From the Department of Pharmacology and Center for Molecular
Neuroscience, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-6600
Antidepressant-sensitive serotonin
(5-hydroxytrypta-mine, 5HT) transporters (SERTs) are responsible
for efficient synaptic clearance of extracellular 5HT. Previously
(Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J.,
and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in
transfected HEK-293 cells lead to the internalization of cell-surface
human (h) SERT protein and a reduction in 5HT uptake capacity. In the
present study, we report that PKC activators rapidly, and in a
concentration-dependent manner, elevate the basal level of
hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase
(PP1/PP2A) inhibitors down-regulate 5HT transport and significantly
elevate hSERT 32P incorporation, effects that are additive
with those of PKC activators. Moreover, hSERT phosphorylation induced
by
-phorbol 12-myristate 13-acetate is abolished selectively by the
PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT
phosphorylation induced by phosphatase inhibitors is insensitive to
these agents at comparable concentrations. Protein kinase A and protein
kinase G activators fail to acutely down-regulate 5HT uptake but
significantly enhance hSERT phosphorylation. Basal hSERT and okadaic
acid-induced phosphorylation were insensitive to chelation of
intracellular calcium and
Ca2+/calmodulin-dependent protein kinase
inhibitors. Together these results reveal hSERT to be a phosphoprotein
whose phosphorylation state is likely to be tightly controlled by
multiple kinase and phosphatase pathways that may also influence the
transporter's regulated trafficking.
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INTRODUCTION |
The biogenic amine, serotonin (5-hydroxytryptamine,
5HT),1 is a neurotransmitter
in the central nervous system and peripheral nervous system (2, 3) as
well as a major secretory product of activated platelets (4). Synaptic
clearance of 5HT following release and accumulation of 5HT by platelets
is determined in large part by the action of the plasma membrane 5HT
transporter (SERT (5, 6)). SERT-mediated 5HT accumulation is driven by
transmembrane ion gradients (Na+, Cl
, and
K+) (7-9) and is effectively blocked by indoleamine
derivatives, addictive amphetamines, cocaine, and, most selectively, by
SSRIs such as fluoxetine (ProzacTM) (5, 10-12). At
synapses, SERT blockade in vivo elevates extracellular 5HT,
permitting spillover of the amine and increased stimulation of 5HT
receptors (13, 14). Selective blockade of central nervous system SERTs
in humans is thought to represent the initial step in the pharmacologic
amelioration of a wide spectrum of affective disorders, including major
depression, anxiety disorders, appetite disorders, and
obsessive-compulsive disorder (5, 15, 16).
The biochemical and behavioral effects of SERT modulation of SERT
modulation using exogenous agents suggest that SERT expression may be
tightly regulated in vivo and, perhaps, altered in its regulation in disease states (17). The cloning and functional characterization of SERT cDNAs (18-20) has revealed the carrier to
belong to a gene family of Na+ and
Cl
-dependent transporters, containing
homologous ion-coupled dopamine and norepinephrine (NE) transporters
(DATs and NETs, respectively) among others (21-23). A single hSERT
gene is located at chromosome locus 17q11.2 (20) and has recently been
implicated in anxiety traits (24), major depression (25), and autism
(26). hSERT gene expression is significantly regulatable by activation
of both PKA and PKC pathways (17), and potential target sites for second messenger-mediated regulation of gene expression have been identified at or near transcription initiation and mRNA splicing sites (27-29). Recent findings suggest that the more delayed and long
term changes in 5HT uptake activity mediated by altered SERT gene
expression are mirrored by rapid, posttranscriptional events that alter
5HT uptake capacity at sites of expression (17). For example, platelet,
endothelial, and brain SERTs are down-regulated within minutes by PKC
activation (30-32), phenomena recapitulated in transfected COS (33),
HEK-293 cells (1), and LLC-PK1
cells.2 We have recently
shown that PKC-mediated down-regulation of 5HT uptake in stably
transfected HEK-293 cells (HEK-hSERT) occurs via a specific reduction
in cell-surface transporter protein (1). The presence of multiple,
canonical serine and threonine phosphorylation sites on SERT
cytoplasmic domains (18-20, 34) and the ability of NH2 and
COOH termini, where most of these sites lie, to serve as substrates for
purified protein kinases (e.g. PKC, PKA (17, 35)) suggests
that rapid kinase-mediated regulation of 5HT uptake may occur, in part,
as a consequence of transporter phosphorylation. Direct protein
phosphorylation is known to regulate the activity and/or surface
distribution of many ion channels, receptors, and transporters;
however, to date, SERT phosphorylation has not been described.
To explore regulatory posttranslational processing of SERT proteins, we
have generated and characterized SERT-specific antibodies that
immunoprecipitate and immunoblot SERT polypeptides in vitro and in vivo (1, 36). We now report the use of these
antibodies to establish the direct phosphorylation of hSERT proteins
using 293-hSERT cells. hSERT proteins in this system are phosphorylated under basal conditions, and phosphorylation can be significantly elevated by both PKC and cyclic nucleotide (cAMP and cGMP)-activated protein kinases. In addition, studies with phosphatase inhibitors reveal endogenous pathways leading to SERT phosphorylation independent of PKC, PKA, and PKG. These findings reveal a highly dynamic, and
potentially complex, process of SERT phosphorylation and
dephosphorylation whose regulation coincides, in part, with altered
trafficking of SERT proteins. We propose that direct SERT
phosphorylation may be a determinant of receptor and second
messenger-mediated changes in 5HT transport capacity and discuss the
potential roles of SERT phosphorylation in altered plasma membrane
expression.
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EXPERIMENTAL PROCEDURES |
Materials--
293-hSERT cells were previously generated and
characterized in this laboratory (1). Trypsin, glutamine, penicillin,
streptomycin, G418, and phosphate-free Dulbecco's modified Eagle's
medium (DMEM) were purchased from Life Technologies, Inc., or obtained
from the Vanderbilt Media Core. Cys/Met-free DMEM was obtained from Cellgro. PMA and phorbol 12,13-dibutyrate isomers, staurosporine, cholera toxin, dialyzed fetal bovine serum, and protease inhibitors were obtained from Sigma. Okadaic acid, KT5720, KN-62, KN-93, 8-(4-chlorophenylthio)adenosine 3
,5
-cyclic monophosphate,
8-(4-chlorophenylthio)guanosine 3
,5
-cyclic monophosphate were
purchased from LC Laboratories/Alexis Biochemicals. Bisindolylmaleimide
I, calyculin A, cyclosporin A, (
)indolactam V, BAPTA-AM, and
microcystin were purchased from Calbiochem. [3H]5-HT
(5-hydroxy-[3H]tryptamine trifluoroacetate (~100
Ci/mmol)) and [32P]orthophosphate (10 mCi/ml) were
obtained from Amersham Corp. Trans35S-label (~1209
Ci/mmol) was obtained from ICN Pharmaceuticals. Protein A-Sepharose was
obtained from Pharmacia Biotech Inc. All other reagents were of the
highest grade possible from standard commercial sources.
Cell Culture--
293-hSERT and parental HEK-293 lines were
maintained in monolayer culture in 75-cm2 flasks in an
atmosphere of 5% CO2 at 37 °C as described previously (1). Both lines were grown in DMEM containing 10% dialyzed fetal
bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Medium for the transfected line was
supplemented with G418 (250 µg/ml). Use of dialyzed serum at a 1000 molecular weight cutoff was necessary to prevent loss of expression of
5HT uptake and SERT protein expression through, as yet, undefined mechanisms.
Assay of 5-HT Transport--
[3H]5-HT transport
activity was assayed in monolayer cultures for the times indicated at
37 °C as described previously (1). Briefly, cells were plated on
poly-D-lysine (0.1 mg/ml)-coated 6-well (500,000 cells/well) or 24-well (100,000 cells/well) plates 48 h before
experiments. At assay, the medium was removed by aspiration, and the
cells were washed with 2 ml of Krebs-Ringer's (KRH) buffer containing
130 mM NaCl, 1.3 mM KCl, 2.2 mM
CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.8 g/liter glucose, 10 HEPES, pH 7.4. Cells were then incubated in KRH buffer containing 100 µM pargyline and 100 µM ascorbic acid for
10 min at 37 °C with or without various modulators. 5HT uptake
assays were initiated by the addition of [3H]5HT (1 µM final concentration), and the assays were terminated by three rapid washes (2 ml each) with KRH buffer at room temperature containing 100 µM imipramine. Cells were then solubilized
in 1% SDS or Optiphase Supermix scintillation mixture (Wallac,
Gaithersburg, MD), and [3H]5HT accumulation was
determined by liquid scintillation spectrometry. Specific 5HT uptake
was determined by subtracting the amount accumulated [3H]5HT in the presence of 1 µM paroxetine.
Statistical analyses comparing vehicle and modulator-modified uptake
were performed using Student's paired t tests.
Metabolic Labeling and Immunoprecipitations--
For
phosphorylation studies, 293-hSERT cells were seeded on
poly-D-lysine-coated 6-well plates at 5 × 105 cells/well. After 48 h, monolayers were washed
once in phosphate-free DMEM and incubated for 1 h at 37 °C.
Typically, cells were then incubated at 37 °C with the same medium
containing 1 mCi/ml carrier-free [32P]orthophosphate for
1 h to equilibrate the intracellular ATP pools with labeled
phosphate. Effectors at various concentrations (see figure legends) or
vehicles were added to the medium, and the incubation was continued at
37 °C. Kinase inhibitors were preincubated for 30 min prior to
addition of kinase activators or phosphatase inhibitors for the times
indicated. Experiments to test the role of intracellular calcium in
okadaic acid and CaM kinase II in okadaic acid-induced phosphorylation
were conducted in calcium-free media in the presence of BAPTA-AM or in
the presence of the CaM kinase II inhibitors, KN-62 and KN-93. The
adherent cells were washed three times with phosphate-buffered saline
and lysed by the addition of 400 µl/well ice-cold modified
radioimmunoprecipitation (RIPA, 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4) buffer containing protease (1 µM pepstatin A, 250 µM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) and phosphatase
inhibitors (10 mM sodium fluoride, 50 mM sodium
pyrophosphate, and 1 µM okadaic acid) for 1 h at
4 °C with agitation. RIPA extracts were centrifuged at 20,000 × g for 30 min at 4 °C. Protein content of supernatant was assessed using the DC protein assay (Bio-Rad) with bovine serum
albumin as the standard. Protein content between wells and experiments
showed <5% variability. Labeling with Trans35S-label was
carried out in Cys/Met-free DMEM as described previously (36, 37).
Supernatants were precleared by the addition of 100 µl (3 mg) of
Protein A-Sepharose beads for 1 h at 4 °C. hSERT protein was
immunoprecipitated overnight at 4 °C by the addition of
SERT-specific antibody, CT-2 (10 µl of antisera) on end-over-end continuous mixing, followed by 1-h incubation with Protein A-Sepharose beads (3 mg in 100 µl in RIPA buffer) at 22 °C. Additional
experiments to test specificity were carried out with the hNET-specific
antibody N430 (37), CT-2B preimmune serum, or a second SERT-specific serum S365 (36). The immunoadsorbents were washed three times with
ice-cold RIPA buffer prior to the addition to 50 µl of Laemmli sample
buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 5%
-mercaptoethanol, and 0.01% bromphenol blue), incubated for 30 min at 22 °C, and then resolved by SDS-PAGE (10%), with
radiolabeled proteins detected by autoradiography or direct
PhosphorImager (Molecular Dynamics) analysis. The relative amounts of
32P incorporated into hSERT protein were estimated using
ImageQuant software (Molecular Dynamics). Quantitation from digitized
autoradiograms was evaluated on multiple film exposures to ensure
quantitation within the linear range of the film and gave identical
results to estimations achieved with direct PhosphorImager
quantitation.
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RESULTS |
Immunoprecipitation of Phosphorylated hSERT Protein--
To
determine whether hSERT proteins are subject to phosphorylation, we
metabolically labeled stably transfected 293-hSERT cells with
[32P]orthophosphate and immunoprecipitated detergent
extracts with a set of SERT-specific and control antisera. In these
initial experiments, the Ser/Thr phosphatase inhibitor okadaic acid (1 µM) was applied to intact cells before extraction in an
attempt to preserve labeling from endogenous kinases. A more systematic analysis of the effects of okadaic acid-induced labeling is presented later in this report. SDS-PAGE/autoradiography of immunoprecipitates from labeled 293-hSERT cells reveals a broad band centered at ~96 kDa
(Fig. 1), the size expected from
immunoblots for mature, N-glycosylated hSERT protein (1,
36). The 96-kDa species is absent from immunoprecipitates of parental
HEK-293 cells, metabolically labeled, and extracted in parallel. The
96-kDa band is also not immunoprecipitated from transfected cell
extracts if CT-2 preimmune serum, the NET-specific antibody N430, or
CT-2 serum preabsorbed with CT-2/GST fusion protein are
utilized for immunoprecipitations. The 96-kDa band is retained,
however, if CT-2 antiserum is preabsorbed with GST, the protein carrier
for the fusion protein utilized to raise the CT-2 antibody. The SERT
antipeptide antibody S365 (36), like CT-2 antiserum, immunoprecipitates
the same 96-kDa band, although the S365 antipeptide antibody displays
consistently lower recovery in both 32P- and
35S-metabolic labeling paradigms (data not shown). In
addition, we have raised another two polyclonal antisera against the
COOH terminus of SERT and found that, like CT-2 and S-365 sera, they also immunoprecipitate a phosphorylated 96-kDa band selectively from
293-hSERT cell extracts (data not shown). Together these findings
support the contention that the phosphorylated 96-kDa species
immunoprecipitated from 293-hSERT extracts represents posttranslationally modified hSERT protein. CT-2 antisera also immunoprecipitates a minor species at ~76 kDa (Fig. 1, *) that we
suspect is a less heavily glycosylated or partially degraded form of
hSERT protein. Together, these data suggest that hSERT protein is a
target for direct phosphorylation by endogenous protein kinases and
phosphatases in stably transfected HEK-293 cells.

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Fig. 1.
Phosphorylation of hSERT in 293-hSERT
cells. Parental HEK-293 and 293-hSERT cells were metabolically
labeled with [32P]orthophosphate in phosphate-free DMEM
for 2 h at 37 °C in the presence of 1 µM -PMA
and 1 µM okadaic acid. RIPA extraction, immunoprecipitation, SDS-PAGE, and autoradiography were performed as
described under "Experimental Procedures." N430 antisera
immunoprecipitates NET proteins but not SERT proteins. S365 antisera,
like CT-2, is SERT-specific although of lower titer than CT-2.
Preabsorption of SERT CT-2 antisera with either GST protein or GST-COOH
fusion protein (the CT-2 immunogen) was performed as described
previously (36). An autoradiogram of a 10% SDS-PAGE is shown which is
representative of three experiments. The location of protein molecular
mass standards in kDa electrophoresed in parallel is shown to the
left of the figure. The position marked for
32P-labeled hSERT (~96 kDa, arrow) matches
that observed with direct immunoblotting. An asterisk
denotes the position of a labeled coprecipitating product believed to
be a degradation product or lightly glycosylated form of SERT
protein.
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Stimulation of hSERT Phosphorylation by Activation of
PKC--
293-hSERT cells display PKC-dependent
down-regulation of 5HT uptake capacity and hSERT-associated currents
associated with a reduction in surface transporter protein (1). We
tested whether PKC activation affects the phosphorylation state of
hSERT protein. In the absence of phosphatase inhibitors or PKC
activators (Fig. 2, 0 min time point), we
immunoprecipitate phosphorylated hSERT protein from 293-hSERT cells.
This basal level of labeling is certainly lower than described
previously (and in subsequent figures) with okadaic acid treatment but
is nonetheless readily apparent when compared with immunoprecipitations
from nontransfected HEK-293 cells that are electrophoresed in parallel.
Treatment of transfected cells with the PKC activator
-PMA induces a
time- (Fig. 2) and concentration (Fig.
3)-dependent augmentation of
basal hSERT phosphorylation, reaching levels 5-6-fold that of basal
hSERT phosphorylation at maximal time and concentration points. The
effects of
-PMA are rapid, as 1 µM
-PMA increases
hSERT phosphorylation by more than 2-fold within the first 5 min of
application (Fig. 2, A and B). We repeated our
published analyses of
-PMA-induced inhibition of 5HT uptake so that
transport and phosphorylation analyses could be followed in parallel,
and we can demonstrate that the elevation in basal hSERT
phosphorylation induced by
-PMA displays a similar time course to
the
-PMA-induced losses in 5HT transport (Fig. 2C). As
with phosphorylation, the rate of change in uptake is greatest after
the first 5 min of
-PMA treatment and then rises gradually
thereafter to essentially plateau by 60 min. Low concentrations of
-PMA were required to effect an increase in hSERT
phosphorylation.

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Fig. 2.
Time-course effect of -PMA on
phosphorylation of hSERT and 5-HT uptake. 293-hSERT cells were
labeled with [32P]orthophosphate in phosphate-free DMEM
for 1 h at 37 °C and incubated with 1 µM -PMA
for various times. Control cells received the same volume of vehicle
(ethanol). RIPA extraction, immunoprecipitation, SDS-PAGE, and
autoradiography were performed as described under "Experimental
Procedures." A, an autoradiogram of immunoprecipitates, representative of three experiments, is shown. Molecular mass standards
are indicated on the left, and the position of hSERT is
marked on the right (arrow). B,
quantitation of hSERT labeling. Radioactive bands corresponding to
hSERT from three independent experiments were quantified by
densitometric analysis of scanned x-ray film using ImageQuant
(Molecular Dynamics), and normalized to the response to vehicle (taken
as 100%), and the mean values ± S.E. are given. C,
time course of the inhibition of 5-HT uptake by -PMA. 293-hSERT
cells were preincubated with 1 µM -PMA for the times
indicated and then assayed for 5-HT (1 µM, 10 min) uptake as described under "Experimental Procedures." Data are the average from three separate experiments. Nonspecific uptake was defined as the
uptake in the presence of 0.1 µM paroxetine and
subtracted from the total accumulation to yield specific uptake.
Asterisks denote values significantly different from vehicle
controls, p < 0.05, Student's paired t
test.
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Fig. 3.
Dose dependence of -PMA on the
phosphorylation of hSERT and 5-HT uptake. 293-hSERT cells were
labeled with [32P]orthophosphate in phosphate-free DMEM
for 1 h at 37 °C and incubated with various concentrations of
-PMA for 1 h at 37 °C. Control cells received the same
volume of vehicle (ethanol). RIPA extraction, immunoprecipitation,
SDS-PAGE, and autoradiography were performed as described under
"Experimental Procedures." A, an autoradiogram of
immunoprecipitates, representative of three experiments, is shown.
Molecular mass standards are indicated on the left, and the
position of hSERT is marked on the right (arrow).
B, quantitation of hSERT labeling. Autoradiograms of three
experiments were analyzed as described in Fig. 2B.
C, dose-response inhibition of 5-HT uptake by -PMA. Cells
were preincubated with various concentrations of -PMA for 1 h
followed by a 10-min 5-HT uptake as described in Fig. 2C.
Asterisks denote values significantly different from vehicle
controls, p < 0.05, Student's paired t
test.
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Both hSERT phosphorylation and 5HT uptake inhibition are observed at
low concentrations of
-PMA (Fig. 3, A and C).
A greater than 2-fold increase in phosphorylation is evident with 1 nM
-PMA (60-min assay), and the EC50 for
phosphorylation was 1.5 nM. A similar concentration
dependent in
-PMA regulation of [3H]5HT uptake was
observed with reductions in [3H]5HT transport observed
with 1 nM
-PMA. Like
-PMA, the PKC activators,
-PDBu and indolactam V, stimulated 32P incorporation
into hSERT protein 4-6-fold (Fig. 4).
Moreover, the phorbol ester isomers that are inactive for PKC
activation (
-PMA and
-PDBu) failed to elevate hSERT
phosphorylation at equivalent concentrations. The PKC inhibitors
staurosporine (200 nM) and bisindolylmaleimide I (1 µM) do not perturb basal hSERT phosphorylation but
completely block hSERT phosphorylation induced by
-PMA (Fig. 4).
KT5720, a selective PKA inhibitor blocks neither basal nor
-PMA-induced hSERT phosphorylation. These findings indicate that
-PMA-induced hSERT phosphorylation is mediated by PKC activation and
that neither PKC nor PKA-dependent pathways are responsible
for basal hSERT phosphorylation.

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Fig. 4.
Stereospecificity and specificity of
PKC-mediated hSERT phosphorylation. 293-hSERT cells were labeled
with [32P]orthophosphate for 1 h and incubated for
an additional 1 h with or without PKC modulators and inhibitors.
Kinase inhibitors were added 30 min prior to the addition of -PMA.
The concentrations used were -PMA, -PDBu, -PMA, -PDBu at 1 µM each; ( )-indolactam V, 10 µM;
staurosporine, 200 nM; bisindolylmaleimide I
(BIM), 1 µM, and KT5720, 1 µM.
Immunoprecipitations, autoradiography, and densitometry of x-ray film
were performed as described under "Experimental Procedures" and
Fig. 2B, and results are the mean ± S.E. of three
separate experiments. Inset shows a portion of a
representative autoradiogram showing hSERT labeling in response to
various activators and inhibitors. Asterisks denote values significantly different from vehicle controls, p < 0.05, Student's paired t test.
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Analysis of hSERT Phosphorylation following Endogenous Phosphatase
Inhibition--
As described initially, basal hSERT phosphorylation is
significantly augmented if cells are pretreated with the PP1/PP2A
phosphatase inhibitor okadaic acid, thereby preserving the labeling
derived from endogenous protein kinases. Since okadaic acid might be
stabilizing the effects of basal PKC activity or reflect labeling of
hSERT by distinct kinases, we sought to characterize the effects of phosphatase inhibition more carefully. First, we determined time and
dose dependences of okadaic acid-stimulated hSERT phosphorylation. Like
PKC-mediated phosphorylation of hSERT, incorporation of 32P
into hSERT following okadaic acid (1 µM) treatment is
rapid, with a nearly 3-fold increase over basal phosphorylation
achieved within 5 min (Fig. 5,
A and B) and a gradual increase evident to 1 h of treatment. However, unlike hSERT phosphorylation following
-PMA
treatment, a pronounced rise in SERT phosphorylation is evident between
60 and 120 min after okadaic acid treatment. Like
-PMA, okadaic acid
treatment reduces 5HT uptake in 293-hSERT cells, although the time
course of the response is significantly distinct from that of phorbol
esters (Fig. 5C). As with okadaic acid-stimulated hSERT
phosphorylation, we observe the appearance of enhanced down-regulation
of [3H]5HT uptake from 60 to 120 min of treatment.
Concentration-response studies with okadaic acid (Fig.
6) reveal maximal effects (6-9-fold) observed by 1 µM (Fig. 5) and an EC50 for
hSERT phosphorylation of ~350 nM, similar to that found
for the concentration dependence of okadaic acid-triggered reductions
in 5HT uptake (Fig. 6C). We also found the potent PP1/2A
inhibitor calyculin A to augment hSERT phosphorylation to a similar
extent as okadaic acid, but we found the PP2B inhibitor microcystin to
be significantly less efficacious and the calcineurin inhibitor
cyclosporin A to be relatively ineffective (Table
I).

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Fig. 5.
Time-course effect of okadaic acid on
phosphorylation of hSERT and 5-HT uptake. 293-hSERT cells were
labeled with [32P]orthophosphate in phosphate-free DMEM
for 1 h at 37 °C and incubated with 1 µM okadaic
acid for various times. Control cells received same volume of
vehicle (ethanol). RIPA extraction, immunoprecipitation, SDS-PAGE,
and autoradiography were performed as described under "Experimental
Procedures." A, representative autoradiogram of labeling results. B, quantitation of hSERT labeling.
Autoradiograms of three experiments were analyzed as described in Fig.
2B and results presented as mean ± S.E. C,
time course of inhibition of 5-HT uptake by okadaic acid. Cells were
preincubated with 1 µM okadaic acid for various times
followed by a 10-min 5-HT uptake as described under "Experimental
Procedures." Asterisks denote values significantly
different from vehicle controls, p < 0.05, Student's
paired t test.
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Fig. 6.
Dose response of okadaic acid for the
phosphorylation of hSERT and 5-HT uptake. 293-hSERT cells were
labeled with [32P]orthophosphate in phosphate-free DMEM
for 1 h at 37 °C and incubated with various concentrations of
okadaic acid for 120 min. Control cells received same volume of vehicle
(ethanol). RIPA extraction, immunoprecipitation, SDS-PAGE, and
autoradiography were performed as described under "Experimental
Procedures." A, representative autoradiogram of labeling
results. B, quantitation of hSERT labeling. Autoradiograms
of three experiments were analyzed as described in Fig. 2B.
C, dose-response inhibition of 5-HT uptake by okadaic acid.
Cells were preincubated with 1 µM okadaic acid for
various concentrations followed by 10-min 5-HT uptake as described
under "Experimental Procedures." Asterisks denote values
significantly different from vehicle controls, p < 0.05, Student's paired t test.
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Table I
Effect of phosphoprotein phosphatase inhibitors and its kinase
selectivity on hSERT phosphorylation
293-hSERT cells were metabolically labeled with
[32P]orthophosphate and incubated at 37 °C for 1 h in
the absence (none) and presence of the following agents: okadaic acid,
1 µM; calyculin A, 1 µM; microcystin, 5 µM; cyclosporin A, 5 µM; staurosporine, 200 nM; bisindolylmalcimide I, 500 nM KT5720, 1 µM; KN-62, 2 µM; KN 93, 2 µM,
and BAPTA-AM, 10 µM. Protein kinase inhibitors
(staurosporine, bisindolyl- maleimide I, KT5720, KN-62, KN-93, and
BAPTA-AM) were added 30 min prior to the addition of okadaic acid. RIPA
extraction, immunoprecipitation, SDS-PAGE, and autoradiography were
performed as described under "Experimental Procedures."
Autoradiograms from three different experiments were scanned
densitometrically, and the mean values ± S.E. were given.
Statistical significance was calculated comparing treated to untreated
samples using the paired Student's t test.
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Next we asked whether
-PMA-stimulated and okadaic acid-induced hSERT
phosphorylation act through a common mechanism by coapplication studies
and cross-inhibitor studies. As shown in Fig.
7,
-PMA and okadaic acid each at
maximally efficacious concentrations yield additive hSERT
phosphorylation when coapplied, achieving more than a 10-fold increase
in 32P incorporation over basal conditions in these
experiments. Note again that okadaic acid achieves an increase in hSERT
phosphorylation between 60 and 120 min that is not observed with
-PMA. These findings suggest that PKC and okadaic acid-inhibitable
phosphatases in HEK cells phosphorylate hSERT through distinct
pathways. Further evidence in support of this contention was gathered
when we tested the ability of PKC inhibitors to block okadaic
acid-induced phosphorylation (Table I). Staurosporine and
bisindolylmaleimide I at concentrations that fully block the
-PMA
triggered phosphorylation of hSERT (refer to Fig. 4) fail to alter
significantly okadaic acid-induced hSERT phosphorylation.
Phosphorylation of hSERT revealed by okadaic acid treatment was also
insensitive to intracellular calcium chelation as well as inhibition of
CaM kinase II by KN-93 and KN-62 (Table I).

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Fig. 7.
Additivity of -PMA and okadaic
acid-induced hSERT phosphorylation. 293-hSERT cells were labeled
with [32P]orthophosphate for 1 h at 37 °C and
then incubated with -PMA (1 µM) and okadaic acid (1 µM), either separately or in combination, for indicated
times. The concentrations of -PMA and okadaic acid used are the
maximally effective doses on the phosphorylation of hSERT. RIPA
extraction, immunoprecipitation, SDS-PAGE, and autoradiography were
performed as described under "Experimental Procedures." A
representative autoradiogram of four separate experiments corresponding
to the 32P-labeled hSERT is shown. A,
representative autoradiogram of time-dependent phosphorylations of agents added separately or together. B,
quantitation of phosphorylation data averaged across three experiments
presented as means ± S.E. Asterisks denote values
significantly different (p < 0.05, Student's paired
t test) in coapplication experiments from -PMA treatment
alone.
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PKA-mediated hSERT Phosphorylation Is Distinct from PKC and
Phosphatase Pathways--
Differences in the time course and inhibitor
sensitivity of
-PMA- and okadaic acid-induced hSERT phosphorylations
suggest the involvement of one or more kinases distinct from PKC.
Purified cytoplasmic NH2 and COOH termini of rSERT are
substrates for PKA as well as PKC in vitro (17, 35). We
found the PKA activators forskolin and cholera toxin elevate hSERT
phosphorylation 4-5-fold (Fig. 8). A
similar level of hSERT phosphorylation is achieved with the
membrane-permeant cAMP analog, 8-pCT-cAMP. The effect of cholera toxin
is dose- and time-dependent with significant effects
(3-fold) on incorporation of 32P in hSERT observed at 10 ng/ml (data not shown). In time course studies, the cholera
toxin-induced phosphorylation of hSERT is more delayed and gradual than
observed for PKC activators, with a doubling of hSERT labeling not
observed for 45 min and another doubling evident by 90 min of
incubation (data not shown). Furthermore, unlike
-PMA and okadaic
acid, PKA activators did not alter 5HT transport levels (data not
shown). The PKC inhibitors staurosporine and bisindolylmaleimide I, at
concentrations that block
-PMA effects on hSERT phosphorylation
(Fig. 4), fail to reduce cholera toxin and forskolin-triggered
phosphorylation (Fig. 8). However, the specific PKA inhibitor KT5720,
which does not block the
-PMA-triggered labeling of hSERT (Fig. 4),
blocks the effects of PKA activators completely (Fig. 8). At this same
concentration, KT5720 blocks neither the basal level of hSERT
phosphorylation nor the okadaic acid-augmented phosphorylation (Table
I).

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Fig. 8.
Protein kinase A-dependent and
PKC-independent phosphorylation of hSERT. 293-hSERT cells were
labeled with [32P]orthophosphate in phosphate-free DMEM
for 1 h at 37 °C and then incubated for an additional 120 min
with or without activators of adenylyl cyclase and/or PKA or PKC
inhibitors. Kinase inhibitors were added 30 min prior to the addition
of cholera toxin or forskolin. The concentrations used were cholera
toxin, 1 µg/ml; forskolin, 1 µM; 8-pCPT-cAMP, 1 mM; KT5720, 1 µM; staurosporine, 200 nM; and bisindolylmaleimide I, 1 µM. RIPA
extraction, immunoprecipitation, SDS-PAGE, and autoradiography were
performed as described under "Experimental Procedures."
Inset shows a representative autoradiogram of labeling
experiments. Averaged data from four separate experiments are
presented ± S.E. Asterisks denote values significantly
different (p < 0.05, Student's paired t
test) from treatments with no activator or vehicle, performed in
parallel.
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Consistent with the inability of PKA inhibitors to mimic the effects of
PKC inhibitors or attenuate the effects of okadaic acid,
phosphorylation levels are found to be additive when cholera toxin and
-PMA were coapplied or when cholera toxin and okadaic acid were
coapplied at maximally effective concentrations (Table II). Although coapplied
-PMA and
okadaic acid yield additive reductions in 5HT uptake, cholera toxin
does not potentiate the effects of either agent on uptake (data not
shown). Interestingly, coapplication of
-PMA, cholera toxin, and
okadaic acid is no more effective at augmenting hSERT phosphorylation
than the coapplication of any two of these modulators (Table II).
Although we did not explore cGMP-linked pathways in detail in the
present studies, we also wish to note that the membrane-permeant cGMP
analog and activator of PKG, 8-pCT-cGMP (1 mM, 60 min),
also significantly elevated hSERT phosphorylation in 293-hSERT cells
with little or no effect on 5HT transport (data not shown). Together
these findings reveal the capacity for cyclic nucleotide-activated
phosphorylation of hSERT that is uncoupled to regulatory changes in 5HT
transport activity in 293-hSERT cells and which appear to be distinct
from transporter phosphorylation pathways modulated by PKC and
PP1/2A.
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Table II
Coordinate effects of -PMA, cholera toxin, and okadaic
acid-induced phosphorylation of hSERT
293-hSERT cells were metabolically labeled with
[32P]orthophosphate and exposed for 2 h to -PMA,
cholera toxin, and okadaic acid, either separately or in combination,
at their maximally effective doses as follows: -PMA, 1 µM; cholera toxin, 1 µg/ml, and okadaic acid, 1 µM. RIPA extraction, immunoprecipitation, SDS-PAGE and
autoradiography were performed as described under "Experimental
Procedures." Autoradiogram from three different experiments were
scanned densitometrically, and the mean ± S.E. values were given.
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DISCUSSION |
The transport of 5HT across plasma membranes of platelets,
pulmonary endothelium, placenta, and neurons is increasingly described as a tightly regulated process involving both transcriptional and
posttranscriptional mechanisms (17). In the nervous system, presynaptic
sites of SERT expression are significant distances from nuclear sites
of SERT gene transcription, imposing temporal constraints on the
ability of cells to modulate transport capacity and thereby affect
extracellular 5HT clearance. Even in transfected cells, maturation and
surface expression of NE and 5HT transporters takes several hours
(37-40); recent in vivo covalent labeling studies on the
DATs reveal a delay of more than a week to resupply striatal transport
sites once they have been irreversibly inactivated (41).
Evidence has accumulated to indicate that receptor and kinase-linked
pathways may rapidly up- and down-regulate endogenous SERTs as well as
SERTs expressed in heterologous systems (17). We have recently shown
(1) that HEK-293 cells stably transfected with hSERT cDNA, like
endothelial cells (30), platelets (31), JAR (42, 43), and RBL-2H3 cells
(34), and serotonergic neurons (32) support rapid modulation of 5HT
transport following activation of PKC. Phorbol esters capable of
activating PKC activity rapidly diminish 5HT uptake capacity in
293-hSERT cells, kinetically revealed as a change in 5HT transport
Vmax, and this down-regulation is blocked by the
PKC inhibitor staurosporine (1). hSERT-mediated currents are also
down-regulated by
-PMA in a staurosporine-sensitive manner in
voltage clamped 293-hSERT cells, suggesting that regulation is not due
to changes in ionic driving forces or the membrane potential (1).
Furthermore, reductions in transport capacity are paralleled by a
quantitatively similar reduction in cell-surface SERT protein (1).
Recently, 5HT uptake capacity changes have been reported with the rat
SERT cDNA transiently transfected into COS-7 cells (33), suggesting
that this modulation is not unique to the HEK-293 context of our
transfection studies. Analogous findings of PKC-dependent
modulation of transport capacity have been obtained in studies of
native and heterologously expressed dopamine (44-46), NE (47),
-aminobutyric acid (48-50), and glycine (51) transporters and thus
mechanisms for acute regulation of members of the
Na+/Cl-dependent neurotransmitter transporter
gene family may possess many similarities. Members of the gene family
share gross topological properties including cytoplasmic
NH2 and COOH termini. These latter domains possess a large
number of potential targets for protein phosphorylation. Moreover, we
have found that the NH2 and COOH termini of SERTs,
expressed and purified as GST fusion proteins, are substrates for
protein kinases including PKC and PKA (17, 35). These findings
increased our interest in exploring the question of whether direct
protein phosphorylation of SERTs occurs and how this phosphorylation
might effect transporter activity and surface expression.
The present studies demonstrate for the first time the existence of
phosphorylated hSERT proteins and support the existence of multiple
kinetically and molecularly distinct pathways leading to hSERT
phosphorylation. We observe 1) that hSERT protein in 293-hSERT cells is
phosphorylated under basal conditions and 2) this phosphorylation is
potentiated by addition of the PP1/2A protein phosphatase inhibitors
okadaic acid and calyculin A. Although basal phosphorylation is modest
with respect to that achieved with phosphatase inhibition, it can be
readily perceived when nontransfected cells are immunoprecipitated in
parallel. In addition, we document the labeling of hSERT by 3) PKC- and
4) cyclic nucleotide activated kinase (PKA and PKG)-linked pathways.
PKC- and PKA-induced hSERT phosphorylation appear additive with each
other and with okadaic acid-induced labeling, suggesting that the
endogenous phosphorylation protected by phosphatase inhibition is not
mediated by either PKC or PKA. Indeed, neither PKC nor PKA inhibitors
block the okadaic acid-induced phosphorylation of hSERT at
concentrations required to fully abolish PKC or PKA labeling,
respectively. Moreover, the kinetics of phosphorylation can be
distinguished by activators of these pathways, and distinct effects are
observed on transport. Like PKC activation, okadaic acid action leads
to a reduction in transport capacity, but a loss of 5HT transport is
not seen with PKA activators that phosphorylate hSERT to the same
extent. Intracellular calcium and the activity of Ca2+/CaM
kinase II have been implicated in acute SERT regulation (42, 52);
however, our studies with BAPTA and specific Ca2+/CaM
kinase II inhibitors fail to implicate these pathways in okadaic
acid-induced phosphorylation. These data lead us to conclude that
distinct sites are being phosphorylated as a result of PKC, PKA, and an
as yet unidentified endogenous kinase(s).
PKA and PKG activation in 293-hSERT cells leads to rapid
phosphorylation of hSERT protein yet no observable functional change in
5HT transport capacity. Although chronically administered
cAMP-elevating agents have significant effects on hSERT gene
transcription in JAR cells (53, 54), a role for PKA-linked pathways in
acute SERT regulation is not evident. PKA-mediated phosphorylation of SERT proteins may reflect artifactual labeling in this heterologous system that never occurs to any significant extent in vivo.
However, it is also possible that PKA-mediated labeling is facilitatory to other regulatory signals that we have failed to activate (or cannot)
in these cells. In RBL-2H3 cells (34), brain (55, 56), and platelets
(57), both nitric oxide and PKG-linked pathways have been implicated in
acute, receptor-mediated regulation of 5HT uptake though as yet SERTs
have not been shown to be phosphorylated under similar conditions. We
find the potent, membrane-permeant analog 8-pCT-cGMP to elevate SERT
phosphorylation although changes in uptake are not evident. As with PKA
activation, this labeling may not translate into functional changes due
to the heterologous or overexpression nature of the current system.
Further studies are warranted to determine whether PKA and PKG
activation promote the phosphorylation and regulation of SERTs in other
cell contexts and whether the sites of phosphorylation overlap with
those labeled following PKC activation.
PKC activation leads to both transporter phosphorylation (this study)
and transporter redistribution from the cell surface (1). It is not
possible, however, to conclude from our findings that phosphorylation
of transporter protein is causal in the reduction of 5HT
uptake since other proteins that may play important roles in
transporter trafficking are likely to be targets of PKC-linked pathways
as well. However, the close correspondence between dose and time
dependence for transport reductions and transporter phosphorylation forces us to consider that at least a signal for membrane
redistribution may be direct transporter phosphorylation. Rat and human
SERT proteins possess multiple canonical sites for Ser/Thr
phosphorylation among which are several PKC sites (18-20, 34). Our own
studies with purified cytoplasmic NH2 and COOH termini (35)
reveal both domains to be substrates for PKC (and PKA but not PKG).
However, mutation of canonical PKC phosphorylation sites individually
did not reduce this phosphorylation but rather enhanced it in several cases (58). Similarly, Sakai and co-workers (33) have described PKC and
PP1/2A-mediated reductions in 5HT transport capacity in transiently
transfected COS-7 cells, effects that were not abolished by mutation of
canonical PKC sites. Mutation of PKC sites in GAT1
-aminobutyric
acid transporters (49) and GLYT1 glycine transporters (51) also fails
to diminish PKC-mediated changes in transport capacity. We suspect that
multiple sites may be phosphorylated on cytoplasmic domains by
activated PKC (and these sites may interact), such that individual
mutations are incapable of diminishing ultimate functional effects.
Alternatively, PKC may phosphorylate other kinases and phosphatases
that ultimately affect the hSERT phosphorylation state. Currently, we
are exploring the merits of these arguments by trying to identify the
sites of 32P incorporation and reconstituting the labeling
patterns with in vitro extracts to permit kinase
identification.
Striking similarities are evident in comparison of our findings with
phosphorylation studies of native (45) or heterologously expressed rat
DAT proteins (46). DATs expressed in these environments, like hSERTs,
exhibit basal phosphorylation that can be augmented by phosphatase
inhibition and PKC activation. For both transporters, activity changes
are manifested primarily as a reduction in transport Vmax. As in our studies, DATs are less sensitive
to the PP1/2A/2B inhibitor microcystin than okadaic acid and calyculin
A, although this may reflect differential permeability of the compounds
in HEK-293 cells. However, both SERT and DAT are insensitive to the calcineurin (PP2B) antagonist cyclosporin A. Presently, we are attempting to define the specific phosphatase complex modulating SERT
phosphorylation to differentiate between PP1 and PP2A. An important
difference between the DAT and SERT studies is our finding that okadaic
acid-induced SERT phosphorylation is staurosporine-insensitive at
concentrations required to block
-PMA effects. This might represent
a specificity of SERT for a distinct kinase or simply an inability to
distinguish PKC and the endogenous kinase in LLC-PK1 cells. Consistent
with the latter explanation, Huff and co-workers (45, 46) found
additive phosphorylation of DAT proteins with coapplication of
-PMA
and okadaic acid. Another striking difference between the DAT and hSERT
studies is the evidence we present that PKA and PKG activation,
independent of PKC or okadaic acid-linked pathways, leads to SERT
phosphorylation. DATs are not phosphorylated by treatment of cells with
forskolin; the activation of cGMP-linked pathways has not been
reported. Phosphorylation of SERT by PKA- or PKG-linked pathways may
reveal modulatory pathways not shared with catecholamine
transporters.
Phosphorylation of hSERT induced by activated PKC parallels in time and
concentration the loss of 5HT uptake capacity and loss of surface
protein. How might the phosphorylation of hSERT protein affect a
redistribution of transporter protein from the cell surface? In
G-protein-coupled receptors, agonist occupancy and heterologous stimuli
lead to the phosphorylation, desensitization, and often, the
down-regulation of receptors mediated by internalization (59, 60).
Phosphorylated
adrenergic receptors are recognized by arrestin
proteins, targeting them to plasma membrane domains that support
clathrin- and dynamin-mediated endocytosis (61). Removal of phosphate
from receptors by Ser/Thr phosphatases is thought to assist in the
return of fully functional receptors to the cell surface (62). We
suspect we have identified pathways that may be analogous to those used
for heterologous receptor desensitization. Thus, receptors activating
PKC may, in addition to other actions, affect surface distribution of
SERT proteins by phosphorylating the transporter and enhancing its
internalization rate. Alternatively, PKC activation could slow the rate
of transporter insertion in the plasma membrane. Presently, little is
known of the dynamics of transporter membrane trafficking. In oocytes, heterologously expressed
-aminobutyric acid GAT1 transporters appear
to be shuttled in and out of the plasma membrane by membrane vesicles
dependent on SNARE proteins, a process that is regulated by PKC (50).
Interestingly, different pools of PKC appear to be available in oocytes
such that exogenous (48) and intracellular (50) PMA treatments yield
opposite changes in GAT1 expression. These findings remind us of the
potential complexities associated with multiple PKC isoforms (63) and
our present lack of knowledge of the cellular compartments where
phosphorylation proceeds. It should be very informative to determine
whether surface or intracellular SERT proteins are targets of
phosphorylation. In 293-hSERT cells, we estimate ~75% SERT proteins
are on the cell surface, but without additional studies, we cannot
claim that it is the transporter in the plasma membrane that becomes
phosphorylated in response to kinase activation or phosphatase
inhibition. Interestingly, in preliminary studies, we find that 5HT
itself can perturb both basal and PKC-mediated phosphorylation in these
cells, suggesting that some or all of the phosphorylation observed may
occur on surface resident SERTs accessible to transmitter. In summary, our studies suggest that endogenous transporter phosphorylation may
contribute to the rapid regulation of SERT expression and reinforces
the idea that phosphorylation may control synaptic signaling by
modifying both neurotransmitter release and clearance.
We thank D. Malone for laboratory assistance,
R. Delplane for help with manuscript production, and S. Apparsundaram for careful review of the manuscript.