The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary targets for antidepressants for therapeutic intervention of mood disorders. Our previous studies have identified the involvement of several signalling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether Akt/PKB (protein kinase) regulates SERT function is not known. In the present study, we made the novel observation that inhibition of Akt resulted in the down-regulation of SERT function through the regulation of SERT trafficking and phosphorylation. Akt inhibitor Akt X {10-(4′-[N-diethylamino)butyl]-2-chlorophenoxazine} reduced the endogenously phosphorylated Akt and significantly decreased 5-HT uptake and 5-HT-uptake capacity. Furthermore, SERT activity is also reduced by siRNA down-regulation of total and phospho-Akt levels. The reduction in SERT activity is paralleled by lower levels of cell-surface SERT protein, reduced SERT exocytosis with no effect on SERT endocytosis and accumulation of SERT in intracellular endocytic compartments with the most prominent localization to late endosomes and lysosomes. Akt2 inhibitor was more effective than Akt1 inhibitor in inhibiting SERT activity. Inhibition of downstream Akt kinase GSK3α/β (glycogen synthase kinase α/β) stimulates SERT function. Akt inhibition leads to a decrease in SERT basal phosphorylation. Our results provide evidence that Akt regulates SERT function and cell-surface expression by regulating the intracellular SERT distribution and plasma membrane availability, which perhaps may be linked to SERT phosphorylation state. Thus any changes in the activation of Akt and/or GSK3α/β could alter SERT-mediated 5-HT clearance and subsequently serotonergic neurotransmission.

INTRODUCTION

The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) expressed in presynaptic serotonergic neurons controls 5-HT neurotransmission by rapid reuptake of released 5-HT [13]. 5-HT transport is of clinical relevance because SERT is a high-affinity target for antidepressants such as SSRIs (selective serotonin-reuptake inhibitors) [3,4]. SSRIs are used successfully to treat several psychiatric disorders. Furthermore, SERT is one of the high-affinity molecular targets for drugs of abuse such as cocaine and MDMA (3,4-methylenedioxymethampetamine) (‘ecstasy’) [5]. Documentation of altered SERT expression in various types of psychopathology indicates the importance of SERT in maintaining normal brain function [69]. Studies from our laboratory and others have revealed that the SERT-mediated 5-HT clearance is orchestrated through multiple regulatory pathways that dictate SERT gene transcription, expression, catalytic activity, trafficking, phosphorylation, protein–protein interactions and degradation (reviewed in [10] and see references therein). For example, the function of native and heterologously expressed SERT is rapidly inhibited in response to acute depletion of intracellular Ca2+, inhibition of calmodulin, CaMKII (Ca2+/calmodulin-dependent protein kinase II), Src kinase, p38 MAPK (mitogen-activated protein kinase) and activation of PKC (protein kinase C) [1117]. On the other hand, increased intracellular Ca2+, activation of NOS (nitric oxide synthase)/cGMP, Src kinase and MAPK pathways stimulate SERT activity [16,1821]. Receptor-mediated regulation of SERT has also been documented. Activation of ARs (adenosine receptors), 5-HT1B, atypical histamine receptors and BDNF (brain-derived neurotrophic factor)/TrkB (tropomyosin kinase B) stimulates SERT activity [2227], whereas stimulation of α2-adrenergic receptor signalling cascades reduces 5-HT uptake [28]. In addition, SERT substrates and inhibitors can also influence kinase/phosphatase-mediated SERT regulation [29].

The serine/threonine-specific protein kinase Akt/PKB (protein kinase B), has been implicated in the pathogenesis of serotonin-related disorders [3032] Activated Akt phosphorylates GSK3α/β (glycogen synthase kinase 3α/β), resulting in the inactivation of GSK3 [33]. SSRIs and other drugs acting on 5-HT neurotransmission have been shown to inhibit GSK3β by an Akt-linked phosphorylation site [3436]. Interestingly, altered Akt/GSK3β pathways have been implicated in major depressive disorders [32,37]. Given the pivotal role of SERT activity in regulating 5-HT neurotransmission, it is possible that the physiological effect of 5-HT may also be regulated through Akt-linked SERT regulation. However, it is not currently known whether Akt regulates 5-HT uptake through regulating SERT. In the present study, we explored the role of Akt in SERT regulation and demonstrate the time- and dose-dependent reduction in SERT activity by the Akt phospho-inhibitor Akt X {10-(4′-[N-diethylamino)butyl]-2-chlorophenoxazine}. Both pharmacological and genetic reductions in Akt phosphorylation reduced SERT activity. The reduction in SERT activity is correlated with reduced cell-surface expression and basal phosphorylation of SERT that could be linked to reduced plasma membrane delivery. These results suggest that constitutively active Akt controls SERT expression and function by a phosphorylation-dependent trafficking mechanism.

EXPERIMENTAL

Materials

Akt inhibitor Akt X was purchased from EMD Millipore. Akt1 inhibitor A-674563 {(2S)-1-[5-(3-methyl-1H-indazol-5-yl)pyridin-3-yloxy]-3-phenylpropan-2-amine} and Akt2 inhibitor CCT128930 {4-(4-chlorobenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine} were purchased from Selleckchem. ECL reagents, EZ link sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate], sulfo-NHS acetate (sulfo-N-hydroxysuccinimide acetate) and NeutrAvidin Agarose were from Pierce. [3H]5-HT creatinine sulfate (28.1 Ci/mmol), 32PO4 carrier-free orthophosphate, Optiphase Supermix and Kodak BioMax MS-1 films were from PerkinElmer. Reagents for SDS/PAGE and Bradford protein assay were obtained from Bio-Rad Laboratories. Lipofectamine™ 2000, DMEM (Dulbecco's modified Eagle's medium) and all other cell culture media were purchased from Invitrogen/Life Technologies. FBS was from Hyclone/GE Healthcare Life Sciences. ON-TARGETplus SMARTpool specific to Akt1 and ON-TARGETplus non-targeting siRNA were purchased from Thermo Scientific Dharmacon. SuperSignal West Pico Chemiluminescent substrate was from Thermo Fisher Scientific. Protein A–Sepharose was from GE Healthcare Life Sciences. GSK3α/β inhibitors BIO (6-bromoindirubin-3′-oxime), CHIR-99021 {6-[(2-{[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino}ethyl)amino]-3-pyridinecarbonitrile} and TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) were obtained from Sigma–Aldrich. All other chemicals were obtained from Sigma–Aldrich or Fisher Scientific unless indicated otherwise. The following antibodies were used: anti-calnexin from BD Biosciences, monoclonal anti-rabbit Akt antibody (clone AW24) and anti-phospho-Akt (Ser473) antibody (clone 11E6) from EMD Millipore, anti-GSK3 antibody sampler kit from Cell Signaling Technology, rabbit anti-ERGIC53 (endoplasmic recticulum–Golgi intermediate compartment 53) from Sigma–Aldrich, rabbit anti-Lamp1 (lysosome-associated membrane protein 1) from Abcam, polyclonal rabbit anti-giantin from Covance Research Products, polyclonal goat anti-SERT ST(C-20) from Santa Cruz Biotechnology, HRP (horseradish peroxidase)-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories, fluorescently tagged antibodies (Alexa Fluor® 488-conjugated donkey anti-rabbit and Alexa Fluor® 568 anti-goat) from Invitrogen, and rabbit polyclonal anti-SERT antibody (SR-12) generated by our laboratory. In our previous studies, the use and specificity of SR-12 anti-SERT antibody were characterized thoroughly [15,16,21].

Molecular biology

The coding sequence of hSERT (human SERT) was inserted into the mammalian expression vector pcDNA3.1(−) (Invitrogen) using XbaI and HindIII restriction sites. The EGFP-tagged Rab5, Rab7 and Rab11 constructs (pEGFP-C1-Rab5, pEGFP-C1-Rab7, and pEGFP-C1-Rab11) were a gift from Dr Kathrine W. Roche (NINDS, National Institutes of Health, Bethesda, MD, U.S.A.) [38]. The pEGFP-Rab4 plasmid was a gift from Dr Jose A. Esteban (Universidad Autonoma de Madrid, Madrid, Spain) [39]

Cell culture and transfections

HEK (human embryonic kidney)-293 cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1% (w/v) penicillin/streptomycin and 1% (w/v) glutamine. For transient transfection experiments, cells were trypsinized and seeded in poly-D-lysine coated 24-well plates (100000 cells/well) or 12-well plates (250000 cells/well) and incubated at 37°C for 24 h in a humidified CO2 incubator (5% CO2). The cells were transfected with 150 ng/well (24-well plate) or 500 ng/well (24-well plate) hSERT cDNA and/or empty pcDNA3.0 vector using Lipofectamine™ 2000 according to the manufacturer's instructions. Experiments were performed 24–28 h after transfection [16]. For microscopy, cells were transfected 48 h before experiments using 1 μg of pcDNA3 hSERT+1 μg of empty vector pcDNA3 in 75 cm2 flasks or 0.5 μg of pcDNA3 hSERT+0.25 μg of empty vector pcDNA3+0.25 μg of pEGFP-Rab4, pEGFP-Rab5a, pEGFP-Rab7 or pEGFP-Rab11 in 25 cm2 flasks using Lipofectamine™ 2000. On the day before experiments, 750000 cells/well were seeded on polyornithine-coated coverslips in six-well plates.

[3H]5-HT uptake (SERT) assay

5-HT uptake was performed as described previously [16,21]. The transfected cells grown in 24-well plates were washed and incubated with 1 ml of serum-free DMEM at 37°C for 2 h. Cells were then washed with 1 ml of pre-warmed (37°C) KRH (Krebs–Ringer Hepes) buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4 and 10 mM Hepes, pH 7.4) on a heating block (37°C) and treated with modulators in 500 μl of assay buffer (KRH solution containing 0.1 mM ascorbic acid and 0.1 mM pargyline) at 37°C for various periods or concentrations as indicated in the Figure legends. The uptake assay was started by incubating the cells with 50 nM [3H]5-HT at 37°C for 5 min. The uptake was terminated by removing the radiolabelled substrate followed by washing the cells twice with KRH buffer. Non-specific [3H]5-HT uptake was defined as the accumulation in the presence of 10 nM fluoxetine and was subtracted from the total counts. Non-specific background was also compared with cells transfected with pcDNA vector alone. The cells were lysed with 400 μl of Optiphase Supermix by shaking the plate for 30 min. The radioactivity was measured using a MicroBeta2 LumiJET liquid-scintillation counter (PerkinElmer). Results are given as means±S.E.M. from three experiments performed in duplicate or triplicate on different batches of HEK-293 cells.

SERT kinetics

The HEK-293 cells transfected with hSERT cDNA were pre-treated with Akt X (20 μM) for 30 min at 37°C. The cells were incubated with the mixture of [3H]5-HT and unlabelled 5-HT from 50 nM to 5 μM for 5 min and washed with KRH solution twice. Non-specific [3H]5-HT uptake was determined as the accumulation in the presence of 10 nM fluoxetine and was subtracted from total uptake. The radioactivity was measured using a liquid-scintillation counter. For kinetic analysis of 5-HT uptake, the values were plotted as fmol of 5-HT uptake against the concentration of 5-HT and results are means±S.E.M. from three experiments performed in triplicate on different batches of HEK-293 cells as described previously by our laboratory [16,21]. Substrate Km and Vmax values for 5-HT uptake were determined by non-linear least-square fits using Prism 6 (GraphPad Software) with the generalized Michaelis–Menten equation: V=Vmax·[S]h/(Khm+[S]h), where V is the transport velocity, [S] is substrate (5-HT) concentration, and h is the Hill coefficient.

Cell-surface biotinylation to determine cell-surface SERT density

Cell-surface biotinylation and immunoblot analyses were utilized to determine the plasma membrane density of SERT protein as described previously [16,21]. hSERT cDNA-transfected HEK-293 cells grown in 12-well plates were pre-treated with Akt X (20 μM) for 45 min at 37°C. The cells were washed with 1 ml of ice-cold PBS/Ca2+-Mg2+buffer (138 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 9.6 mM Na2HPO4, 1 mM MgCl2 and 0.1 mM CaCl2, pH 7.3) and incubated twice with 1 ml of EZ-link sulfo-NHS-SS-biotin (0.5 mg/ml) solution on ice for 20 min. The cells were washed with 1 ml of ice-cold PBS/Ca2+-Mg2+ buffer and the biotinylation was quenched by incubating the cells with 100 mM glycine in PBS/Ca2+-Mg2+ buffer for 15 min at 4°C. The cells were washed again with ice-cold PBS/Ca2+-Mg2+ buffer. The cells were lysed with 400 μl of cold RIPA lysis buffer [10 mM Tris/HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1% (w/v) SDS and 0.1% sodium deoxycholate] supplemented with protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μM pepstatin, and 250 μM PMSF) and phosphatase inhibitors (10 mM sodium fluoride, 50 mM sodium pyrophosphate and 5 mM activated sodium orthovanadate) by shaking the plate at 4°C for 1 h and by passing through a 26-gauge needle six times. The lysed cells were centrifuged at 25000 g for 30 min. The supernatant was collected in fresh microtubes and concentration of the protein was measured using Bradford assay. NeutrAvidin beads were washed twice with ice-cold PBS/Ca2+-Mg2+ buffer and once with ice-cold RIPA buffer. Equal amounts of biotinylated samples were incubated with 60 μl of NeutrAvidin beads at 4°C on a nutator overnight. The next day, the samples were washed three times with RIPA buffer. The biotinylated proteins were eluted in 45 μl of 2× Laemmli buffer [62.5 mM Tris/HCl, pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 0.01% Bromophenol Blue and 5% (v/v) 2-mercaptoethanol] and separated by SDS/PAGE (10% gel) along with total extracts and unbound fractions, transferred on to a PVDF membrane and probed with SERT-specific SR-12 antibody as described previously [21]. The immunoreactive proteins were visualized using ECL or ECL plus reagent. Subsequently, the blots were stripped and reprobed with anti-calnexin antibody to validate the cell-surface biotinylation of plasma membrane proteins as well as loading protein levels. SERT densities from total, non-biotinylated (representing the intracellular pool) and biotinylated (representing the surface pool) fractions were normalized using total levels of SERT as well as levels of calnexin in the total extract. Multiple exposures were evaluated by digital quantification using NIH ImageJ (version 1.48v) software to ensure that results were within the linear range of the film exposure.

Assay of SERT delivery into the plasma membrane

The plasma membrane insertion of SERT (exocytosis) assay was performed using biotinylation and immunoblotting as described previously [40,41]. Briefly, HEK-293 cells transfected with hSERT cDNA were grown in 12-well plates, cooled rapidly to 4°C by washing with ice-cold PBS/Ca2+-Mg2+ buffer and incubated with 1 mg/ml sulfo-NHS acetate in ice-cold PBS/Ca2+-Mg2+ buffer for 1 h at 4°C (trafficking-non-permissive conditions) to block all free amino groups [42,43]. After washing away the sulfo-NHS acetate with ice-cold PBS/Ca2+-Mg2+ buffer, the cell- membrane-impermeant sulfo-NHS-SS-biotin (1 mg/ml) in PBS/Ca2+-Mg2+ containing the vehicle or Akt X (pre-warmed at 37°C) was added to the cells and incubated further for the indicated periods at 37°C (trafficking-permissive conditions). At the end of each period, the cells were cooled rapidly to 4°C and incubated twice with 100 mM glycine in ice-cold PBS/Ca2+-Mg2+ buffer for 15 min. One dish of cells was incubated at 4°C as an experimental control. Biotinylated SERTs inserted into the plasma membrane (surface) and non-biotinylated (intracellular) SERTs were analysed by immunoblotting with SR-12 anti-SERT antibody and quantified as described in the ‘Cell-surface biotinylation to determine cell-surface SERT density’ section.

Reversible biotinylation to determine SERT internalization

A reversible biotinylation technique was performed to determine the effect of Akt X on internalization (endocytosis) of SERT as described previously [40,41]. hSERT cDNA-transfected HEK-293 cells grown in a 30-mm-diameter dish were cooled rapidly to 4°C to inhibit endocytosis by washing with ice-cold PBS/Ca2+-Mg2+ buffer and cell-surface-biotinylated with a disulfide-cleavable biotin (sulfo-NHS-SS-biotin), and free biotinylating reagent was removed by quenching with glycine. SERT endocytosis was initiated by incubating the cells with pre-warmed PBS/Ca2+-Mg2+ buffer with vehicle or Akt X (20 μM) at 37°C for the indicated periods. At the end of each period, the cells were cooled rapidly to 4°C and washed with ice-cold PBS/Ca2+-Mg2+ buffer to stop the endocytosis. The cells were then washed and incubated twice with 50 μM sodium 2-mercaptoethanesulfonate, a reducing agent in PBS/Ca2+-Mg2+ buffer for 20 min to dissociate the biotin from cell-surface-resident proteins via disulfide exchange. To define total biotinylated SERTs, one dish of biotinylated cells was not subjected to reduction with sodium 2-mercaptoethanesulfonate, and processed directly for extraction followed by isolation by NeutrAvidin beads. To define sodium 2-mercaptoethanesulfonate-accessible SERTs, another dish of cells was treated with sodium 2-mercaptoethanesulfonate immediately (at zero time) following biotinylation at 4°C to reveal the quantity of cell-surface SERT biotinylation that sodium 2-mercaptoethanesulfonate can reverse efficiently. The cells were lysed, and biotinylated proteins were separated using NeutrAvidin beads and subjected to immunoblotting as described in the ‘Cell-surface biotinylation to determine cell-surface SERT density’ section.

siRNA interference of Akt

The Akt interference was performed in HEK-293 cells with ON-TARGETplus SMARTpool siRNAs specific to Akt. The non-specific scrambled siRNA was used as a control. siRNA duplexes (30 nM) were transfected into cells using Lipofectamine™ 2000 according to the manufacturer's instructions. At 16 h after siRNA transfection, HEK-293 cells were then transfected with hSERT cDNA. After 24 h, a 5-HT uptake assay was performed as described in the ‘[3H]5-HT uptake (SERT) assay’ section. In parallel, immunoblot analysis of total and phospho-Akt was carried out to verify the suppression of Akt levels. The same blot was reprobed for calnexin antibody to determine the non-specific effect of siRNAs as described previously [16,21].

Phospho- and total Akt and GSK3α/β levels

After pre-treatment of HEK-293 cells expressing hSERT with modulators as indicated in the Figure legends, or the HEK-293 cells that were transfected with siRNA specific to Akt or scrambled siRNA, were lysed with RIPA buffer containing phosphatase inhibitors (10 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 μM okadaic acid) and 0.1% protease inhibitor cocktail to quantify total and phospho-Akt and GSK3α/β levels. Lysates were centrifuged at 25000 g for 30 min at 4°C, and supernatant was collected for immunoblotting analysis. Proteins were incubated with Laemmli sample buffer and separated by SDS/PAGE (10% gel). The separated proteins were transferred on to a PVDF membrane and probed with anti-phospho-Akt or anti-phospho-GSK3α/β antibodies to detect phospho-Akt and phospho-GSK3α/β levels. The same blots were then stripped and reprobed with anti-Akt/PKBα or anti-GSK3α/β antibody to determine the total Akt and GSK3α/β levels. The immunoreactive proteins were visualized using SuperSignal West Pico Chemiluminescent substrate. Protein bands were scanned and the band densities were quantified using ImageJ software.

Metabolic labelling to detect SERT phosphorylation

Phosphorylated [32P]SERT was immunoisolated and quantified as described previously [29,44]. Briefly, HEK-293 cells expressing hSERT were metabolically labelled with [32P]orthophosphate in phosphate-free DMEM for 1 h and incubated with Akt X (20 μM) for 15 min at 37°C. The cells were washed and solubilized with RIPA buffer containing protease and phosphatase inhibitors (composition given above). The SERT proteins were immunoprecipitated using the SERT-specific antibody SR-12. The immunoprecipitated proteins were isolated by the addition of Protein A–Sepharose beads, washed three times with RIPA buffer and eluted in 45 μl of Laemmli sample buffer for 30 min at 22°C. The eluted proteins were separated by SDS/PAGE (10% gel). Gels were dried and exposed to Kodak BioMax MS-1 films and 32P-radiolabelled SERT was visualized by autoradiography. The SERT bands were quantified using ImageJ software as described previously [29,44].

Immunostaining

Transiently transfected HEK-293 cells were pre-treated with 20 μM Akt X in KRH buffer as described above for the uptake assay. Subsequently, the cells were washed in PBS and fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. The samples were washed in PBS before blocking with 1% (w/v) BSA in PBS, and permeabilized using 0.2% saponin. The sample was stained for hSERT using goat anti-SERT (1:100 dilution), lysosome using rabbit anti-Lamp1 (1:800 dilution), Golgi using rabbit anti-giantin (1:800 dilution), and ERGIC53 using rabbit anti-ERGIC53 (1:500 dilution) as well as Alexa Fluor® 488-conjugated donkey anti-rabbit and Alexa Fluor® 568-conjugated donkey anti-goat antibodies. The samples were then mounted in ProLong Gold Antifade reagent (Molecular Probes/Invitrogen).

Confocal microscopy

Confocal microscopy was performed with an inverted Zeiss LSM 510 laser-scanning unit through an oil immersion ×63 1.4 numerical aperture objective (Carl Zeiss). Alexa Fluor® 488 and EGFP were excited using a 488 nm argon/krypton laser and the emitted light was filtered through a 505–530 nm bandpass filter. The Alexa Fluor® 568 was excited using a 543 nm helium/neon laser and the emitted light was filtered using a 560 nm long-pass filter. For co-localization studies, images were acquired using line shift, and an averaging of eight. Images were collected in 1024 pixels×1024 pixels and pinholes were set to achieve optical sections of 1 μm.

Quantitative co-localization microscopy

The amount of internalization was calculated using ImageJ software. A mask was created to remove background using permanent settings for each image. The remaining intracellular fluorescence was normalized to the total remaining fluorescence. In each individual experiment, the average intracellular fraction of vehicle-treated cells was normalized to 1. Statistical significance was determined by a two-sample Student's t test with a Welch correction. Results are means±S.E.M. from 52–69 cells from three independent experiments. Co-localization was studied utilizing Van Steensel's cross-correlation fraction with the JACoP plug-in [45]. The red and green image was split into the two channels, and a mask was generated for each colour to remove background light before measuring the cross-correlation. Van Steensel's cross-correlation function was determined by calculating the Pearson's coefficient in a given overlay of the two images, and then shifting one of the colours 20 pixels in each x direction compared with the other, plotting Pearson's coefficient as a function of the pixel shift (Δx). Complete overlay gives a cross-correlation function of 1 at Δx=0. Co-localization was determined on the basis of 18 cells from three independent experiments. Results are means±S.E.M.

Statistical analyses

Results are expressed as means±S.E.M. As noted in the Figure legends, one-way or two-way ANOVA was used followed by post-hoc testing (Bonferroni) for multiple comparisons. Student's t test was performed for comparison between two groups. Prism 6 was used for data analysis. A P value <0.05 was considered statistically significant.

RESULTS

Akt inhibition reduces SERT activity

Using Akt X, which inhibits Akt activity [46] we asked whether Akt/PKB regulates SERT function. Akt X treatment of the HEK-293 cells, transiently expressing hSERT, reduced 5-HT uptake in a dose- and time-dependent manner (Figures 1A and 1B). When various concentrations of Akt X (1, 2.5, 5, 10 and 20 μM) were tested for 30 min, a dose-dependent decrease in 5-HT uptake is evident [F(5,42)=12.95; P<0.0001] (Figure 1A). A Bonferroni's post-hoc analysis revealed that 5, 10 and 20 μM Akt X produced a significant decrease in 5-HT uptake (P≤0.01) compared with vehicle-treated cells. Pre-incubation of cells with 2.5, 5, 10 and 20 μM Akt X for 30 min produced 16%, 27%, 32% and 55% inhibition of 5-HT uptake respectively (Figure 1A). When cells were treated with 10 μM Akt X for 5, 15, 30 and 60 min at 37°C before the [3H]5-HT uptake assay, a time-dependent decrease in 5-HT uptake was observed [F(4,44)=9.04; P<0.0001] (Figure 1B). A Bonferroni's post-hoc analysis revealed that a significant decrease in 5-HT uptake is evident after 5 min of pre-treatment of Akt X (20%; P≤0.05) and reached maximum inhibition after 30 min of pre-treatment (38%; P≤0.0001). To verify that Akt X inhibits Akt activity that is correlated with SERT activity inhibition, we examined the phosphorylation status of Akt (Ser473). As shown in Figure 1(C), immunoblotting of cell lysates showed significantly reduced levels of phospho-Akt (P≤0.02; n=3) in cells treated with Akt X (20 μM, 30 min) with no significant change in the total Akt protein. These results indicate that acute inhibition of Akt activity leads to decreased SERT activity.

Regulation of 5-HT uptake by Akt X

Figure 1
Regulation of 5-HT uptake by Akt X

(A) Dose-dependence. Transiently transfected HEK-293 cells with hSERT were pre-treated with assay buffer alone (vehicle) and different concentration of Akt X for 30 min at 37°C followed by 5 min of [3H]5-HT (50 nM) uptake. Results are means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001 compared with vehicle (one-way ANOVA with Bonferroni's multiple comparison test). (B) Time course. Transiently transfected HEK-293 cells were pre-treated with 20 μM Akt X for the times indicated and assayed for [3H]5-HT (50 nM) uptake for 5 min at 37°C. Results are means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001 compared with vehicle (one-way ANOVA with Bonferroni's multiple comparison test). Results are shown as percentages of specific SERT-mediated uptake values relative to the uptake observed in vehicle control as described in the Experimental section. (C) Representative immunoblot (upper panel) showing phospho-Akt, total Akt in vehicle- and Akt X- (20 μM, 30 min) treated cells. The results in the lower panel are a statistical analysis of normalized (phospho-Akt/total Akt) band densities from three separate experiments. *P<0.05 compared with vehicle (Student's t test). Veh, vehicle.

Figure 1
Regulation of 5-HT uptake by Akt X

(A) Dose-dependence. Transiently transfected HEK-293 cells with hSERT were pre-treated with assay buffer alone (vehicle) and different concentration of Akt X for 30 min at 37°C followed by 5 min of [3H]5-HT (50 nM) uptake. Results are means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001 compared with vehicle (one-way ANOVA with Bonferroni's multiple comparison test). (B) Time course. Transiently transfected HEK-293 cells were pre-treated with 20 μM Akt X for the times indicated and assayed for [3H]5-HT (50 nM) uptake for 5 min at 37°C. Results are means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001 compared with vehicle (one-way ANOVA with Bonferroni's multiple comparison test). Results are shown as percentages of specific SERT-mediated uptake values relative to the uptake observed in vehicle control as described in the Experimental section. (C) Representative immunoblot (upper panel) showing phospho-Akt, total Akt in vehicle- and Akt X- (20 μM, 30 min) treated cells. The results in the lower panel are a statistical analysis of normalized (phospho-Akt/total Akt) band densities from three separate experiments. *P<0.05 compared with vehicle (Student's t test). Veh, vehicle.

Specific knockdown of Akt by siRNA reduces SERT activity

To support our findings that Akt regulates SERT activity, we used siRNA targeting Akt and determined the effect of loss of function of Akt on 5-HT uptake. At 40 h after transfection of siRNAs, total Akt (t=3.72, df=11, P<0.003) as well as phospho-Akt (Ser473) (t=3.66, df=7, P<0.008) were significantly reduced in cells transfected with Akt siRNA/hSERT compared with cells transfected with non-specific scrambled siRNA/hSERT (Figures 2A and 2B). Akt siRNA and/or scrambled siRNA transfection did not affect the total expression of calnexin (Figure 2A), ruling out any non-specific effects. In parallel experiments, a significant reduction in 5-HT uptake was observed in cells transfected with Akt siRNA/hSERT compared with that observed in cells transfected with non-specific scrambled siRNA/hSERT (t=4.86, df=16, P<0.0002) (Figure 2C). Consistent with Akt X results (Figure 1), the results from the specific genetic knockdown of Akt (Figure 2) strongly support the idea that endogenous Akt plays a crucial role in the regulation of SERT activity.

Effect of siRNA knockdown of Akt expression on 5-HT transport

Figure 2
Effect of siRNA knockdown of Akt expression on 5-HT transport

HEK-293 cells were transfected with 30 nM siRNAs targeted to Akt or control scrambled siRNAs before the transfection of hSERT as described in the Experimental section. Transfected cells were used for 5-HT transport, total Akt and phospho-Akt expression. (A) Representative Western blot from separate experiments showing the expression levels of total Akt (n=6) and phospho-Akt (n=5) and calnexin. (B) Mean±S.E.M. band densities of total and phospho-Akt. **P<0.01 compared with scrambled siRNAs plus hSERT-transfected cells (Student's t test). (C) Mean±S.E.M. SERT-specific 5-HT uptake values. SERT-specific 5-HT uptake was performed as described in the Experimental section and in the legend to Figure 1. ***P<0.0002 (n=9) significantly different scrambled siRNAs plus hSERT-transfected cells (Student's t test).

Figure 2
Effect of siRNA knockdown of Akt expression on 5-HT transport

HEK-293 cells were transfected with 30 nM siRNAs targeted to Akt or control scrambled siRNAs before the transfection of hSERT as described in the Experimental section. Transfected cells were used for 5-HT transport, total Akt and phospho-Akt expression. (A) Representative Western blot from separate experiments showing the expression levels of total Akt (n=6) and phospho-Akt (n=5) and calnexin. (B) Mean±S.E.M. band densities of total and phospho-Akt. **P<0.01 compared with scrambled siRNAs plus hSERT-transfected cells (Student's t test). (C) Mean±S.E.M. SERT-specific 5-HT uptake values. SERT-specific 5-HT uptake was performed as described in the Experimental section and in the legend to Figure 1. ***P<0.0002 (n=9) significantly different scrambled siRNAs plus hSERT-transfected cells (Student's t test).

Akt inhibition reduces SERT-mediated 5-HT uptake capacity

The kinetic parameters (Michaelis–Menten constant, Km, and Vmax) of SERT were determined using vehicle and Akt X (20 μM, 30 min) -treated HEK-293 cells expressing hSERT (Figure 3). Akt X treatment produced a significant reduction (~26%) in the maximal velocity (Vmax) compared with vehicle-treated cells (t=2.6, df=6, P<0.04). The Vmax in vehicle-treated cells is 3263±249 fmol/min per 106 cells and in Akt X-treated cells is 2408±215 fmol/min per 106 cells. Interestingly, Akt X exposure did not change SERT's affinity for its substrate 5-HT (P<0.99). Km values obtained were 180±52.67 and 180.8±38.87 nM for vehicle-treated and Akt X-treated cells respectively (Figure 3).

Effect of Akt X on SERT-mediated 5-HT-uptake kinetics

Figure 3
Effect of Akt X on SERT-mediated 5-HT-uptake kinetics

HEK-293 cells transiently transfected with hSERT were pre-treated with vehicle or Akt X (20 μM, 30 min). After treatment, uptake of 5-HT was quantified over the concentration range 25–2500 nM using a 5 min uptake period. Non-specific uptake at each concentration of 5-HT used (in the presence of 0.01 μM fluoxetine) was subtracted from total uptake (measured in the absence of fluoxetine) to calculate SERT-mediated 5-HT uptake. Results are means±S.E.M. from three different experiments. Non-linear curve fits of data for uptake used the generalized Michaelis–Menten equation.

Figure 3
Effect of Akt X on SERT-mediated 5-HT-uptake kinetics

HEK-293 cells transiently transfected with hSERT were pre-treated with vehicle or Akt X (20 μM, 30 min). After treatment, uptake of 5-HT was quantified over the concentration range 25–2500 nM using a 5 min uptake period. Non-specific uptake at each concentration of 5-HT used (in the presence of 0.01 μM fluoxetine) was subtracted from total uptake (measured in the absence of fluoxetine) to calculate SERT-mediated 5-HT uptake. Results are means±S.E.M. from three different experiments. Non-linear curve fits of data for uptake used the generalized Michaelis–Menten equation.

Akt inhibition reduces cell-surface density of SERT

Next we sought to determine whether Akt X-mediated down-regulation of SERT activity is attributable to reduced cell-surface SERT levels, using membrane-impermeant protein-biotinylating agents. Cell-surface SERT levels were determined using HEK-293 cells expressing hSERT following treatment with vehicle or Akt X. Figure 4 shows that the Akt X treatment (20 μM, 45 min) decreased cell-surface expression of SERT. Quantification of cell-surface SERT immunoreactive band densities revealed a significant decrease (~38%, t=3.51, df=8, P<0.008) in the amount of cell-surface SERT (Figure 4). Total SERT level did not change following Akt X exposure. Calnexin protein expression was not affected by Akt X treatment or not present in biotinylated fractions (results not shown). Thus the reduction in cell-surface SERT protein correlated with the reduction in the transport capacity Vmax as measured by 5-HT uptake (Figure 3).

Cell-surface SERT density

Figure 4
Cell-surface SERT density

HEK-293 cells transiently transfected with hSERT were pre-treated with vehicle or Akt X (20 μM, 45 min). After the treatment, biotinylation with EZ-link sulfo-NHS-SS-biotin was performed. Biotinylated fractions, and total fractions were subjected to SDS/PAGE followed by immunoblotting with SERT-specific SR-12 antibody as described in the Experimental section. A representative SERT immunoblot of four different experiments is shown in the upper panel. Quantified mean±S.E.M. SERT band densities are shown in the lower panel. **P<0.008 compared with vehicle control (Student's t test).

Figure 4
Cell-surface SERT density

HEK-293 cells transiently transfected with hSERT were pre-treated with vehicle or Akt X (20 μM, 45 min). After the treatment, biotinylation with EZ-link sulfo-NHS-SS-biotin was performed. Biotinylated fractions, and total fractions were subjected to SDS/PAGE followed by immunoblotting with SERT-specific SR-12 antibody as described in the Experimental section. A representative SERT immunoblot of four different experiments is shown in the upper panel. Quantified mean±S.E.M. SERT band densities are shown in the lower panel. **P<0.008 compared with vehicle control (Student's t test).

Akt inhibition reduces SERT plasma membrane delivery without affecting the internalization

The Akt X-induced decrease in SERT cell-surface expression could arise from reduced plasma membrane insertion or enhanced endocytosis of SERT, or a combination of both. We therefore investigated the effect of Akt X on SERT plasma membrane insertion (exocytosis) and SERT internalization using a biotinylation strategy as described previously [40]. Figure 5(A) shows the results of biotinylation experiments in which newly inserted SERT levels to the plasma membrane following Akt X or vehicle treatment. To evaluate the delivery of SERT to the cell surface, first pre-existing cell-surface SERTs were blocked from biotinylation by treating cells with sulfo-NHS acetate. Sulfo-NHS acetate treatment at 4°C blocks all the free amino groups on the proteins that would be biotinylated otherwise [42,43]. No biotinylated SERT was present at the zero time point in sulfo-NHS acetate-treated cells, suggesting that all pre-existing cell-surface SERTs are blocked (from modification by biotinylation). Therefore any biotinylated SERT observed at subsequent time points after warming the cells to 37°C represents newly delivered SERT only. In vehicle-treated cells there was a gradual time-dependent increase in SERT plasma membrane insertion which plateaued at the 30 min time point [F(3,25)=4.36; P<0.02]. This increase represents constitutive transporter delivery to the plasma membrane. Akt X treatment significantly decreased constitutive plasma membrane recycling of SERT [F(7,39)=5.05; P<0.0004]. Although a Bonferroni's post-hoc analysis revealed that a significant decrease in SERT delivery was apparent at 15 and 30 min following Akt X treatment (P<0.03 and <0.04 respectively), a trend in non-significant decrease in SERT delivery was evident at 2.5 and 5 min following Akt X treatment (Figure 5A). These results indicate that Akt activity regulates SERT plasma membrane delivery.

Effect of Akt X on SERT exocytosis and endocytosis

Figure 5
Effect of Akt X on SERT exocytosis and endocytosis

(A) SERT exocytosis/plasma membrane insertion. HEK-293 cells expressing hSERT were treated with sulfo-NHS acetate to block all free amino groups before biotinylation with sulfo-NHS-SS-biotin in the presence of vehicle or Akt X (20 μM, 45 min) for the indicated periods. Isolation of biotinylated and non-biotinylated proteins and immunoblotting of SERT were performed as described in the Experimental section. A representative blot (upper panel) shows changes in biotinylated SERT following Akt X and vehicle treatments. Biotinylated SERT bands were quantified using ImageJ, and mean±S.E.M. band densities are shown in the lower panel. *P<0.05 indicates significant changes in the SERT exocytosis following Akt X treatment compared with corresponding vehicle treatment at each time point (one-way ANOVA with Bonferroni's multiple comparison test). (B) SERT endocytosis/internalization. Cells were biotinylated with sulfo-NHS-SS-biotin and incubated with vehicle or 20 μM Akt X for the times indicated. Following sodium 2-mercaptoethanesulfonate (MesNa) treatment, biotinylated (internalized) SERTs were isolated and analysed as described in the Experimental section. Representative immunoblots from three separate experiments are given in the upper panel. Mean±S.E.M. SERT band densities quantifying biotinylated SERT levels are shown in the lower panel. P=0.39, no significant effect on SERT internalization following Akt X treatment (one-way ANOVA with Bonferroni's multiple comparison test).

Figure 5
Effect of Akt X on SERT exocytosis and endocytosis

(A) SERT exocytosis/plasma membrane insertion. HEK-293 cells expressing hSERT were treated with sulfo-NHS acetate to block all free amino groups before biotinylation with sulfo-NHS-SS-biotin in the presence of vehicle or Akt X (20 μM, 45 min) for the indicated periods. Isolation of biotinylated and non-biotinylated proteins and immunoblotting of SERT were performed as described in the Experimental section. A representative blot (upper panel) shows changes in biotinylated SERT following Akt X and vehicle treatments. Biotinylated SERT bands were quantified using ImageJ, and mean±S.E.M. band densities are shown in the lower panel. *P<0.05 indicates significant changes in the SERT exocytosis following Akt X treatment compared with corresponding vehicle treatment at each time point (one-way ANOVA with Bonferroni's multiple comparison test). (B) SERT endocytosis/internalization. Cells were biotinylated with sulfo-NHS-SS-biotin and incubated with vehicle or 20 μM Akt X for the times indicated. Following sodium 2-mercaptoethanesulfonate (MesNa) treatment, biotinylated (internalized) SERTs were isolated and analysed as described in the Experimental section. Representative immunoblots from three separate experiments are given in the upper panel. Mean±S.E.M. SERT band densities quantifying biotinylated SERT levels are shown in the lower panel. P=0.39, no significant effect on SERT internalization following Akt X treatment (one-way ANOVA with Bonferroni's multiple comparison test).

We next examined the internalization of SERT under basal/constitutive conditions and following treatment with Akt X using the reversible biotinylation strategy and by quantifying the fraction of cell-surface biotinylated SERT that moves in a time-dependent manner to an intracellular compartment. Biotin from remaining biotinylated proteins on the cell surface at the end of a particular treatment protocol was removed by treatment with sodium 2-mercaptoethanesulfonate, a non-membrane-permeant reducing agent that reduces disulfide bonds and liberates biotin from biotinylated proteins at the cell surface. Thus the amount of biotinylated proteins inaccessible to sodium 2-mercaptoethanesulfonate action is defined as ‘the amount of protein endocytosed or internalized’. The amount of SERT biotinylated in the absence of sodium 2-mercaptoethanesulfonate represents total biotinylated transporter. Sodium 2-mercaptoethanesulfonate treatment immediately after biotinylation showed less than 2–3% of total biotinylated SERT indicating very little internalization at 4°C (Figure 5B, compare lanes 1 and 2 from the left). Following vehicle treatment, a gradual time-dependent increase in biotinylated SERT was observed in vehicle-treated cells (Figure 5B). This increase in internalized SERT represents basal or constitutive SERT endocytosis. However, Akt X did not significantly affect SERT endocytosis when compared with vehicle treatment (Figure 5B). The results from the exocytosis and endocytosis assays collectively demonstrate that inhibition of Akt decreases cell-surface SERT and that reduced SERT plasma membrane insertion contributes to reduced SERT-mediated 5-HT uptake.

Intracellularly accumulated hSERT co-localizes with markers of lysosomal degradation

In order to investigate the intracellular accumulation of hSERT in Akt X-treated cells, we performed immunocytochemistry on hSERT in transiently transfected HEK-293 cells. The cells were treated with 20 μM Akt X for 45 min at 37°C or vehicle before immunocytochemistry. Confocal imaging of the cells showed that hSERT primarily localized to the plasma membrane of the HEK-293 cells with only low levels of intracellular transporter (Figure 6A, left). In agreement with the biochemical data, we also observed that treatment with Akt X led to the appearance of multiple hSERT-positive vesicular structures in the cytoplasm of the transfected cells (Figure 6A, right, yellow arrowheads). Notably, quantification of this intracellular accumulation suggested a significant 6-fold increase (P<0.0001; 52–69 cells from three independent experiments) in intracellular hSERT accumulation in the Akt X-treated cells compared with vehicle-treated cells (Figure 6B).

Effect of Akt X on hSERT cellular distribution

Figure 6
Effect of Akt X on hSERT cellular distribution

HEK-293 cells transiently transfected with hSERT were serum-starved for 2 h and treated with vehicle or Akt X (20 μM, 45 min at 37°C) before fixation. The samples were visualized following antibody labelling targeting the C-terminal tail of SERT using confocal microscopy. (A) Images of hSERT in vehicle- or Akt X-treated HEK-293 cells. Images are representative of three independent experiments. Yellow arrowheads mark intracellular accumulation. (B) Quantification of the intracellular levels of hSERT. A mask was generated for each cell based on a fixed intensity, in order to remove background fluorescence. An ROI (region of interest) was generated measuring the intracellular levels as a ratio of the total fluorescence from each cell. In each individual experiment the average intracellular level of vehicle-treated cells was normalized to 1. Significance was determined by a two-sample Student's t test with a Welch correction (P<0.0001). Results are means±S.E.M. from 52–69 cells from three independent experiments. A.U., arbitrary units.

Figure 6
Effect of Akt X on hSERT cellular distribution

HEK-293 cells transiently transfected with hSERT were serum-starved for 2 h and treated with vehicle or Akt X (20 μM, 45 min at 37°C) before fixation. The samples were visualized following antibody labelling targeting the C-terminal tail of SERT using confocal microscopy. (A) Images of hSERT in vehicle- or Akt X-treated HEK-293 cells. Images are representative of three independent experiments. Yellow arrowheads mark intracellular accumulation. (B) Quantification of the intracellular levels of hSERT. A mask was generated for each cell based on a fixed intensity, in order to remove background fluorescence. An ROI (region of interest) was generated measuring the intracellular levels as a ratio of the total fluorescence from each cell. In each individual experiment the average intracellular level of vehicle-treated cells was normalized to 1. Significance was determined by a two-sample Student's t test with a Welch correction (P<0.0001). Results are means±S.E.M. from 52–69 cells from three independent experiments. A.U., arbitrary units.

To investigate the nature of the intracellular compartment to which hSERT co-localized upon Akt X treatment, we first performed co-staining with antibodies targeting ERGIC53, the Golgi apparatus (giantin) and lysosomes (Lamp1). Our imaging suggested no accumulation of hSERT positive vesicles in either ERGIC53 or Golgi (Figure 7A, white arrows). However, a marked number of hSERT-positive vesicles were positive also for Lamp1, indicating partial accumulation of hSERT in the lysosomal pathway upon treatment with Akt X (Figure 7A, yellow arrowheads). For quantification of the imaging, we used Van Steensel's cross correlation function, which calculates the Pearson coefficient as it shifts the two colour images across each other and plots the cross-correlation. No peak was observed for ERGIC53 and Golgi in agreement with no co-localization. In contrast, there was for Lamp1 a sharp peak at Δx=0 (cross-correlation value 0.15), suggesting partial, but specific, overlap between hSERT and Lamp1 (Figure 7B). One way ANOVA with Bonferroni's post-hoc multiple comparisons established a significant difference between Lamp1 and ERGIC as well as giantin (P<0.0001)

Co-localization of intracellular hSERT with ERGIC, Golgi and lysosome

Figure 7
Co-localization of intracellular hSERT with ERGIC, Golgi and lysosome

HEK-293 cells transiently expressing hSERT were serum-starved and treated with Akt X (20 μM, 45 min at 37°C) before fixation. (A) The cells were stained with antibodies targeting hSERT using Alexa Fluor® 568-labelled secondary antibody and ERGIC53, Golgi (giantin) and lysosome (Lamp1) using Alexa Fluor® 488-labelled antibody. Images are representative of three independent experiments. White arrows mark hSERT positive compartments that do not co-localize, and yellow arrowheads mark co-localization between hSERT and Lamp1. (B) Quantification of the hSERT co-localization with ERGIC53, Golgi, and lysosome using Van Steensel's cross-correlation function. This approach determines the cross-correlation for the movement of two images relative to each other. A sharp peak at Δx=0 represents co-localization. Values less than 1 show partial co-localization. Results are for 18 cells for each cellular marker from three independent experiments.

Figure 7
Co-localization of intracellular hSERT with ERGIC, Golgi and lysosome

HEK-293 cells transiently expressing hSERT were serum-starved and treated with Akt X (20 μM, 45 min at 37°C) before fixation. (A) The cells were stained with antibodies targeting hSERT using Alexa Fluor® 568-labelled secondary antibody and ERGIC53, Golgi (giantin) and lysosome (Lamp1) using Alexa Fluor® 488-labelled antibody. Images are representative of three independent experiments. White arrows mark hSERT positive compartments that do not co-localize, and yellow arrowheads mark co-localization between hSERT and Lamp1. (B) Quantification of the hSERT co-localization with ERGIC53, Golgi, and lysosome using Van Steensel's cross-correlation function. This approach determines the cross-correlation for the movement of two images relative to each other. A sharp peak at Δx=0 represents co-localization. Values less than 1 show partial co-localization. Results are for 18 cells for each cellular marker from three independent experiments.

Next, we performed co-transfections with small Rab GTPases tagged with EGFP. The EGFP–Rab constructs can be used as markers of distinct endocytic compartments. Rab4 mediates fast recycling from the early endosome to the plasma membrane and has therefore been utilized as a marker for the early endosome and the ‘short recycling loop’. Rab5 is involved in fusion with the early endosome and is thus also used as a marker for the early endosome. Because Rab7 is enriched in compartments of the late endosome and mediates the maturation of the late endosome as well as the fusion with the lysosome it is used as a marker of the late endosome/lysosome and lysosomal degradation. Finally, Rab11 marks the ‘long recycling loop’ through the recycling endosomes (reviewed in [47]). The confocal imaging showed some overlay of the hSERT immunosignal with the markers of the early endosome (Rab4 and Rab5a) and the short recycling loop (Rab4) (Figure 8A, yellow arrowheads). However, the co-localization of hSERT with EGFP–Rab7, and thus late endosomes, appeared more prominent, whereas the co-localization of the hSERT signal and EGFP–Rab11 was very limited (Figure 8A, yellow arrowheads). One-way ANOVA with Bonferroni's post-hoc multiple comparisons revealed a significant difference between Rab7 and Rab11 (P<0.01). This pattern was supported by the cross-correlation analysis supporting the strongest specific co-localization of hSERT with EGFP–Rab7 (cross-correlation value ~0.17) followed by EGFP–Rab5 (cross-correlation value ~0.125) and EGFP–Rab4 (cross-correlation value ~0.10). For EGFP–Rab11, the cross-correlation value was only 0.05 (Figure 8B). Taken together, the data suggest that hSERT accumulates upon Akt X treatment in intracellular endocytic compartments with the most prominent localization to late endosomes and lysosomes.

Co-localization of intracellular hSERT with EGFP-labelled markers of endocytic compartments

Figure 8
Co-localization of intracellular hSERT with EGFP-labelled markers of endocytic compartments

HEK-293 cells transiently expressing hSERT and EGFP-labelled Rab4, Rab5a, Rab7 or Rab11 were serum-starved and treated with Akt X (20 μM, 45 min at 37°C) before fixation. (A) The cells were stained with antibodies targeting hSERT using Alexa Fluor® 568-labelled secondary antibody. Images are representative of three independent experiments. Yellow arrowheads mark co-localization between hSERT and endocytic markers. (B) Quantification of the hSERT co-localization with EGFP–Rab4, EGFP–Rab5a, EGFP–Rab7 or EGFP–Rab11 using Van Steensel's cross-correlation function. This approach determines the cross-correlation for the movement of two images relative to each other. A sharp peak at Δx=0 represents co-localization. Values less than 1 show partial co-localization. The peak becomes taller as the amount of co-localization increases. Results are for 18 cells for each cellular marker from three independent experiments

Figure 8
Co-localization of intracellular hSERT with EGFP-labelled markers of endocytic compartments

HEK-293 cells transiently expressing hSERT and EGFP-labelled Rab4, Rab5a, Rab7 or Rab11 were serum-starved and treated with Akt X (20 μM, 45 min at 37°C) before fixation. (A) The cells were stained with antibodies targeting hSERT using Alexa Fluor® 568-labelled secondary antibody. Images are representative of three independent experiments. Yellow arrowheads mark co-localization between hSERT and endocytic markers. (B) Quantification of the hSERT co-localization with EGFP–Rab4, EGFP–Rab5a, EGFP–Rab7 or EGFP–Rab11 using Van Steensel's cross-correlation function. This approach determines the cross-correlation for the movement of two images relative to each other. A sharp peak at Δx=0 represents co-localization. Values less than 1 show partial co-localization. The peak becomes taller as the amount of co-localization increases. Results are for 18 cells for each cellular marker from three independent experiments

Akt2/PKBβ inhibition reduces SERT activity

Three mammalian isoforms of Akt have been identified (reviewed in [48]). We took a pharmacological approach to identify the involvement of Akt1 and Akt2 by treating hSERT-expressing HEK-293 cells with different concentrations of Akt1 inhibitor A-674563 and Akt2 inhibitor CCT128930 for 45 min at 37°C [49,50]. SERT activity measured from Akt1 and Akt2 inhibitor-treated cells showed a dose-dependent decrease in 5-HT uptake at different degrees (Figure 9). The decrease in SERT activity was influenced by dose and its interaction with Akt1 or Akt2 inhibitor [two-way ANOVA; interaction: F(5,64)=5.94; P<0.0001; dose: F(1,64)=45.11; P<0.0001 and drug (Akt1, Akt2 inhibitors): F(5,64)=30.56; P<0.0001)]. Subsequent Bonferroni's post-hoc multiple analysis revealed that whereas 1 μM Akt2 inhibitor CCT128930 caused a significant inhibitory effect on SERT activity (~31%; P≤0.01), Akt1 inhibitor A-674563 produced an equivalent inhibitory effect (~36%, P≤0.01) only at higher (5 μM) concentrations. In addition, the results revealed that the Akt2 inhibitor CCT128930 was more potent than the Akt1 inhibitor A-674563 in inhibiting SERT activity (Figure 9).

Effect of Akt1 and Akt2 inhibitors on SERT activity

Figure 9
Effect of Akt1 and Akt2 inhibitors on SERT activity

Transiently transfected HEK-293 cells with hSERT were pre-treated with vehicle (DMSO) or different concentrations of Akt1 inhibitor A-674563 or Akt2 inhibitor CCT128930 for 40 min at 37°C followed by 5 min of [3H]5-HT (50 nM) uptake. Results are means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001 compared with corresponding vehicle, P<0.05, ∧∧P<0.01, ∧∧∧P<0.0001 compared with equivalent Akt1 inhibitor A-674563 concentration (two-way ANOVA with Bonferroni's multiple comparison test).

Figure 9
Effect of Akt1 and Akt2 inhibitors on SERT activity

Transiently transfected HEK-293 cells with hSERT were pre-treated with vehicle (DMSO) or different concentrations of Akt1 inhibitor A-674563 or Akt2 inhibitor CCT128930 for 40 min at 37°C followed by 5 min of [3H]5-HT (50 nM) uptake. Results are means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001 compared with corresponding vehicle, P<0.05, ∧∧P<0.01, ∧∧∧P<0.0001 compared with equivalent Akt1 inhibitor A-674563 concentration (two-way ANOVA with Bonferroni's multiple comparison test).

GSK3α/β inhibition stimulates SERT activity

Because activated Akt phosphorylates and inactivates GSK3α/β [33,51], we examined whether Akt regulates SERT function through modulating GSK3α/β. We assessed the effect of three known inhibitors specific to GSK3α/β (TDZD-8, CHIR-99021 and BIO [5254]) on SERT-mediated 5-HT uptake in HEK-293 cells expressing hSERT. Figure 10(A) shows that treatment with various concentrations of GSK3α/β inhibitors for 45 min resulted in significantly higher SERT activity [Bonferroni's post-hoc multiple analysis: F(9,55)=7.10; P<0.0001]. In parallel, we tested the effect of CHIR-99021 on phospho-GSK3α/β levels. It has been reported that GSK3 inhibitors cause an increase in the phosphorylation of GSK3α/β (Ser21 and Ser9 respectively) to inhibit GSK3α/β activity [55]. Consistent with published studies, our representative Western blot (Figure 10B) shows that treatment of CHIR-99021 increased phospho-levels of GSK3α/β suggesting decreased GSK3α/β activities. These results demonstrated that inhibition of GSK3α/β activity enhances SERT activity.

Effect of GSK3α/β inhibitors on SERT activity

Figure 10
Effect of GSK3α/β inhibitors on SERT activity

(A) Transiently transfected HEK-293 cells with hSERT were pre-treated with vehicle or different concentrations of GSK3α/β inhibitors, BIO, CHIR-99021 and TDZD-8 for 40 min at 37°C followed by 5 min of [3H]5-HT (50 nM) uptake (A). Results are means±S.E.M. *P<0.05, **P<0.01 compared with the respective vehicle control (one-way ANOVA with Bonferroni's multiple comparison test). (B) Representative Western blots from three separate experiments show the levels of total and phospho-GSK3α/β using specific antibodies. Veh, vehicle.

Figure 10
Effect of GSK3α/β inhibitors on SERT activity

(A) Transiently transfected HEK-293 cells with hSERT were pre-treated with vehicle or different concentrations of GSK3α/β inhibitors, BIO, CHIR-99021 and TDZD-8 for 40 min at 37°C followed by 5 min of [3H]5-HT (50 nM) uptake (A). Results are means±S.E.M. *P<0.05, **P<0.01 compared with the respective vehicle control (one-way ANOVA with Bonferroni's multiple comparison test). (B) Representative Western blots from three separate experiments show the levels of total and phospho-GSK3α/β using specific antibodies. Veh, vehicle.

Akt inhibition reduces SERT basal phosphorylation

Since SERT regulation is linked to transporter phosphorylation [10,15,16,19,29,44], we hypothesized that phosphorylation of SERT may contribute to Akt X-induced down-regulation of SERT function and cell-surface expression. To test this, phosphorylation of SERT was examined following treatment with Akt X or vehicle in HEK-293 cells expressing hSERT. Consistent with our previous study [44], using SERT-specific antibody and SDS/PAGE and autoradiography of immunoprecipitates from metabolically 32P-labelled cells expressing hSERT revealed 32P-labelled SERT (~96 kDa) in vehicle-treated cells (Figure 11). This represents basal SERT phosphorylation. Interestingly, treatment with Akt X (20 μM, 30 min) significantly reduced the level of SERT basal phosphorylation (by 58.7%; t=4.71, df=8; P<0.0015), suggesting a role for Akt in regulating SERT basal phosphorylation.

Effect of Akt X on SERT phosphorylation

Figure 11
Effect of Akt X on SERT phosphorylation

HEK-293 cells were transiently transfected with hSERT cDNA. At 24 h after transfection, cells were metabolically labelled with [32P]orthophosphate in phosphate-free DMEM for 1 h and incubated with 20 μM Akt X for 15 min. RIPA extraction, immunoprecipitation, SDS/PAGE and autoradiography were performed as described in the Experimental section. An autoradiogram of 32P-labelled hSERT (~96 kDa), representative of five experiments, is shown (upper panel). Quantification of 32P-labelled SERT bands as mean±S.E.M. arbitrary units is shown in the lower panel. **P<0.001 (n=5) compared with vehicle (Student's t test).

Figure 11
Effect of Akt X on SERT phosphorylation

HEK-293 cells were transiently transfected with hSERT cDNA. At 24 h after transfection, cells were metabolically labelled with [32P]orthophosphate in phosphate-free DMEM for 1 h and incubated with 20 μM Akt X for 15 min. RIPA extraction, immunoprecipitation, SDS/PAGE and autoradiography were performed as described in the Experimental section. An autoradiogram of 32P-labelled hSERT (~96 kDa), representative of five experiments, is shown (upper panel). Quantification of 32P-labelled SERT bands as mean±S.E.M. arbitrary units is shown in the lower panel. **P<0.001 (n=5) compared with vehicle (Student's t test).

DISCUSSION

Depression has been associated with serotoninergic dysfunction in the central nervous system [9] and the major determinant of 5-HT signalling is the SERT. Altered expression and function of SERT is known in many disease states such as anxiety, depression, OCD (obsessive compulsive disorder), autism, irritable bowel syndrome and hypertension [69]. Thus understanding the signalling molecules and pathways involved in the regulation of SERT is of importance. Altered Akt activity and the Akt substrate GSK3β have been implicated in depressed suicide victims [31,32]. It is possible that Akt may regulate SERT function to maintain normal synaptic 5-HT. However, whether Akt activity regulates SERT function has yet to be investigated. Because of the implication of Akt and SERT in mood disorders, it became important to investigate whether Akt-mediated SERT function played any role in 5-HT uptake. The present study, for the first time, identifies the involvement of Akt in SERT regulation. Pharmacological reduction in the activity of Akt with the Akt inhibitor Akt X inhibited SERT activity. Akt X is an N10-substituted phenoxazine, which inhibits Akt activity by blocking Akt phosphorylation [46]. Additionally, genetic knockdown of Akt significantly reduces SERT function. Moreover, Akt X- and siRNA-mediated inhibition of Akt phosphorylation correlated with the inhibition of SERT activity. The combined pharmacological and genetic data indicate that Akt plays a role in the regulation of SERT function.

Accumulating evidence indicates that modulating SERT internalization and membrane insertion are attributable to an altered cell-surface level of SERT expression. In the present study, we found that Akt inhibition is associated with a significant reduction in SERT Vmax without affecting substrate affinity. This suggested the possibility of altered cell-surface abundance of SERT protein. Supporting this, we observed a loss of cell-surface SERT protein after Akt X treatment. Confocal microscopy approach revealed a significant 6-fold increase in intracellular SERT accumulation in Akt X-treated cells supporting further the hypothesis that Akt inhibition decreases 5-HT uptake through decreasing functional SERT protein on the plasma membrane. Greater intracellular SERT is observed in confocal imaging than decreased cell-surface SERT from cell-surface biotinylation/immunoblot. This may be due to the differences associated with different assays employed or sampling bias inherent in the single-cell imaging relative to the population determination of biotinylated cell-surface SERT by immunoblotting. Akt X treatment did not change the total expression of SERT protein, suggesting SERT redistribution rather than loss of total SERT protein via degradation or reduced SERT synthesis. We previously demonstrated that activation of PKC or inhibition of p38 MAPK decreased cell-surface abundance of SERT [12,15,16]. On the other hand, Src-mediated tyrosine phosphorylation of SERT regulates SERT stability and 5-HT transport [17,21]. Perhaps the most compelling cellular events in kinase-mediated amine transporter regulation involve changes in transporter protein internalization and membrane insertion, resulting in the change in cell-surface transporter density. We showed previously that whereas PKC activation triggers SERT internalization, p38 MAPK inhibition impedes the membrane insertion of SERT, suggesting distinct trafficking mechanisms involved in PKC- and p38 MAPK-mediated SERT cell-surface regulation [16]. It is noteworthy that p38 MAPK may also participate in catalytic activation of SERT through an unknown mechanism [18]. Adapting cell-surface biotin labelling of newly delivered SERT to the plasma membrane and reversible biotinylation strategy to follow internalized biotin-tagged SERT, we gathered evidence that Akt inhibition decreases SERT plasma membrane delivery with no significant effect on SERT internalization. Additionally, imaging of co-localization of SERT with intracellular organelle revealed that Akt X treatment increased SERT co-immunosignal with the markers for the lysosome (Lamp1), early endosome (Rab4 and Rab5a), short recycling loop (Rab4) and the late endosomes (Rab7), but not with the Golgi (giantin) or endoplasmic reticulum (ERGIC53) markers. Notably, SERT co-localization with Lamp1 and Rab7 was predominant over the others. These data suggest that activity of Akt regulates SERT trafficking pathways at the level of intracellular organelle such as late endosomes and lysosomes and possibly others and thereby regulating SERT delivery to plasma membrane and subsequent 5-HT transport. Of interest, recent data have suggested that hSERT upon constitutive internalization is sorted primarily to late endosomes/lysosomes [56] and thus it is possible that the Akt-associated SERT subcellular distribution occurs through the same pathway. Pharmacological inhibition of Akt1 and Akt2 indicates that SERT is more sensitive to the Akt2 inhibitor than the Akt1 inhibitor and thus both isoforms are likely to be involved in the regulation of SERT. However, the involvement of the Akt3 isoform cannot be ruled out. Clearly, further studies are needed to firmly confirm the Akt isoforms that are involved in SERT regulation. Akt inactivates its downstream kinase GSK3α/β [33,51]. It is therefore possible that Akt may regulate SERT through inhibiting GSK3α/β activity. Since Akt inhibition inhibits SERT, Akt activation and subsequent inhibition of GSK3α/β should stimulate SERT. Consistent with this notion, all three GSK3α/β inhibitors tested elevated SERT activity, suggesting the involvement of Akt-modulated GSK3α/β in SERT regulation. Since several signalling pathways converge to regulate GSK3α/β (reviewed in [33,5759]), an alternative possible mechanism is that Akt and GSK3α/β may regulate SERT independently through distinct mechanisms or in conjunction with other signalling pathways. Further detailed studies are needed to establish these possibilities.

SERT is a phosphoprotein, and several kinases including PKC, PKA (protein kinase A), PKG (protein kinase G), CaMK, p38 MAPK and Src regulate the phosphorylation of SERT [10,15,16,19,29,44,60]. We have shown that phosphorylation of Thr276 in SERT by activated PKG up-regulates SERT function [19]. Both Akt and GSK3α/β are serine/threonine kinases, and it is possible that Akt and GSK3α/β regulate SERT intracellular trafficking via SERT phosphorylation. The finding that inhibition of Akt decreases SERT basal phosphorylation suggests a role for Akt in the regulation of SERT phosphorylation. Further studies are warranted to determine whether Akt/GSK3α/β phosphorylates SERT directly and to determine the motifs involved and the role of Akt-mediated SERT phosphorylation in SERT trafficking and function.

Autoreceptors (5-HT1A and 5-HT1B) and heteroreceptor (TrkB) exist on serotonergic neurons to activate Akt [6164]. It is well known that activation of 5-HT1A and 5-HT1B influences 5-HT release and firing rates (reviewed in [65] and see reference therein). Furthermore, BDNF/TrkB signalling is critical for synaptic plasticity [66,67]. Although signalling pathways were not described, it has been reported that activation of 5-HT1B or TrkB receptors increases 5-HT clearance [25,26,68]. The observations and findings from the present study suggest that receptors on serotonergic neurons may influence SERT redistribution through Akt/GSK3α/β pathways in order to maintain normal 5-HT neurotransmission. It is therefore tempting to speculate that Akt/GSK3α/β-mediated SERT regulation may serve as one of the mechanisms for functional plasticity at serotonergic terminals. Further studies are needed (i) to identify signalling pathways upstream and downstream of Akt and GSK3α/β, (ii) to delineate how post-translational modifications of SERT are affected by Akt and GSK3α/β signalling, and (iii) to define the role of Akt and GSK3α/β-dependent SERT modifications in SERT trafficking and function.

Abnormal regulations of SERT, 5-HT signalling and BDNF/TrkB signalling have been implicated in the pathophysiology of mood disorders [69,65,6975]. Of interest is that genetic variants of Akt and AKTIP (Akt1-interacting protein) are associated with suicidal behaviour and depression [3032]. Notably, altered GSK3β phosphorylation [76] and polymorphism in the GSK3β promoter [77] have been implicated in depression/suicide and mood disorders. In this regard, the present study identifies the involvement of the Akt and GSK3α/β signalling pathway in SERT regulation and underscores the biological importance because any perturbation in the normal regulation of SERT might have functional consequences for 5-HT-linked signalling and behaviour.

AUTHOR CONTRIBUTION

Jeyaganesh Rajamanickam, Balasubramaniam Annamalai, Troels Rahbek-Clemmensen and Santhanalakshmi Sundaramurthy conducted experiments, acquired data and performed data analysis. Jeyaganesh Rajamanickam and Troels Rahbek-Clemmensen drafted the paper. Lankupalle Jayanthi, Ulrik Gether and Sammanda Ramamoorthy critically revised the paper before submission.

FUNDING

This work was supported by the National Institutes of Health [grant numbers MH062612, MH083928, MH091633] and a start-up fund from Virginia Commonwealth University (to S.R.).

Abbreviations

     
  • BDNF

    brain-derived neurotrophic factor

  •  
  • BIO

    6-bromoindirubin-3′-oxime

  •  
  • CaMK

    Ca2+/calmodulin-dependent protein kinase

  •  
  • DMEM

    Dulbecco’s modified Eagle’s medium

  •  
  • ERGIC53

    endoplasmic recticulum–Golgi intermediate compartment 53

  •  
  • GSK3

    glycogen synthase kinase 3

  •  
  • HEK

    human embryonic kidney

  •  
  • hSERT

    human SERT

  •  
  • 5-HT

    5-hydroxytryptamine

  •  
  • KRH

    Krebs–Ringer Hepes

  •  
  • Lamp1

    lysosome-associated membrane protein

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • PKB

    protein kinase B

  •  
  • PKC

    protein kinase C

  •  
  • PKG

    protein kinase G

  •  
  • SERT

    serotonin transporter

  •  
  • SSRI

    selective serotonin-reuptake inhibitor

  •  
  • sulfo-NHS acetate

    sulfo-N-hydroxysuccinimide acetate

  •  
  • sulfo-NHS-SS-biotin

    sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate

  •  
  • TrkB

    tropomyosin kinase B

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Author notes

1

These authors contributed equally to this work.