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Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina (L.D.J., D.J.S., S.R.)
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee (R.D.B.)
Abstract
The serotonin transporter (SERT) regulates 5-hydroxytryptamine (serotonin) (5-HT) neurotransmission and is a high-affinity target for antidepressants and psychostimulants. In the present study, we investigated the mechanisms that contribute to a previously unidentified biphasic regulation of endogenous SERTs expressed in the platelets. Treatment of rat platelets with -phorbol 12-myristate 13-acetate (PMA) for 5 min or less resulted in a rapid inhibition of SERT involving changes in intrinsic activity of the transporter (increased Km and decreased Vmax). -PMA treatment for 30 min or more produced a sustained inhibition of SERT with a decrease only in the Vmax. Whereas inhibition of SERT activity was detected from 1 to 45 min after phorbol ester addition, the decrease in surface SERT required at least 30 min of phorbol ester incubation. Increased endocytosis of SERT accounted for the decrease in surface SERT at the later point. Protein kinase C (PKC)-mediated phosphorylation of SERT occurs on the plasma membrane during the initial phase of rapid transporter inhibition, and later, the phosphorylated SERT enters the intracellular pool. -PMA-induced phosphorylation of SERT occurs initially on serine residues(s) and then on threonine residue(s). The initial serine phosphorylation corresponded to the first phase of rapid inhibition mediated by changes in intrinsic activity and/or silencing of SERT. The later phosphorylation on threonine residue(s) corresponded to the later phase of sustained inhibition mediated by an enhanced endocytosis of SERT. Together, these data reveal that in platelets, SERT function is regulated by PKC in a biphasic manner involving both trafficking-dependent and independent mechanisms and that these two events occur at distinct phases of transporter phosphorylation.
5-Hydroxytryptamine (serotonin; 5-HT) controls many behavioral and physiological functions (Jacobs and Fornal, 1995). 5-HT uptake via the cocaine- and antidepressant-sensitive serotonin transporter (SERT) is essential for serotonergic neurotransmission termination (Barker and Blakely, 1995). Altered SERT expression and two polymorphic regions in SERT promoter have been implicated in multiple forms of psychopathology, including depression, suicide, anxiety, aggression, schizophrenia, alcoholism, and drug addiction (Murphy et al., 2004). Although clearance of synaptic and extrasynaptic 5-HT seems to be the principal function of SERTs, certain cells, notably platelets, use SERTs to acquire 5-HT from the extracellular environment for subsequent release, involving in the processes of platelet activation (Cirillo et al., 1999). Platelets and 5-HT neurons share many common properties, including vesicular monoamine transporters, 5-HT release, biochemistry, identical SERT sequences, and 5-HT receptors (Owens and Nemeroff, 1994). In addition, alterations in platelet SERT have been observed in many psychiatric disorders and vascular diseases in which 5-HT has been implicated (Meltzer et al., 1981). Therefore, platelets have been widely used as a peripheral indicator of central 5-HT metabolism and SERT function (Wirz-Justice, 1988).
Based on a previous study using human embryonic kidney (HEK-293) cells stably transfected with human SERT (hSERT), we reported that activation of PKC and/or inhibition of protein phosphatases decreases 5-HT uptake. This down-regulation of SERT activity is paralleled by a decrease in SERT surface expression, an increase in SERT phosphorylation, and a decrease in SERT-protein phosphatase 2A catalytic subunit association (Ramamoorthy et al., 1998a; Bauman et al., 2000). We also showed that extracellular SERT substrates attenuate PKC-mediated effects on SERT (Ramamoorthy and Blakely, 1999). We demonstrated recently that in HEK-293 cells, PKC activation stimulated SERT internalization and p38 MAPK inhibition attenuated SERT insertion to the plasma membrane (Samuvel et al., 2005). It is interesting that PKC activation resulted in increased SERT basal phosphorylation and p38 MAPK inhibition resulted in reduced SERT basal phosphorylation. These results suggest that whereas PKC is involved in regulated expression of SERT, p38 MAPK may be involved in maintaining normal/basal expression of SERT. It has been shown recently that p38 MAPK activation also induces a trafficking-independent model of SERT stimulation (Zhu et al., 2005). However, many questions still remain unanswered. Is SERT regulation demonstrated in heterologous model systems similar to that of endogenous model systems Is the PKC-mediated decrease of surface SERT caused by endocytic internalization, decreased exocytic insertion, or both What are the specific PKC isoform(s) involved in SERT regulation What residues represent SERT phosphorylation sites, and are they necessary for regulation In an attempt to define some of the mechanisms by which PKC acutely regulates native SERT, we characterized the time course and pattern of SERT phosphorylation in parallel with SERT functional regulation using rat platelets because they express endogenous SERTs.
In this study, we demonstrate differential time-dependent molecular events associated with PKC-mediated regulation of endogenous SERTs expressed in the platelets. PKC activation in platelets results in reduced SERT function and enhanced SERT basal phosphorylation. It is surprising that PKC activation regulates SERT in a biphasic manner, where the initial phase of inhibition occurs independently of trafficking and the later phase is characterized by an enhanced endocytosis. The biphasic inhibition of SERT is accompanied by a sequential phosphorylation of plasma membrane resident SERT initially on serine residue(s) and then on threonine residue(s). Based on the findings, we conclude that the initial phosphorylation on serine residues might be responsible for changes in the intrinsic properties and/or silencing of SERT and that the phosphorylation on threonine residues later on might trigger internalization of phosphorylated SERT.
Materials and Methods
Materials. Phorbol ester isomers and protease inhibitors were obtained from Sigma-Aldrich (St. Louis, MO). Okadaic acid was purchased from LC Laboratories/Alexis Biochemicals (San Diego, CA). Bisindolylmalemide I was purchased from Calbiochem (San Diego, CA). [3H]5-HT (5-hydroxy-[3H]tryptamine trifluoroacetate), 32PO4 carrier-free orthophosphate, protein A-Sepharose beads, enhanced chemiluminescence (ECL) reagent, and ECL enhanced film were obtained from Amersham Biosciences Inc. (Piscataway, NJ). Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Monomeric streptavidin beads and EZ-link NHS-sulfo-SS-biotin were purchased from Pierce Chemical (Rockford, IL). All other reagents were of the highest grade possible from standard commercial sources.
Isolation of Platelets. Rat platelets were collected in a tube containing acid-citrate-dextrose from trunk blood after rapid decapitation. All animal procedures were in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Blood was centrifuged immediately at 190g for 15 min at room temperature to obtain platelet-rich plasma. The platelet-rich plasma was centrifuged at 2500g for 15 min at 22°C, and the resulting platelet pellet was gently resuspended in the assay buffer (140 mM NaCl, 5 mM KCl, 10 mM glucose, and 20 mM HEPES, pH 7.2) and used immediately after counting.
Assay of SERT Activity. The activity of SERT in rat platelets was measured by determining the rate of [3H]5-HT accumulation. In brief, 1 x 106 platelets were incubated in assay buffer containing 100 e pargyline and 100 e ascorbic acid with or with out the modulators as indicated previously (Samuvel et al., 2005). 5-HT uptake assays in triplicate were initiated by the addition of 10 nM [3H]5-HT for 3 min at 37°C and terminated by the addition of 1 ml of ice-cold assay buffer containing 100 e imipramine. In some experiments, platelets were washed twice with 10 ml of assay buffer before initiation of 5-HT uptake assay. Assay samples were filtered through GF/F glass filters presoaked in 0.03% polyethylenimine. Radioactivity associated with the filters was counted by liquid scintillation spectrometry. Specific uptake was determined by subtracting the amount of [3H]5-HT accumulated in the presence of 0.01 e fluoxetine.
Metabolic Labeling and Detection of SERT Phosphorylation by Immunoprecipitations. Platelets (1eC2 x 109/ml) were incubated with 5 mCi/ml of [32P]orthophosphate for 60 min at 37°C. Stimulation with protein kinase activators was carried out by adding the agents to the same assay mixture and continuing the incubation for appropriate time periods depending on the experiment. At the end of the incubation, the labeled buffer was removed by centrifugation, and platelet pellets were washed with ice-cold buffer and solubilized with RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and 1% Na deoxycholate) containing protease and phosphatase inhibitors (1 e pepstatin A, 250 e phenylmethylsulfonyl fluoride, 1 e/ml leupeptin, 1 e/ml aprotinin, 10 mM sodium fluoride, 50 mM sodium pyrophosphate, and 1 e okadaic acid). The solubilized extracts were centrifuged at 20,000g for 60 min at 4°C. Supernatants were precleared by the addition of 100 e (3 mg) of protein A-Sepharose beads along with preimmune sera for 1 h at 4°C, and the clear supernatant was subjected to immunoprecipitation. SERT protein was immunoprecipitated overnight at 4°C by the addition of SERT-specific antibody SR-12 (5 e of SERT antiserum), or preabsorbed SR-12, preimmune sera, and/or protein A Sepharose followed by 1-h incubation with protein A-Sepharose beads (3 mg in 100 e in RIPA buffer) at 22°C. SR-12 is a rabbit polyclonal antiserum raised against the C-terminal amino acid (596eC662) sequence of SERT (Ramamoorthy et al., 1998a; Samuvel et al., 2005). The immunoadsorbents were washed and eluted with the addition of 50 e of Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.05% bromphenol blue). Proteins were separated by 6 to 14% SDS-polyacrylamide gel electrophoresis, and 32P-labeled SERT protein was detected by autoradiography. Quantitation from digitized autoradiograms was evaluated on multiple film exposures to insure quantitation within the linear range of the film. Band density was quantified using NIH Image 1.60.
Phosphoamino Acids Analysis. Phosphoamino acid analysis was performed as described previously (Ramamoorthy and Balasubramanian, 1989). The region of the gel corresponding to 32P-labeled SERT was excised and incubated in 0.5 ml of 0.1 M sodium phosphate buffer, pH 7.0, containing 1% SDS, 3 mM -mercaptoethanol, and 100 e of histone (as carrier) with continues overnight shaking at 22°C. The gel was washed once with 0.5 ml of the same buffer without carrier histone. The protein from the combined washings was precipitated with 20% trichloroacetic acid. The precipitated protein was washed once with 10% trichloroacetic acid, twice with acetone and subjected to acid hydrolysis for 90 min in 5.7 N HCl at 110°C. The HCl was removed by evaporation in vacuo and the samples were subjected to high-voltage electrophoresis on cellulose thin layer plates as described previously (Ramamoorthy and Balasubramanian, 1989). Standard phosphoamino acids were added to the radioactive samples during hydrolysis and located by ninhydrin spray. The [32P]phosphoamino acids from SERT were located by autoradiography and aligned with standards.
Quantification of Surface SERT by Biotinylation. Cell surface biotinylation was performed as described previously (Ramamoorthy et al., 1998b; Jayanthi et al., 2004) with the following modifications. Suspended (1 x 109) platelets were incubated with 0.1 e -PMA or vehicle (ethanol) for indicated time periods. Ice-cold assay buffer containing 0.2 mg/ml NHS-SS-biotin was added to the platelet pellet and incubated for 15 min at 4°C. The platelets were washed with ice-cold assay buffer containing 100 mM glycine followed by solubilization with RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% Na deoxycholate) containing protease and phosphatase inhibitors (1 e pepstatin A, 250 e phenylmethylsulfonyl fluoride, 1 e/ml leupeptin, 1 e/ml aprotinin, 10 mM sodium fluoride, 50 mM sodium pyrophosphate, and 1 e okadaic acid). The surface-biotinylated proteins were isolated using monomeric avidin beads. SERT levels from total extract, eluted fractions from monomeric avidin beads (surface-biotinylated fraction), and nonbound fractions (intracellular fractions) were analyzed by SDS-PAGE (10%), electroblotted to polyvinylidene difluoride membranes (Amersham), and probed with SERT antibody as described above. Immunoreactive bands were detected by ECL (Amersham Biosciences Inc.). Thereafter, the blots were stripped and probed with anti-calnexin to validate the surface biotinylation, equal protein load and transfer, and platelet integrity. Values of total, nonbiotinylated, and surface SERT protein were normalized using the levels of calnexin in total extract, and values were averaged across three or more experiments. Multiple exposures of immunoblots were obtained to ensure development within the linear range of the film (Kodak X-AR). The band densities were quantified by integrating band density using computer-assisted densitometry (NIH Image 1.60).
To determine the subcellular location of SERT phosphorylation, platelets were metabolically labeled with [32P]orthophosphate (see above for details) followed by biotinylation as described above, except that bound proteins from monomeric avidin beads were eluted using 2 mM free d-biotin. The eluate, unbound fractions, and total extractions were subjected to SERT immunoprecipitations and autoradiography.
Assay of SERT Endocytosis by Reversible Biotinylation. Reversible biotinylation strategy was used as described recently by us (Jayanthi et al., 2004). Isolated platelets (1 x 109) were suspended in 1 ml of ice-cold assay buffer containing 0.1 mg/ml sulfo-NHS-SS-biotin for 20 min at 4°C. After two washes in assay buffer, two tubes were kept at 4°C with the ice-cold assay buffer with or with out -PMA (to estimate total surface SERT and efficiency of surface biotin removal), and the other tubes were incubated for different times at 37°C in prewarmed assay buffer containing -PMA (0.1 e) or vehicle to initiate endocytosis. For each incubation time, samples were kept at 4°C to estimate total surface SERT at a given time period of incubation. Other samples were incubated with 1 ml of ice-cold cleaving solution [50 mM 2-mercaptoethanesulfonic acid (MesNa), 100 mM NaCl, and 50 mM Tris-HCl, pH 8.9] (to estimate internalized SERT) for 20 min and washed three times with assay buffer containing 5 mg/ml iodoacetamide. Because MesNa does not penetrate the plasma membrane, the internalized biotinylated SERT is protected from surface-resident SERT and can be selectively detected. The internalized, biotinylated SERT was quantitated based on the loss of sensitivity to MesNa. Biotinylated SERTs that were resistant to reversal of biotinylation were defined as "the amount of SERT endocytosed or internalized". All samples were solubilized using RIPA buffer, and biotinylated SERTs were isolated using monomeric avidin beads and then blotted with SERT-specific antibody. To calculate the percentage of SERT internalization, the intensity of the band in the lane where biotinylated platelets stored at 4°C were immediately exposed to MesNa is subtracted as background from all other band densities. In our experimental stripping conditions, the biotinylated SERT present in avidin-bound fractions after MesNa treatment was less than 5% of total biotinylated fraction. The level of SERT in MesNa-resistant fraction was not altered by keeping cell dishes in ice slurry up to 45 min and between the vehicle and -PMA treatment. In initial experiments, experiments were optimized for dose, time, and pH of the buffer used in stripping procedure with MesNa. We found that MesNa treatment did not completely remove SERT from avidin bound fractions. We always found less than 5% of SERT in avidin-bound fractions after MesNa treatment at 4°C. This residual signal may be arising because of the inability of MesNa to access disulfide bonds for reaction as a result of SERT embedding in the plasma membrane.
To determine 32P-labeled SERT internalization, platelets were metabolically labeled with 32P-orthophosphate (see above for details) followed by reversible biotinylation as described above, except that bound proteins from monomeric avidin beads were eluted using 2 mM d-biotin and subjected to immunoprecipitation of SERT followed by SDS-PAGE and autoradiography.
Statistical Analyses. Values are expressed as mean ± S.E.M. Analysis by one-way analysis of variance was used followed by post hoc testing (Tukey-Kramer, Bonferroni, and Dunnett's test). Student's t test was performed for paired observations. A value of p < 0.05 was considered statistically significant.
Results
PKC Activation Inhibits SERT Activity. Treatment of purified platelets with -PMA (PKC activator) decreased basal 5-HT uptake. The dose and time dependence of this inhibition is summarized in Fig. 1, A and B, and shows that -PMA inhibited SERT activity as rapidly as 1 min and over the concentration from 1 to 100 nM. The functional effects of -PMA were also rapid with 60 to 70% of the maximal inhibition of SERT activity occurring within 5 min. An additional modest decrease in SERT activity was observed after 5 min of incubation with 100 nM -PMA. Higher concentrations of -PMA (10eC500 nM) showed no further decrease in SERT activity and showed a similar time course as observed with 100 nM -PMA. Therefore, a 100 nM concentration of -PMA was chosen for subsequent experiments. -PMA (100 nM), an inactive analog of -PMA, did not affect the basal 5-HT uptake (Table 1). It is noteworthy that the inhibitory effect of -PMA on SERT activity was effectively blocked by the PKC inhibitors staurosporine and bisindolylmaleimide I but not by the PKA inhibitor KT5720 (Table 1). In addition, these inhibitors alone had no effect on 5-HT uptake.
SERT activity and SERT phosphorylation were performed as described under Materials and Methods. Platelets were exposed to -PMA for 5 and 30 min at 37°C. Kinase inhibitors were added to the incubation mixture 15 min before the addition of -PMA. The concentrations used were 100 nM -PMA, 100 nM -PMA, 500 nM staurosporine, 250 nM bisindolylmaleimide I, and 1 e KT5720. Controls (vehicle) received same concentrations of vehicles. Experiments for SERT activity were conducted in triplicates and the mean values ± S.E.M. from three different experiments were given. For SERT phosphorylation, autoradiograms from three different experiments were scanned, and the mean values ± S.E.M. were given as arbitrary units. Data are expressed as percentage of vehicle. Data were analyzed using one-way factorial ANOVA with post hoc Tukey-Kramer tests.
We documented previously that although SERT substrates such as 5-HT attenuate PKC-mediated SERT surface down-regulation and SERT phosphorylation, SERT antagonists, such as selective serotonin reuptake inhibitors and cocaine, block the effect of 5-HT (Ramamoorthy and Blakely, 1999). Because platelets store and release 5-HT, a possibility of released 5-HT from platelets during -PMA treatment could dilute radiolabeled 5-HT, leading to a rapid loss of radiolabeled 5-HT uptake observed at early treatments of -PMA (Fig. 1A). To determine whether released 5-HT has any effect on PKC-mediated rapid inhibition of 5-HT uptake during early stage, platelets were treated with vehicle or -PMA in the presence and absence of cocaine followed by thorough washings before the initiation of 5-HT uptake. Washing vehicle and 100 nM -PMA-treated (5 min) platelets before 5-HT transport assay exhibited similar effect of -PMA on 5-HT uptake compared with unwashed platelets (Fig. 1C). In addition, incubation of cocaine did not influence 5-HT uptake in the presence and/or absence of -PMA (Fig. 1C).
Kinetic analysis indicated that -PMA treatment of platelets affected SERT kinetics differentially based on the time of -PMA treatment, as shown in Table 2. -PMA treatment for 5 min significantly decreased apparent substrate affinity for 5-HT (Km) and Vmax. However, -PMA exposure for 30 min decreased Vmax, but had no significant effect on Km (Table 2). Similar results were observed when kinetic analysis was performed on data obtained from using washed platelets (data not shown).
Platelets were exposed to 0.1 e -PMA for 5 and 30 min at 37°C. Controls (vehicle) received same concentrations of vehicles. Experiments for SERT activity were conducted in triplicates as described under Materials and Methods, and the mean values ± S.E.M. from three different experiments are given. Nonlinear curve fits of data for uptake used the generalized Michaelis-Menten equation to obtain Vmax and Km values for 5-HT uptake. Data were analyzed using one-way ANOVA with post hoc Tukey-Kramer tests.
PKC Activation Increases SERT Phosphorylation. To determine whether native SERT proteins expressed in platelets are phosphorylated, platelets were metabolically labeled with 32P and immunoprecipitated with SERT-specific antibody (SR-12) and control antisera (Fig. 2A). SDS-PAGE/autoradiography of immunoprecipitates from 0.1 e -PMA (30 min)-exposed platelets revealed a broad band centered at 100 kDa, the size similar from immunoblots for mature SERT proteins in platelets. The 32P-labeled 100 kDa was not immunoprecipitated with preabsorbed SR-12, preimmune sera, and or protein A-Sepharose (Fig. 2A). Our studies rely on the specificity of our immunoprecipitations. Although there is no SERT knockout rat model available to use as a control or no phospho-specific SERT-antibody available for these studies, we find primarily a single band with direct immunoblotting of platelet extracts. A band of similar size was previously reported in rat midbrain synaptosomes (Samuvel et al., 2005) (data not shown). Moreover, immunoprecipitation studies in transfected cells reveal an absence of phosphorylation signal with vector transfections and a band of similar size as seen in platelets when cells are transfected with rat SERT cDNA (data not shown). In addition, immunoblot detections show a band of similar size as seen in platelets when cells are transfected with rat SERT cDNA but not in vector transfections. Thus, this SERT-specific antibody (SR-12) has been tested rigorously with respect to its ability to isolate and detect SERT protein.
We tested whether the time course of -PMA effect shows any correlation between SERT activity and SERT phosphorylation in platelets. Platelets in the assay buffer without any externally added regulators, showed very low levels SERT phosphorylation (Fig. 2F). Treatment of platelets with 0.1 e -PMA increased SERT phosphorylation. The effect of -PMA was rapid and showed a gradual increase in SERT phosphorylation for up to 10 min, followed by a steep increase at 15 min, and reached a plateau phase thereafter (Fig. 2, F and G). The time course of increased SERT phosphorylation paralleled that of SERT inhibition in early time points (1eC10 min) of -PMA treatment. Major (60eC70%) inhibition of SERT activity was achieved within this early time point (1eC10 min) after -PMA treatment and thereafter, only a moderate decrease in SERT activity was observed. The initial gradual increase in SERT phosphorylation corresponded to the sharp decrease in SERT activity achieved within 5 to 10 min, and the later steep increase in SERT phosphorylation, reaching a plateau, corresponded to the later moderate inhibition of 5-HT uptake (Fig. 2, F and G). A similar time-course effect of -PMA on SERT activity and SERT phosphorylation was observed when higher concentrations of 1 e -PMA were used (data not shown). -PMA (100 nM), an inactive analog of -PMA, did not affect SERT phosphorylation (Table 1). The effect of -PMA was blocked by the PKC inhibitors staurosporine and bis-indolylmaleimide I but not by PKA inhibitor KT5720 (Table 1). Parallel Western blot analysis of SERT and calnexin at each time of -PMA treatments before immunoprecipitation showed the presence of equal amount of SERT and calnexin proteins in all samples (Fig. 2, B and C). Furthermore, Western blot analysis of SERT from immunodepleted (after removal of protein A-Sepharose-SERT immunocomplex) fractions revealed the absence of SERT protein in the immunodepleted fractions. Western blot analysis of SERT immunocomplexes (immunoprecipitated SERT eluted from protein A-Sepharose) showed the presence of equal SERT band intensity in the immunoprecipitated fractions at all time points of -PMA treatments (Fig. 2, D and E). These results suggest that the changes in 32P-labeled SERT observed at different times of -PMA treatment are not caused by uneven use and/or uneven immunoprecipitation of SERT.
PKC Activation Induces Phosphorylation Initially on Serine Residues Corresponding to Early Phase of SERT Inhibition and Then on Threonine Residues Corresponding to Later Phase of SERT Inhibition. Because we observed a biphasic reduction in SERT activity after -PMA treatment, we asked whether -PMA induces phosphorylation of SERT on different amino acid(s) at different time intervals after treatment. 32P-Labeled SERT proteins were immunoisolated from platelets treated with -PMA either for 5 or 30 min in the presence of phosphatase and protease inhibitors and subjected to gel separation, acid hydrolysis, and phophoamino acid analyses. As shown in Fig. 3, phosphoserine residue(s) but not phosphothreonine or phosphotyrosine residues were detected on phospho-SERT isolated from platelets that were incubated with -PMA for 5 min. However, both phosphoserine and phosphothreonine residues were detected from platelets that were incubated with -PMA for 30 min. Densitometry as well as counting of phosphoserine spot showed that there was no increase in the intensity or counts of phosphoserine level between 5 and 30 min of -PMA treatment (data not shown). Vehicle-treated platelets showed very less SERT phosphorylation and showed no detectable level of phosphoamino acids under 5- or 30-min incubation.
Biphasic Regulation of SERT Function. Unlike in 293-hSERT cells, where PKC activation reduces only SERT Vmax without affecting the Km (Qian et al., 1997), in platelets, PKC activation reduced both Vmax and apparent affinity for 5-HT. This could arise as a result of altered catalytic properties or functional silencing of plasma membrane resident SERT and/or altered surface SERT density. The initial increase in SERT phosphorylation (phosphorylation on serine residues) was correlated with a dramatic decrease in SERT activity after the 5-min treatment with -PMA. There was only a little reduction in SERT activity thereafter, although SERT phosphorylation (phosphorylation on threonine residues) kept increasing during this time (Fig. 2, F and G). These results suggest that the phosphorylation of serine residue(s) influences SERT activity by mechanisms that are different from those mediated by threonine phosphorylation. To examine the mechanisms underlying this biphasic regulation of SERT activity, surface biotinylation experiments were performed to quantitate SERT levels at the cell surface and in the cell interior, both in control and -PMA-treated platelets after 5- and 30-min incubations. SR-12 SERT antibody detects essentially equally both biotinylated and nonbiotinylated SERT in platelets (data not shown). Figure 4, A and B, shows the density of SERT immunoreactivity from biotinylated and nonbiotinylated fractions from platelets treated with 100 nM -PMA or vehicle. The amount of SERT immunoreactivity from the total extract did not change after 5- or 30-min treatment with -PMA. Although, a decrease in SERT activity (Figs. 1A and 2G) was evident after 5-min -PMA treatment, there was no change either in the cell surface SERT (biotinylated fraction) or in the intracellular SERT (nonbiotinylated) compared with control vehicle treatment. However, a decrease in cell surface SERT (80% of control) and a simultaneous increase in intracellular SERT immunoreactivity was evident in platelets after 30-min -PMA treatment (Fig. 4, A and B). Subsequent stripping and reprobing with calnexin, an intracellular marker, revealed less than 2% of total calnexin in biotinylated fractions (Fig. 4A).
Enhanced SERT Endocytosis Accounts for Decreased Surface SERT Density and Later Phase of SERT Inhibition. Changes in the rate of endocytosis can alter surface expression and hence functional properties of a protein. To determine whether the -PMA induced decrease in surface SERT occurring in the later phase was caused by increased SERT endocytotic internalization, a reversible biotinylation strategy was exploited. The amount of biotinylated SERT protein was determined for the surface (platelets left at 4°C without cleavage of surface biotin, total biotinylated SERT) and intracellular (after MesNa treatment, internalized biotinylated SERT) pools (Fig. 5). Biotinylation of platelets using NHS-SS-Biotin before -PMA exposure did not affect SERT activity or -PMA-mediated SERT inhibition or SERT immunoreactivity (data not shown). Control experiments performed at 4°C, where endocytosis was arrested, revealed a background signal of approximately 4 to 8% compared with platelets incubated at 37°C (Fig. 5A). This probably represents biotinylated surface proteins that were not completely cleaved by MesNa. This background signal did not seem to be consistently altered by -PMA treatment. After 5-min treatment with -PMA, there was no significant change in the level of biotinylated SERT in MesNa protected fractions compared with vehicle-treated control (Fig. 5, A and B). However, a significant increase in the amount of biotinylated SERT in MesNa-resistant fractions was observed from platelets treated with -PMA for 30 min at 37°C compared with respective vehicle-treated control (Fig. 5, A and B). The percentage of internalization shown in Fig. 5B is consistent with a decrease in surface SERT that occurred only at the later phase of SERT inhibition (Fig. 4, A and B).
PKC-Mediated SERT Phosphorylation Occurs at the Plasma Membrane. Based on the above-mentioned results, we predict that the early (5-min) serine phosphorylation of SERT might be occurring at the cell surface, and subsequent threonine phosphorylation may provide a signal for SERT endocytotic internalization and/or occur in parallel with SERT internalization. To test this hypothesis, using metabolically 32P-labeled platelets, we specifically isolated surface-biotinylated, 32P-labeled SERT from intracellular SERT after -PMA treatment by sequential SERT isolation using streptavidin-agarose beads and SR-12 SERT antibody. We found 32P-labeled SERT in the plasma membrane (biotinylated) fraction with no detectable 32P-labeled SERT in the intracellular pool (nonbiotinylated fraction) after 5-min treatment with -PMA (Fig. 6, A and B). In contrast, after 30 min of -PMA exposure, little or no 32P-labeled SERT was detected in the plasma membrane fraction with a parallel increase in the 32P-labeled SERT in the intracellular pool (Fig. 6, A and B). The amount of SERT phosphorylation observed in the total extract at 30 min was approximately equal to the sum of the SERT phosphorylation observed at the cell surface and in the intracellular pool (data not shown). To test whether phosphorylated SERT from the plasma membrane indeed is internalized, 32P-labeled SERT was isolated from MesNa-resistant fractions as described previously (see Materials and Methods) using reversible biotinylation method followed by sequential isolation using streptavidin-agarose beads and SR-12 SERT antibody (Fig. 6, C and D). MesNa treatment in control experiments performed at 4°C where endocytosis was arrested revealed a background signal of approximately 5 to 10% of that from platelets incubated at 37°C or from total amount of 32P-labeled SERT (Fig. 6C). This probably represents biotinylated surface proteins that were not completely cleaved by MesNa. This background signal was unaltered by -PMA (5- or 30-min) treatment. Although, a severalfold increase in total 32P-labeled SERT was observed after 5-min -PMA treatment, no significant increase in 32P-labeled SERT was detected from MesNa-resistant fractions compared with the background signal (Fig. 6, C and D). Almost 70 to 80% of 32P-labeled SERT was detected in MesNa-resistant fraction after treatment with -PMA for 30 min at 37°C compared with total 32P-labeled SERT (Fig. 6, C and D).
Discussion
The elucidation of the mechanisms underlying SERT regulation and signal transduction pathways are crucial to the understanding of SERT function in 5-HT homeostasis. PKC is a well established regulator for SERT activity (Anderson and Horne, 1992; Qian et al., 1997; Ramamoorthy et al., 1998a). Early studies using heterologously expressed hSERT in HEK-293 cells showed that -PMA increased SERT phosphorylation with parallel decreases in the Vmax for uptake of 5-HT and surface SERT density (Qian et al., 1997; Ramamoorthy et al., 1998a). Triggering SERT activity by providing SERT substrates and co-ions attenuated this PKC-mediated decrease in SERT phosphorylation and SERT surface density (Ramamoorthy and Blakely, 1999). 5-HT suppresses SERT phosphorylation, suggesting the surface pool is phosphorylated, but this has not been confirmed directly. The results of the present study demonstrate that, similar to heterologously expressed SERT, native SERT expressed in platelets is inhibited by PKC activation, and this inhibition is accompanied by a decrease in surface expression of SERT protein. However, analysis of 5-HT transport kinetics indicated a significant decrease in both the transport capacity (Vmax) and the apparent affinity for 5-HT after 5-min -PMA treatment. It is interesting that 30 min of -PMA treatment caused a reduction only in Vmax. The change in the apparent substrate affinity suggests that PKC activation results in altered intrinsic transport properties. Future studies examining endocytotic-signal and/or PKC-phosphorylation defective SERTs should reveal the difference between intrinsic and trafficking mediated SERT regulation.
Surface biotinylation studies indicated no change in cell surface SERT level after 5-min exposure to -PMA but a significant decrease in cell surface SERT level after a 30-min incubation. These results further support the idea that early inhibition of SERT is caused by altered intrinsic transport properties and/or silencing of 5-HT transport rather than by trafficking changes. The later inhibition was caused by a redistribution of SERT protein from the plasma membrane to cytosolic fraction via increased SERT endocytosis. Although we observed a rapid 90% inhibition of 5-HT uptake after -PMA treatment, kinetic analysis showed a 50 to 60% reduction in Vmax values. It is probable that -PMA treatment could cause release of 5-HT from platelets and influence the effect of -PMA on SERT regulation. Previous studies have shown that SERT substrates such as 5-HT attenuate PKC effects on SERT regulation and that the effect of 5-HT is blocked by SERT antagonists such as cocaine (Ramamoorthy and Blakely, 1999; Whitworth et al., 2002). Thus, the observed rapid decrease in 5-HT uptake, changes in 5-HT uptake kinetics, SERT surface expression, and SERT phosphorylation within the early -PMA treatment could represent a composite of influence of released 5-HT on PKC-mediated SERT effect from platelets. However, in our current study with washed platelets and/or -PMA treatment in the presence of SERT blocker, cocaine does not significantly affect the -PMA effect on 5-HT uptake (Fig. 1C). If the rapid, early inhibition of 5-HT by -PMA were the result of enhancement of release of 5-HT from platelets by -PMA influencing PKC-mediated regulation of 5-HT uptake, there would have been a lesser inhibition in washed platelets and that cocaine should prevent this effect. Although the possibility that the influence of released 5-HT from platelets on SERT as well as 5-HT receptors could not be completely ruled out in the current study, the data support the assertion that the early rapid inhibition of 5-HT uptake caused by -PMA treatment may not be associated with release of 5-HT from platelets.
It is interesting that the early inhibition was accompanied by an increase in SERT phosphorylation on serine residues with the later inhibition being associated with an increase in SERT phosphorylation on threonine residues. Phosphorylation of serine and threonine residues has been described for DAT expressed in rat brain synaptosomes and cells transfected with DAT after PKC activation (Foster et al., 2002; Granas et al., 2003; Lin et al., 2003). Although it is not known whether direct SERT phosphorylation modulates SERT activity, trafficking, or both, the current data suggest that the dual-stage SERT phosphorylation may have functional importance in SERT regulation. Changes in transport kinetics (both transport capacity and substrate apparent affinity) and the dual-stage phosphorylation of SERT suggest that the early phosphorylation on serine residues might be involved in changing the intrinsic transport properties having a major role in SERT inhibition. The later phosphorylation on threonine residues might be involved in SERT internalization with less direct effect on transport function. Examination of the time course of internalization of phosphorylated SERT indicated that up to 5 min, phosphorylated SERTs are present only at the plasma membrane level, and internalized phospho-SERT could be isolated after 5 min. It is therefore possible that early SERT phosphorylation on serine residues may not only perturb SERT function (render SERT nonfunctional) but also bring about conformational changes exposing threonine residues on SERT proteins for later phosphorylation by PKC or another kinase downstream of PKC. Thus, phosphorylation on threonine residues may provide a signal for SERT internalization.
Activation of PKC generally leads to inhibition of transporter-mediated uptake and sequestration of transporter protein from the cell surface to intracellular compartment (Beckman and Quick, 1998; Zahniser and Doolen, 2001; Ramamoorthy, 2002; Robinson, 2003). However, recently, Lin et al. (2003) showed a change in DAT cell surface expression after phosphoinositide 3-kinase and mitogen-activated protein kinase kinase 1/2 kinase modulation but not by PKC activation, even though there was an 30% inhibition of DA uptake. This suggests that changes in transporter activity cannot always be accounted for by changes in surface transporter level (Lin et al., 2003). PKC activation also results in transporter phosphorylation, suggesting an association between transporter phosphorylation and internalization (Ramamoorthy et al., 1998a; Foster et al., 2003; Lin et al., 2003; Vaughan, 2004). It has been shown recently that DAT exhibits normal PKC-mediated transporter kinetics and trafficking patterns even when PKC-mediated DAT phosphorylation is eliminated by mutation of predicted intracellular PKC sites on DAT (Granas et al., 2003), suggesting that PKC-mediated DAT phosphorylation and down-regulation are two independent phenomena. It is possible that PKC-mediated SERT phosphorylation and down-regulation may be independent events occurring in parallel. Although it is not known whether direct SERT phosphorylation is essential for transporter down-regulation, it is possible that transporter phosphorylation may have distinct roles in different transporters. Indeed, the N and C termini that contain the majority of consensus phosphorylation sites are poorly conserved among the members of monoamine transporter family (Vaughan, 2004). SERT and DAT also differ in the effect of substrate and inhibitors on their PKC-mediated down-regulation (Daniels and Amara, 1999; Ramamoorthy and Blakely, 1999; Saunders et al., 2000; Chi and Reith, 2003). Experiments with molecular mutant(s) of SERT phosphate acceptor sites are currently underway to establish any direct link between PKC-linked SERT phosphorylation and inhibition of 5-HT transport.
One quantitative aspect of the data in the present study is noteworthy. More than two-thirds (majority) of the inhibition of SERT activity was achieved within 5 min of -PMA treatment (Fig. 1), a time when no detectable decreases in surface SERT protein were evident (Fig. 4). After 30 min, when PKC activation decreased SERT surface expression (Fig. 4), a modest additional decrease in SERT activity occurred (Figs. 1 and 2). Although the activity and surface density data were not obtained in the same platelets, it is evident that the decrease in SERT activity at 5 min is caused by decreased intrinsic SERT transport activity. There is convincing evidence that transporter proteins exist in association with partner proteins such as protein phosphatase 2A catalytic subunit, PICK1, Hic-5, syntaxin 1A (Bauman et al., 2000; Torres et al., 2001; Carneiro et al., 2002; Quick, 2003; Sung et al., 2003). There is no current evidence that binding of these partner (transporter-associated) proteins to transporter per se alters transporter phosphorylation. However, it is possible that association of these proteins with the transporter can be a mechanism of altering transporter activity without changing transporter abundance in the plasma membrane. As indicated above, this may be mediated by SERT phosphorylation on serine residues, binding to associated proteins, or both. It has recently been demonstrated that activation of p38 MAPK-linked pathways in multiple models, including platelets, increases 5-HT transport activity without enhancing SERT trafficking, an effect linked to an increase in 5-HT binding affinity (Zhu et al., 2005). Our studies with PKC activation may represent antagonism of this same process or may involve population of a novel inactive state. Our studies also indicate the existence of a correlation between SERT activity, SERT phosphorylation and SERT internalization. However, the stoichiometry of SERT phosphorylation and the mechanisms underlying this relationship remain to be elucidated.
In summary, the data from the present study suggest a model of biphasic regulation of SERT activity by PKC activation involving dual mechanisms. Immediate inhibition is mediated by changes in intrinsic activity of SERT and/or silencing SERT function and is associated with serine phosphorylation. A more sustained inhibition is produced by enhanced endocytic removal of SERT from the plasma membrane. At present, the fates of the remaining surface transporters and the transporters that have undergone endocytosis are unknown. We speculate two possible ways of biphasic SERT regulation that are consistent with the empirical observations. One possibility is that serine residues on certain fractions of SERT undergo phosphorylation resulting in dramatic reductions in transport via yet unidentified mechanisms at 5 min. At 30 min, the serine-phosphorylated SERT fraction undergoes further phosphorylation on threonine residues triggering SERT endocytosis. Internalized SERT may be dephosphorylated by an associated phosphatase-triggering exocytotic insertion into the plasma membrane. The rate of exocytotic insertion of SERT (now dephosphorylated and reactivated) might balance the rate of endocytosis of SERT, resulting in very little additional decrease in SERT function. The second possibility is that serine residues on all of SERT undergo phosphorylation, resulting in dramatic reduction in transport. A restricted population of serine-phosphorylated SERT then undergoes phosphorylation on threonine residues, triggering internalization. The remainder of serine phosphorylated SERT might recover its function via reversal of phosphorylation, binding to associated proteins, or both. In both ways, total cellular SERT phosphorylation remains elevated at 30 min due mainly to the intracellular endocytosed pool of SERT.
Platelets express 5-HT1A receptors and thus precludes an examination of the influence of SERT-mediated 5-HT effect on PKC-mediated SERT sequential phosphorylation and trafficking. Further studies are needed to understand the time-dependent differential influence of -PMA on this complex SERT regulation in the presence of SERT substrates via SERT and 5-HT receptors. The active transport of 5-HT by platelet SERT is thought to be important in maintaining the circulating concentration of 5-HT below the levels required to activate vascular smooth cells and platelet 5-HT receptors (Stoltz, 1985; Nemeroff et al., 1998; Musselman et al., 2002). Therefore, the above-mentioned findings suggest that SERT function in platelets may be regulated by kinases and phosphatases linked to endogenous receptors and that this regulation may play a role in maintaining blood 5-HT levels. Any perturbations in this homeostatic regulation may contribute to elevated platelet 5-HT levels reported in some autistic subjects (Anderson et al., 1990; Cook and Leventhal, 1996; Cook et al., 1997).
Acknowledgements
We thank Dr. John Woodward (Medical University of South Carolina, Charleston, SC) for critical review and helpful comments on this manuscript.
L.D.J. and D.J.S. contributed equally to this work.
doi:10.1124/mol.104.009555.
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