEM) were purchased from Life Technologies, Inc., or obtained from the Vanderbilt Media Core. Cys/Met-free DMEM was obtained from Cellgro. PMA and phorbol 12,13-dibutyrate isomers, staurosporine, cholera toxin, dialyzed fetal bovine serum, and protease inhibitors were obtained from Sigma. Okadaic acid, KT5720, KN-62, KN-93, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate, 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate were purchased from LC Laboratories/Alexis Biochemicals. Bisindolylmaleimide I, calyculin A, cyclosporin A, (−)indolactam V, BAPTA-AM, and microcystin were purchased from Calbiochem. [3H]5-HT (5-hydroxy-[3H]tryptamine trifluoroacetate (∼100 Ci/mmol)) and [32P]orthophosphate (10 mCi/ml) were obtained from Amersham Corp. Trans35S-label (∼1209 Ci/mmol) was obtained from ICN Pharmaceuticals. Protein A-Sepharose was obtained from Pharmacia Biotech Inc. All other reagents were of the highest grade possible from standard commercial sources. 293-hSERT and parental HEK-293 lines were maintained in monolayer culture in 75-cm2 flasks in an atmosphere of 5% CO2 at 37 °C as described previously (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar). Both lines were grown in DMEM containing 10% dialyzed fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Medium for the transfected line was supplemented with G418 (250 μg/ml). Use of dialyzed serum at a 1000 molecular weight cutoff was necessary to prevent loss of expression of 5HT uptake and SERT protein expression through, as yet, undefined mechanisms. [3H]5-HT transport activity was assayed in monolayer cultures for the times indicated at 37 °C as described previously (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar). Briefly, cells were plated on poly-d-lysine (0.1 mg/ml)-coated 6-well (500,000 cells/well) or 24-well (100,000 cells/well) plates 48 h before experiments. At assay, the medium was removed by aspiration, and the cells were washed with 2 ml of Krebs-Ringer's (KRH) buffer containing 130 mm NaCl, 1.3 mm KCl, 2.2 mmCaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, 1.8 g/liter glucose, 10 HEPES, pH 7.4. Cells were then incubated in KRH buffer containing 100 μm pargyline and 100 μm ascorbic acid for 10 min at 37 °C with or without various modulators. 5HT uptake assays were initiated by the addition of [3H]5HT (1 μm final concentration), and the assays were terminated by three rapid washes (2 ml each) with KRH buffer at room temperature containing 100 μm imipramine. Cells were then solubilized in 1% SDS or Optiphase Supermix scintillation mixture (Wallac, Gaithersburg, MD), and [3H]5HT accumulation was determined by liquid scintillation spectrometry. Specific 5HT uptake was determined by subtracting the amount accumulated [3H]5HT in the presence of 1 μm paroxetine. Statistical analyses comparing vehicle and modulator-modified uptake were performed using Student's paired t tests. For phosphorylation studies, 293-hSERT cells were seeded on poly-d-lysine-coated 6-well plates at 5 × 105 cells/well. After 48 h, monolayers were washed once in phosphate-free DMEM and incubated for 1 h at 37 °C. Typically, cells were then incubated at 37 °C with the same medium containing 1 mCi/ml carrier-free [32P]orthophosphate for 1 h to equilibrate the intracellular ATP pools with labeled phosphate. Effectors at various concentrations (see figure legends) or vehicles were added to the medium, and the incubation was continued at 37 °C. Kinase inhibitors were preincubated for 30 min prior to addition of kinase activators or phosphatase inhibitors for the times indicated. Experiments to test the role of intracellular calcium in okadaic acid and CaM kinase II in okadaic acid-induced phosphorylation were conducted in calcium-free media in the presence of BAPTA-AM or in the presence of the CaM kinase II inhibitors, KN-62 and KN-93. The adherent cells were washed three times with phosphate-buffered saline and lysed by the addition of 400 μl/well ice-cold modified radioimmunoprecipitation (RIPA, 10 mm Tris, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4) buffer containing protease (1 μm pepstatin A, 250 μm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin) and phosphatase inhibitors (10 mm sodium fluoride, 50 mm sodium pyrophosphate, and 1 μm okadaic acid) for 1 h at 4 °C with agitation. RIPA extracts were centrifuged at 20,000 × g for 30 min at 4 °C. Protein content of supernatant was assessed using the DC protein assay (Bio-Rad) with bovine serum albumin as the standard. Protein content between wells and experiments showed <5% variability. Labeling with Trans35S-label was carried out in Cys/Met-free DMEM as described previously (36Qian Y. Melikian H.E. Rye D.B. Levey A.I. Blakely R.D. J. Neurosci. 1995; 15: 1261-1274Crossref PubMed Google Scholar, 37Melikian H.E. McDonald J.K. Gu H. Rudnick G. Moore K.R. Blakely R.D. J. Biol. Chem. 1994; 269: 12290-12297Abstract Full Text PDF PubMed Google Scholar). Supernatants were precleared by the addition of 100 μl (3 mg) of Protein A-Sepharose beads for 1 h at 4 °C. hSERT protein was immunoprecipitated overnight at 4 °C by the addition of SERT-specific antibody, CT-2 (10 μl of antisera) on end-over-end continuous mixing, followed by 1-h incubation with Protein A-Sepharose beads (3 mg in 100 μl in RIPA buffer) at 22 °C. Additional experiments to test specificity were carried out with the hNET-specific antibody N430 (37Melikian H.E. McDonald J.K. Gu H. Rudnick G. Moore K.R. Blakely R.D. J. Biol. Chem. 1994; 269: 12290-12297Abstract Full Text PDF PubMed Google Scholar), CT-2B preimmune serum, or a second SERT-specific serum S365 (36Qian Y. Melikian H.E. Rye D.B. Levey A.I. Blakely R.D. J. Neurosci. 1995; 15: 1261-1274Crossref PubMed Google Scholar). The immunoadsorbents were washed three times with ice-cold RIPA buffer prior to the addition to 50 μl of Laemmli sample buffer (62.5 mm Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.01% bromphenol blue), incubated for 30 min at 22 °C, and then resolved by SDS-PAGE (10%), with radiolabeled proteins detected by autoradiography or direct PhosphorImager (Molecular Dynamics) analysis. The relative amounts of32P incorporated into hSERT protein were estimated using ImageQuant software (Molecular Dynamics). Quantitation from digitized autoradiograms was evaluated on multiple film exposures to ensure quantitation within the linear range of the film and gave identical results to estimations achieved with direct PhosphorImager quantitation. To determine whether hSERT proteins are subject to phosphorylation, we metabolically labeled stably transfected 293-hSERT cells with [32P]orthophosphate and immunoprecipitated detergent extracts with a set of SERT-specific and control antisera. In these initial experiments, the Ser/Thr phosphatase inhibitor okadaic acid (1 μm) was applied to intact cells before extraction in an attempt to preserve labeling from endogenous kinases. A more systematic analysis of the effects of okadaic acid-induced labeling is presented later in this report. SDS-PAGE/autoradiography of immunoprecipitates from labeled 293-hSERT cells reveals a broad band centered at ∼96 kDa (Fig. 1), the size expected from immunoblots for mature, N-glycosylated hSERT protein (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar,36Qian Y. Melikian H.E. Rye D.B. Levey A.I. Blakely R.D. J. Neurosci. 1995; 15: 1261-1274Crossref PubMed Google Scholar). The 96-kDa species is absent from immunoprecipitates of parental HEK-293 cells, metabolically labeled, and extracted in parallel. The 96-kDa band is also not immunoprecipitated from transfected cell extracts if CT-2 preimmune serum, the NET-specific antibody N430, or CT-2 serum preabsorbed with CT-2/GST fusion protein are utilized for immunoprecipitations. The 96-kDa band is retained, however, if CT-2 antiserum is preabsorbed with GST, the protein carrier for the fusion protein utilized to raise the CT-2 antibody. The SERT antipeptide antibody S365 (36Qian Y. Melikian H.E. Rye D.B. Levey A.I. Blakely R.D. J. Neurosci. 1995; 15: 1261-1274Crossref PubMed Google Scholar), like CT-2 antiserum, immunoprecipitates the same 96-kDa band, although the S365 antipeptide antibody displays consistently lower recovery in both 32P- and35S-metabolic labeling paradigms (data not shown). In addition, we have raised another two polyclonal antisera against the COOH terminus of SERT and found that, like CT-2 and S-365 sera, they also immunoprecipitate a phosphorylated 96-kDa band selectively from 293-hSERT cell extracts (data not shown). Together these findings support the contention that the phosphorylated 96-kDa species immunoprecipitated from 293-hSERT extracts represents posttranslationally modified hSERT protein. CT-2 antisera also immunoprecipitates a minor species at ∼76 kDa (Fig. 1, *) that we suspect is a less heavily glycosylated or partially degraded form of hSERT protein. Together, these data suggest that hSERT protein is a target for direct phosphorylation by endogenous protein kinases and phosphatases in stably transfected HEK-293 cells. 293-hSERT cells display PKC-dependent down-regulation of 5HT uptake capacity and hSERT-associated currents associated with a reduction in surface transporter protein (1Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-47Crossref PubMed Google Scholar). We tested whether PKC activation affects the phosphorylation state of hSERT protein. In the absence of phosphatase inhibitors or PKC activators (Fig. 2, 0 min time point), we immunoprecipitate phosphorylated hSERT protein from 293-hSERT cells. This basal level of labeling is certainly lower than described previously (and in subsequent figures) with okadaic acid treatment but is nonetheless readily apparent when compared with immunoprecipitations from nontransfected HEK-293 cells that are electrophoresed in parallel. Treatment of transfected cells with the PKC activator β-PMA induces a time- (Fig. 2) and concentration (Fig.3)-dependent augmentation of basal hSERT phosphorylation, reaching levels 5–6-fold that of basal hSERT phosphorylation at maximal time and concentration points. The effects of β-PMA are rapid, as 1 μm β-PMA increases hSERT phosphorylation by more than 2-fold within the first 5 min of application (Fig. 2, A and B). We repeated our published analyses of β-PMA-induced inhibition of 5HT uptake so that transport and phosphorylation analyses could be followed in parallel, and we can demonstrate that the elevation in basal hSERT phosphorylation induced by β-PMA displays a similar time course to the β-PMA-induced losses in 5HT transport (Fig. 2 C). As with phosphorylation, the rate of change in uptake is greatest after the first 5 min of β-PMA treatment and then rises gradually thereafter to essentially plateau by 60 min. Low concentrations of β-PMA were required to effect an increase in hSERT phosphorylation.Figure 3Dose dependence of β-PMA on the phosphorylation of hSERT and 5-HT uptake. 293-hSERT cells were labeled with [32P]orthophosphate in phosphate-free DMEM for 1 h at 37 °C and incubated with various concentrations of β-PMA for 1 h at 37 °C. Control cells received the same volume of vehicle (ethanol). RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." A, an autoradiogram of immunoprecipitates, representative of three experiments, is shown. Molecular mass standards are indicated on the left, and the position of hSERT is marked on the right (arrow).B, quantitation of hSERT labeling. Autoradiograms of three experiments were analyzed as described in Fig. 2 B.C, dose-response inhibition of 5-HT uptake by β-PMA. Cells were preincubated with various concentrations of β-PMA for 1 h followed by a 10-min 5-HT uptake as described in Fig. 2 C.Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired ttest.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both hSERT phosphorylation and 5HT uptake inhibition are observed at low concentrations of β-PMA (Fig. 3, A and C). A greater than 2-fold increase in phosphorylation is evident with 1 nm β-PMA (60-min assay), and the EC50 for phosphorylation was 1.5 nm. A similar concentration dependent in β-PMA regulation of [3H]5HT uptake was observed with reductions in [3H]5HT transport observed with 1 nm β-PMA. Like β-PMA, the PKC activators, β-PDBu and indolactam V, stimulated 32P incorporation into hSERT protein 4–6-fold (Fig. 4). Moreover, the phorbol ester isomers that are inactive for PKC activation (α-PMA and α-PDBu) failed to elevate hSERT phosphorylation at equivalent concentrations. The PKC inhibitors staurosporine (200 nm) and bisindolylmaleimide I (1 μm) do not perturb basal hSERT phosphorylation but completely block hSERT phosphorylation induced by β-PMA (Fig. 4). KT5720, a selective PKA inhibitor blocks neither basal nor β-PMA-induced hSERT phosphorylation. These findings indicate that β-PMA-induced hSERT phosphorylation is mediated by PKC activation and that neither PKC nor PKA-dependent pathways are responsible for basal hSERT phosphorylation. As described initially, basal hSERT phosphorylation is significantly augmented if cells are pretreated with the PP1/PP2A phosphatase inhibitor okadaic acid, thereby preserving the labeling derived from endogenous protein kinases. Since okadaic acid might be stabilizing the effects of basal PKC activity or reflect labeling of h
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