Phosphorylation and Regulation of Antidepressant-sensitive Serotonin Transporters*

Sammanda Ramamoorthy, Elena Giovanetti, Yan QianDagger , and Randy D. Blakely§

From the Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The biogenic amine, serotonin (5-hydroxytryptamine, 5HT),1 is a neurotransmitter in the central nervous system and peripheral nervous system (2, 3) as well as a major secretory product of activated platelets (4). Synaptic clearance of 5HT following release and accumulation of 5HT by platelets is determined in large part by the action of the plasma membrane 5HT transporter (SERT (5, 6)). SERT-mediated 5HT accumulation is driven by transmembrane ion gradients (Na+, Cl-, and K+) (7-9) and is effectively blocked by indoleamine derivatives, addictive amphetamines, cocaine, and, most selectively, by SSRIs such as fluoxetine (ProzacTM) (5, 10-12). At synapses, SERT blockade in vivo elevates extracellular 5HT, permitting spillover of the amine and increased stimulation of 5HT receptors (13, 14). Selective blockade of central nervous system SERTs in humans is thought to represent the initial step in the pharmacologic amelioration of a wide spectrum of affective disorders, including major depression, anxiety disorders, appetite disorders, and obsessive-compulsive disorder (5, 15, 16).

The biochemical and behavioral effects of SERT modulation of SERT modulation using exogenous agents suggest that SERT expression may be tightly regulated in vivo and, perhaps, altered in its regulation in disease states (17). The cloning and functional characterization of SERT cDNAs (18-20) has revealed the carrier to belong to a gene family of Na+ and Cl--dependent transporters, containing homologous ion-coupled dopamine and norepinephrine (NE) transporters (DATs and NETs, respectively) among others (21-23). A single hSERT gene is located at chromosome locus 17q11.2 (20) and has recently been implicated in anxiety traits (24), major depression (25), and autism (26). hSERT gene expression is significantly regulatable by activation of both PKA and PKC pathways (17), and potential target sites for second messenger-mediated regulation of gene expression have been identified at or near transcription initiation and mRNA splicing sites (27-29). 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 (17). For example, platelet, endothelial, and brain SERTs are down-regulated within minutes by PKC activation (30-32), phenomena recapitulated in transfected COS (33), HEK-293 cells (1), and LLC-PK1 cells.2 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 (1). The presence of multiple, canonical serine and threonine phosphorylation sites on SERT cytoplasmic domains (18-20, 34) 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 (17, 35)) 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 vitro and in vivo (1, 36). 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- 293-hSERT cells were previously generated and characterized in this laboratory (1). 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.

Cell Culture-- 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 (1). 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.

Assay of 5-HT Transport-- [3H]5-HT transport activity was assayed in monolayer cultures for the times indicated at 37 °C as described previously (1). 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 mM CaCl2, 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.

Metabolic Labeling and Immunoprecipitations-- 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 (36, 37). 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 (37), CT-2B preimmune serum, or a second SERT-specific serum S365 (36). 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% beta -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 of 32P 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Immunoprecipitation of Phosphorylated hSERT Protein-- 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 (1, 36). 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 (36), like CT-2 antiserum, immunoprecipitates the same 96-kDa band, although the S365 antipeptide antibody displays consistently lower recovery in both 32P- and 35S-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.


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Fig. 1.   Phosphorylation of hSERT in 293-hSERT cells. Parental HEK-293 and 293-hSERT cells were metabolically labeled with [32P]orthophosphate in phosphate-free DMEM for 2 h at 37 °C in the presence of 1 µM beta -PMA and 1 µM okadaic acid. RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." N430 antisera immunoprecipitates NET proteins but not SERT proteins. S365 antisera, like CT-2, is SERT-specific although of lower titer than CT-2. Preabsorption of SERT CT-2 antisera with either GST protein or GST-COOH fusion protein (the CT-2 immunogen) was performed as described previously (36). An autoradiogram of a 10% SDS-PAGE is shown which is representative of three experiments. The location of protein molecular mass standards in kDa electrophoresed in parallel is shown to the left of the figure. The position marked for 32P-labeled hSERT (~96 kDa, arrow) matches that observed with direct immunoblotting. An asterisk denotes the position of a labeled coprecipitating product believed to be a degradation product or lightly glycosylated form of SERT protein.

Stimulation of hSERT Phosphorylation by Activation of PKC-- 293-hSERT cells display PKC-dependent down-regulation of 5HT uptake capacity and hSERT-associated currents associated with a reduction in surface transporter protein (1). 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 beta -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 beta -PMA are rapid, as 1 µM beta -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 beta -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 beta -PMA displays a similar time course to the beta -PMA-induced losses in 5HT transport (Fig. 2C). As with phosphorylation, the rate of change in uptake is greatest after the first 5 min of beta -PMA treatment and then rises gradually thereafter to essentially plateau by 60 min. Low concentrations of beta -PMA were required to effect an increase in hSERT phosphorylation.


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Fig. 2.   Time-course effect of beta -PMA on 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 1 µM beta -PMA for various times. 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. Radioactive bands corresponding to hSERT from three independent experiments were quantified by densitometric analysis of scanned x-ray film using ImageQuant (Molecular Dynamics), and normalized to the response to vehicle (taken as 100%), and the mean values ± S.E. are given. C, time course of the inhibition of 5-HT uptake by beta -PMA. 293-hSERT cells were preincubated with 1 µM beta -PMA for the times indicated and then assayed for 5-HT (1 µM, 10 min) uptake as described under "Experimental Procedures." Data are the average from three separate experiments. Nonspecific uptake was defined as the uptake in the presence of 0.1 µM paroxetine and subtracted from the total accumulation to yield specific uptake. Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired t test.


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Fig. 3.   Dose dependence of beta -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 beta -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. 2B. C, dose-response inhibition of 5-HT uptake by beta -PMA. Cells were preincubated with various concentrations of beta -PMA for 1 h followed by a 10-min 5-HT uptake as described in Fig. 2C. Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired t test.

Both hSERT phosphorylation and 5HT uptake inhibition are observed at low concentrations of beta -PMA (Fig. 3, A and C). A greater than 2-fold increase in phosphorylation is evident with 1 nM beta -PMA (60-min assay), and the EC50 for phosphorylation was 1.5 nM. A similar concentration dependent in beta -PMA regulation of [3H]5HT uptake was observed with reductions in [3H]5HT transport observed with 1 nM beta -PMA. Like beta -PMA, the PKC activators, beta -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 (alpha -PMA and alpha -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 beta -PMA (Fig. 4). KT5720, a selective PKA inhibitor blocks neither basal nor beta -PMA-induced hSERT phosphorylation. These findings indicate that beta -PMA-induced hSERT phosphorylation is mediated by PKC activation and that neither PKC nor PKA-dependent pathways are responsible for basal hSERT phosphorylation.


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Fig. 4.   Stereospecificity and specificity of PKC-mediated hSERT phosphorylation. 293-hSERT cells were labeled with [32P]orthophosphate for 1 h and incubated for an additional 1 h with or without PKC modulators and inhibitors. Kinase inhibitors were added 30 min prior to the addition of beta -PMA. The concentrations used were alpha -PMA, alpha -PDBu, beta -PMA, beta -PDBu at 1 µM each; (-)-indolactam V, 10 µM; staurosporine, 200 nM; bisindolylmaleimide I (BIM), 1 µM, and KT5720, 1 µM. Immunoprecipitations, autoradiography, and densitometry of x-ray film were performed as described under "Experimental Procedures" and Fig. 2B, and results are the mean ± S.E. of three separate experiments. Inset shows a portion of a representative autoradiogram showing hSERT labeling in response to various activators and inhibitors. Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired t test.

Analysis of hSERT Phosphorylation following Endogenous Phosphatase Inhibition-- 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 hSERT by distinct kinases, we sought to characterize the effects of phosphatase inhibition more carefully. First, we determined time and dose dependences of okadaic acid-stimulated hSERT phosphorylation. Like PKC-mediated phosphorylation of hSERT, incorporation of 32P into hSERT following okadaic acid (1 µM) treatment is rapid, with a nearly 3-fold increase over basal phosphorylation achieved within 5 min (Fig. 5, A and B) and a gradual increase evident to 1 h of treatment. However, unlike hSERT phosphorylation following beta -PMA treatment, a pronounced rise in SERT phosphorylation is evident between 60 and 120 min after okadaic acid treatment. Like beta -PMA, okadaic acid treatment reduces 5HT uptake in 293-hSERT cells, although the time course of the response is significantly distinct from that of phorbol esters (Fig. 5C). As with okadaic acid-stimulated hSERT phosphorylation, we observe the appearance of enhanced down-regulation of [3H]5HT uptake from 60 to 120 min of treatment. Concentration-response studies with okadaic acid (Fig. 6) reveal maximal effects (6-9-fold) observed by 1 µM (Fig. 5) and an EC50 for hSERT phosphorylation of ~350 nM, similar to that found for the concentration dependence of okadaic acid-triggered reductions in 5HT uptake (Fig. 6C). We also found the potent PP1/2A inhibitor calyculin A to augment hSERT phosphorylation to a similar extent as okadaic acid, but we found the PP2B inhibitor microcystin to be significantly less efficacious and the calcineurin inhibitor cyclosporin A to be relatively ineffective (Table I).


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Fig. 5.   Time-course effect of okadaic acid on 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 1 µM okadaic acid for various times. Control cells received same volume of vehicle (ethanol). RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." A, representative autoradiogram of labeling results. B, quantitation of hSERT labeling. Autoradiograms of three experiments were analyzed as described in Fig. 2B and results presented as mean ± S.E. C, time course of inhibition of 5-HT uptake by okadaic acid. Cells were preincubated with 1 µM okadaic acid for various times followed by a 10-min 5-HT uptake as described under "Experimental Procedures." Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired t test.


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Fig. 6.   Dose response of okadaic acid for 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 okadaic acid for 120 min. Control cells received same volume of vehicle (ethanol). RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." A, representative autoradiogram of labeling results. B, quantitation of hSERT labeling. Autoradiograms of three experiments were analyzed as described in Fig. 2B. C, dose-response inhibition of 5-HT uptake by okadaic acid. Cells were preincubated with 1 µM okadaic acid for various concentrations followed by 10-min 5-HT uptake as described under "Experimental Procedures." Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired t test.

                              
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Table I
Effect of phosphoprotein phosphatase inhibitors and its kinase selectivity on hSERT phosphorylation
293-hSERT cells were metabolically labeled with [32P]orthophosphate and incubated at 37 °C for 1 h in the absence (none) and presence of the following agents: okadaic acid, 1 µM; calyculin A, 1 µM; microcystin, 5 µM; cyclosporin A, 5 µM; staurosporine, 200 nM; bisindolylmalcimide I, 500 nM KT5720, 1 µM; KN-62, 2 µM; KN 93, 2 µM, and BAPTA-AM, 10 µM. Protein kinase inhibitors (staurosporine, bisindolyl- maleimide I, KT5720, KN-62, KN-93, and BAPTA-AM) were added 30 min prior to the addition of okadaic acid. RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." Autoradiograms from three different experiments were scanned densitometrically, and the mean values ± S.E. were given. Statistical significance was calculated comparing treated to untreated samples using the paired Student's t test.

Next we asked whether beta -PMA-stimulated and okadaic acid-induced hSERT phosphorylation act through a common mechanism by coapplication studies and cross-inhibitor studies. As shown in Fig. 7, beta -PMA and okadaic acid each at maximally efficacious concentrations yield additive hSERT phosphorylation when coapplied, achieving more than a 10-fold increase in 32P incorporation over basal conditions in these experiments. Note again that okadaic acid achieves an increase in hSERT phosphorylation between 60 and 120 min that is not observed with beta -PMA. These findings suggest that PKC and okadaic acid-inhibitable phosphatases in HEK cells phosphorylate hSERT through distinct pathways. Further evidence in support of this contention was gathered when we tested the ability of PKC inhibitors to block okadaic acid-induced phosphorylation (Table I). Staurosporine and bisindolylmaleimide I at concentrations that fully block the beta -PMA triggered phosphorylation of hSERT (refer to Fig. 4) fail to alter significantly okadaic acid-induced hSERT phosphorylation. Phosphorylation of hSERT revealed by okadaic acid treatment was also insensitive to intracellular calcium chelation as well as inhibition of CaM kinase II by KN-93 and KN-62 (Table I).


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Fig. 7.   Additivity of beta -PMA and okadaic acid-induced hSERT phosphorylation. 293-hSERT cells were labeled with [32P]orthophosphate for 1 h at 37 °C and then incubated with beta -PMA (1 µM) and okadaic acid (1 µM), either separately or in combination, for indicated times. The concentrations of beta -PMA and okadaic acid used are the maximally effective doses on the phosphorylation of hSERT. RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." A representative autoradiogram of four separate experiments corresponding to the 32P-labeled hSERT is shown. A, representative autoradiogram of time-dependent phosphorylations of agents added separately or together. B, quantitation of phosphorylation data averaged across three experiments presented as means ± S.E. Asterisks denote values significantly different (p < 0.05, Student's paired t test) in coapplication experiments from beta -PMA treatment alone.

PKA-mediated hSERT Phosphorylation Is Distinct from PKC and Phosphatase Pathways-- Differences in the time course and inhibitor sensitivity of beta -PMA- and okadaic acid-induced hSERT phosphorylations suggest the involvement of one or more kinases distinct from PKC. Purified cytoplasmic NH2 and COOH termini of rSERT are substrates for PKA as well as PKC in vitro (17, 35). We found the PKA activators forskolin and cholera toxin elevate hSERT phosphorylation 4-5-fold (Fig. 8). A similar level of hSERT phosphorylation is achieved with the membrane-permeant cAMP analog, 8-pCT-cAMP. The effect of cholera toxin is dose- and time-dependent with significant effects (3-fold) on incorporation of 32P in hSERT observed at 10 ng/ml (data not shown). In time course studies, the cholera toxin-induced phosphorylation of hSERT is more delayed and gradual than observed for PKC activators, with a doubling of hSERT labeling not observed for 45 min and another doubling evident by 90 min of incubation (data not shown). Furthermore, unlike beta -PMA and okadaic acid, PKA activators did not alter 5HT transport levels (data not shown). The PKC inhibitors staurosporine and bisindolylmaleimide I, at concentrations that block beta -PMA effects on hSERT phosphorylation (Fig. 4), fail to reduce cholera toxin and forskolin-triggered phosphorylation (Fig. 8). However, the specific PKA inhibitor KT5720, which does not block the beta -PMA-triggered labeling of hSERT (Fig. 4), blocks the effects of PKA activators completely (Fig. 8). At this same concentration, KT5720 blocks neither the basal level of hSERT phosphorylation nor the okadaic acid-augmented phosphorylation (Table I).


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Fig. 8.   Protein kinase A-dependent and PKC-independent phosphorylation of hSERT. 293-hSERT cells were labeled with [32P]orthophosphate in phosphate-free DMEM for 1 h at 37 °C and then incubated for an additional 120 min with or without activators of adenylyl cyclase and/or PKA or PKC inhibitors. Kinase inhibitors were added 30 min prior to the addition of cholera toxin or forskolin. The concentrations used were cholera toxin, 1 µg/ml; forskolin, 1 µM; 8-pCPT-cAMP, 1 mM; KT5720, 1 µM; staurosporine, 200 nM; and bisindolylmaleimide I, 1 µM. RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described under "Experimental Procedures." Inset shows a representative autoradiogram of labeling experiments. Averaged data from four separate experiments are presented ± S.E. Asterisks denote values significantly different (p < 0.05, Student's paired t test) from treatments with no activator or vehicle, performed in parallel.

Consistent with the inability of PKA inhibitors to mimic the effects of PKC inhibitors or attenuate the effects of okadaic acid, phosphorylation levels are found to be additive when cholera toxin and beta -PMA were coapplied or when cholera toxin and okadaic acid were coapplied at maximally effective concentrations (Table II). Although coapplied beta -PMA and okadaic acid yield additive reductions in 5HT uptake, cholera toxin does not potentiate the effects of either agent on uptake (data not shown). Interestingly, coapplication of beta -PMA, cholera toxin, and okadaic acid is no more effective at augmenting hSERT phosphorylation than the coapplication of any two of these modulators (Table II). Although we did not explore cGMP-linked pathways in detail in the present studies, we also wish to note that the membrane-permeant cGMP analog and activator of PKG, 8-pCT-cGMP (1 mM, 60 min), also significantly elevated hSERT phosphorylation in 293-hSERT cells with little or no effect on 5HT transport (data not shown). Together these findings reveal the capacity for cyclic nucleotide-activated phosphorylation of hSERT that is uncoupled to regulatory changes in 5HT transport activity in 293-hSERT cells and which appear to be distinct from transporter phosphorylation pathways modulated by PKC and PP1/2A.

                              
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Table II
Coordinate effects of beta -PMA, cholera toxin, and okadaic acid-induced phosphorylation of hSERT
293-hSERT cells were metabolically labeled with [32P]orthophosphate and exposed for 2 h to beta -PMA, cholera toxin, and okadaic acid, either separately or in combination, at their maximally effective doses as follows: beta -PMA, 1 µM; cholera toxin, 1 µg/ml, and okadaic acid, 1 µM. RIPA extraction, immunoprecipitation, SDS-PAGE and autoradiography were performed as described under "Experimental Procedures." Autoradiogram from three different experiments were scanned densitometrically, and the mean ± S.E. values were given.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The transport of 5HT across plasma membranes of platelets, pulmonary endothelium, placenta, and neurons is increasingly described as a tightly regulated process involving both transcriptional and posttranscriptional mechanisms (17). In the nervous system, presynaptic sites of SERT expression are significant distances from nuclear sites of SERT gene transcription, imposing temporal constraints on the ability of cells to modulate transport capacity and thereby affect extracellular 5HT clearance. Even in transfected cells, maturation and surface expression of NE and 5HT transporters takes several hours (37-40); recent in vivo covalent labeling studies on the DATs reveal a delay of more than a week to resupply striatal transport sites once they have been irreversibly inactivated (41).

Evidence has accumulated to indicate that receptor and kinase-linked pathways may rapidly up- and down-regulate endogenous SERTs as well as SERTs expressed in heterologous systems (17). We have recently shown (1) that HEK-293 cells stably transfected with hSERT cDNA, like endothelial cells (30), platelets (31), JAR (42, 43), and RBL-2H3 cells (34), and serotonergic neurons (32) support rapid modulation of 5HT transport following activation of PKC. Phorbol esters capable of activating PKC activity rapidly diminish 5HT uptake capacity in 293-hSERT cells, kinetically revealed as a change in 5HT transport Vmax, and this down-regulation is blocked by the PKC inhibitor staurosporine (1). hSERT-mediated currents are also down-regulated by beta -PMA in a staurosporine-sensitive manner in voltage clamped 293-hSERT cells, suggesting that regulation is not due to changes in ionic driving forces or the membrane potential (1). Furthermore, reductions in transport capacity are paralleled by a quantitatively similar reduction in cell-surface SERT protein (1). Recently, 5HT uptake capacity changes have been reported with the rat SERT cDNA transiently transfected into COS-7 cells (33), suggesting that this modulation is not unique to the HEK-293 context of our transfection studies. Analogous findings of PKC-dependent modulation of transport capacity have been obtained in studies of native and heterologously expressed dopamine (44-46), NE (47), gamma -aminobutyric acid (48-50), and glycine (51) transporters and thus mechanisms for acute regulation of members of the Na+/Cl-dependent neurotransmitter transporter gene family may possess many similarities. Members of the gene family share gross topological properties including cytoplasmic NH2 and COOH termini. These latter domains possess a large number of potential targets for protein phosphorylation. Moreover, we have found that the NH2 and COOH termini of SERTs, expressed and purified as GST fusion proteins, are substrates for protein kinases including PKC and PKA (17, 35). These findings increased our interest in exploring the question of whether direct protein phosphorylation of SERTs occurs and how this phosphorylation might effect transporter activity and surface expression.

The present studies demonstrate for the first time the existence of phosphorylated hSERT proteins and support the existence of multiple kinetically and molecularly distinct pathways leading to hSERT phosphorylation. We observe 1) that hSERT protein in 293-hSERT cells is phosphorylated under basal conditions and 2) this phosphorylation is potentiated by addition of the PP1/2A protein phosphatase inhibitors okadaic acid and calyculin A. Although basal phosphorylation is modest with respect to that achieved with phosphatase inhibition, it can be readily perceived when nontransfected cells are immunoprecipitated in parallel. In addition, we document the labeling of hSERT by 3) PKC- and 4) cyclic nucleotide activated kinase (PKA and PKG)-linked pathways. PKC- and PKA-induced hSERT phosphorylation appear additive with each other and with okadaic acid-induced labeling, suggesting that the endogenous phosphorylation protected by phosphatase inhibition is not mediated by either PKC or PKA. Indeed, neither PKC nor PKA inhibitors block the okadaic acid-induced phosphorylation of hSERT at concentrations required to fully abolish PKC or PKA labeling, respectively. Moreover, the kinetics of phosphorylation can be distinguished by activators of these pathways, and distinct effects are observed on transport. Like PKC activation, okadaic acid action leads to a reduction in transport capacity, but a loss of 5HT transport is not seen with PKA activators that phosphorylate hSERT to the same extent. Intracellular calcium and the activity of Ca2+/CaM kinase II have been implicated in acute SERT regulation (42, 52); however, our studies with BAPTA and specific Ca2+/CaM kinase II inhibitors fail to implicate these pathways in okadaic acid-induced phosphorylation. These data lead us to conclude that distinct sites are being phosphorylated as a result of PKC, PKA, and an as yet unidentified endogenous kinase(s).

PKA and PKG activation in 293-hSERT cells leads to rapid phosphorylation of hSERT protein yet no observable functional change in 5HT transport capacity. Although chronically administered cAMP-elevating agents have significant effects on hSERT gene transcription in JAR cells (53, 54), a role for PKA-linked pathways in acute SERT regulation is not evident. PKA-mediated phosphorylation of SERT proteins may reflect artifactual labeling in this heterologous system that never occurs to any significant extent in vivo. However, it is also possible that PKA-mediated labeling is facilitatory to other regulatory signals that we have failed to activate (or cannot) in these cells. In RBL-2H3 cells (34), brain (55, 56), and platelets (57), both nitric oxide and PKG-linked pathways have been implicated in acute, receptor-mediated regulation of 5HT uptake though as yet SERTs have not been shown to be phosphorylated under similar conditions. We find the potent, membrane-permeant analog 8-pCT-cGMP to elevate SERT phosphorylation although changes in uptake are not evident. As with PKA activation, this labeling may not translate into functional changes due to the heterologous or overexpression nature of the current system. Further studies are warranted to determine whether PKA and PKG activation promote the phosphorylation and regulation of SERTs in other cell contexts and whether the sites of phosphorylation overlap with those labeled following PKC activation.

PKC activation leads to both transporter phosphorylation (this study) and transporter redistribution from the cell surface (1). It is not possible, however, to conclude from our findings that phosphorylation of transporter protein is causal in the reduction of 5HT uptake since other proteins that may play important roles in transporter trafficking are likely to be targets of PKC-linked pathways as well. However, the close correspondence between dose and time dependence for transport reductions and transporter phosphorylation forces us to consider that at least a signal for membrane redistribution may be direct transporter phosphorylation. Rat and human SERT proteins possess multiple canonical sites for Ser/Thr phosphorylation among which are several PKC sites (18-20, 34). Our own studies with purified cytoplasmic NH2 and COOH termini (35) reveal both domains to be substrates for PKC (and PKA but not PKG). However, mutation of canonical PKC phosphorylation sites individually did not reduce this phosphorylation but rather enhanced it in several cases (58). Similarly, Sakai and co-workers (33) have described PKC and PP1/2A-mediated reductions in 5HT transport capacity in transiently transfected COS-7 cells, effects that were not abolished by mutation of canonical PKC sites. Mutation of PKC sites in GAT1 gamma -aminobutyric acid transporters (49) and GLYT1 glycine transporters (51) also fails to diminish PKC-mediated changes in transport capacity. We suspect that multiple sites may be phosphorylated on cytoplasmic domains by activated PKC (and these sites may interact), such that individual mutations are incapable of diminishing ultimate functional effects. Alternatively, PKC may phosphorylate other kinases and phosphatases that ultimately affect the hSERT phosphorylation state. Currently, we are exploring the merits of these arguments by trying to identify the sites of 32P incorporation and reconstituting the labeling patterns with in vitro extracts to permit kinase identification.

Striking similarities are evident in comparison of our findings with phosphorylation studies of native (45) or heterologously expressed rat DAT proteins (46). DATs expressed in these environments, like hSERTs, exhibit basal phosphorylation that can be augmented by phosphatase inhibition and PKC activation. For both transporters, activity changes are manifested primarily as a reduction in transport Vmax. As in our studies, DATs are less sensitive to the PP1/2A/2B inhibitor microcystin than okadaic acid and calyculin A, although this may reflect differential permeability of the compounds in HEK-293 cells. However, both SERT and DAT are insensitive to the calcineurin (PP2B) antagonist cyclosporin A. Presently, we are attempting to define the specific phosphatase complex modulating SERT phosphorylation to differentiate between PP1 and PP2A. An important difference between the DAT and SERT studies is our finding that okadaic acid-induced SERT phosphorylation is staurosporine-insensitive at concentrations required to block beta -PMA effects. This might represent a specificity of SERT for a distinct kinase or simply an inability to distinguish PKC and the endogenous kinase in LLC-PK1 cells. Consistent with the latter explanation, Huff and co-workers (45, 46) found additive phosphorylation of DAT proteins with coapplication of beta -PMA and okadaic acid. Another striking difference between the DAT and hSERT studies is the evidence we present that PKA and PKG activation, independent of PKC or okadaic acid-linked pathways, leads to SERT phosphorylation. DATs are not phosphorylated by treatment of cells with forskolin; the activation of cGMP-linked pathways has not been reported. Phosphorylation of SERT by PKA- or PKG-linked pathways may reveal modulatory pathways not shared with catecholamine transporters.

Phosphorylation of hSERT induced by activated PKC parallels in time and concentration the loss of 5HT uptake capacity and loss of surface protein. How might the phosphorylation of hSERT protein affect a redistribution of transporter protein from the cell surface? In G-protein-coupled receptors, agonist occupancy and heterologous stimuli lead to the phosphorylation, desensitization, and often, the down-regulation of receptors mediated by internalization (59, 60). Phosphorylated beta  adrenergic receptors are recognized by arrestin proteins, targeting them to plasma membrane domains that support clathrin- and dynamin-mediated endocytosis (61). Removal of phosphate from receptors by Ser/Thr phosphatases is thought to assist in the return of fully functional receptors to the cell surface (62). We suspect we have identified pathways that may be analogous to those used for heterologous receptor desensitization. Thus, receptors activating PKC may, in addition to other actions, affect surface distribution of SERT proteins by phosphorylating the transporter and enhancing its internalization rate. Alternatively, PKC activation could slow the rate of transporter insertion in the plasma membrane. Presently, little is known of the dynamics of transporter membrane trafficking. In oocytes, heterologously expressed gamma -aminobutyric acid GAT1 transporters appear to be shuttled in and out of the plasma membrane by membrane vesicles dependent on SNARE proteins, a process that is regulated by PKC (50). Interestingly, different pools of PKC appear to be available in oocytes such that exogenous (48) and intracellular (50) PMA treatments yield opposite changes in GAT1 expression. These findings remind us of the potential complexities associated with multiple PKC isoforms (63) and our present lack of knowledge of the cellular compartments where phosphorylation proceeds. It should be very informative to determine whether surface or intracellular SERT proteins are targets of phosphorylation. In 293-hSERT cells, we estimate ~75% SERT proteins are on the cell surface, but without additional studies, we cannot claim that it is the transporter in the plasma membrane that becomes phosphorylated in response to kinase activation or phosphatase inhibition. Interestingly, in preliminary studies, we find that 5HT itself can perturb both basal and PKC-mediated phosphorylation in these cells, suggesting that some or all of the phosphorylation observed may occur on surface resident SERTs accessible to transmitter. In summary, our studies suggest that endogenous transporter phosphorylation may contribute to the rapid regulation of SERT expression and reinforces the idea that phosphorylation may control synaptic signaling by modifying both neurotransmitter release and clearance.

    ACKNOWLEDGEMENTS

We thank D. Malone for laboratory assistance, R. Delplane for help with manuscript production, and S. Apparsundaram for careful review of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DA07390 (to R. D. B.) and a National Alliance for Research on Schizophrenia and Depression Young Investigator Award (to S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Graduate student in the Emory University Neuroscience program.

§ To whom correspondence should be addressed: Dept. of Pharmacology, MRBII, Rm. 419, Vanderbilt University School of Medicine, Nashville, TN 37232-6600. Tel.: 615-936-3037; Fax: 615-936-3040; E-mail: randy.blakely{at}mcmail.vanderbilt.edu.

1 The 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.

2 S. Ramamoorthy, unpublished observations.

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Abstract
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Results
Discussion
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