Characterization of Intracellular Regions in the Human Serotonin

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Characterization of Intracellular Regions in the Human Serotonin Transporter for Phosphorylation Sites Lena Sørensen, Kristian Strømgaard, and Anders S. Kristensen* Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark S Supporting Information *

ABSTRACT: In the central nervous system, synaptic levels of the monoamine neurotransmitter serotonin are mainly controlled by the serotonin transporter (SERT), and drugs used in the treatment of various psychiatric diseases have SERT as primary target. SERT is a phosphoprotein that undergoes phosphorylation/dephosphorylation during transporter regulation by multiple pathways. In particular, activation and/or inhibition of kinases including PKC, PKG, p38MAPK, and CaMKII modulate SERT function and trafficking. The molecular mechanisms by which kinase activity is linked to SERT regulation are poorly understood, including the identity of specific phosphorylated residues. To elucidate SERT phosphorylation sites, we have generated peptides corresponding to the entire intracellular region of human SERT and performed in vitro phosphorylation assays with a panel of kinases suggested to be involved in SERT regulation or for which canonical phosphorylation sites are predicted. Peptide analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and quantify site-specific phosphorylation. Five residues located in the N- and C-termini and in intracellular loop 1 and 2 were identified as phosphorylation sites; Ser149, Ser277, and Thr603 for PKC, Ser13 for CaMKII, and Thr616 for p38MAPK. Possible regulatory roles of these potential phosphoacceptors for SERT function and surface expression were investigated using phospho-mimicking and phosphodeficient mutations, coexpression of constitutively active kinases and pharmacological kinase induction in a heterologous expression system. Our results suggest that Ser277 is involved in an initial phase of PKC-mediated down-regulation of SERT. The five identified sites can guide future studies of direct links between SERT phosphorylation and regulatory processes.

Serotonin (5-hydroxytryptamine; 5-HT) is a neurotransmitter in the central nervous system (CNS) involved in control of a variety of behaviors, including mood, sleep, pain, appetite, aggression, and sexual activity. The serotonin transporter (SERT) is an integral membrane protein that facilitates transport of released 5-HT into presynaptic neurons and plays a key role in synaptic 5-HT signaling.1 Inhibitors of SERT such as imipramine and fluoxetine (Prozac) are widely used in the treatment of a variety of psychiatric disorders linked to imbalances in serotonergic signaling such as depression, anxiety, and obsessive-compulsive disorder. Also, psychostimulant drugs of abuse such as amphetamine and its analogue 3,4methylenedioxymethamphetamine (MDMA; ecstasy) have SERT as their primary target.2 Post-translational processes that dynamically regulate SERT surface density, localization, and transport capacity are critical for spatiotemporal regulation of serotonergic signaling.3,4 SERT is a phosphoprotein that undergoes phosphorylation or dephosphorylation in response to activation of multiple intracellular signaling pathways linked to regulation of SERT activity.5−8 Specifically, inhibition or activation of signaling pathways involving the serine/threonine kinases protein kinase C (PKC) and G (PKG), p38 mitogen-activated protein kinase (p38MAPK), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the tyrosine kinase Src modulate SERT function,7−17 trafficking,6,12,18−21 and protein−protein interactions.6,17,22 However, the molecular mechanisms underlying © 2014 American Chemical Society

SERT regulation by kinases are still poorly understood. A central issue is to determine whether kinase-dependent effects are manifested by direct phosphorylation of SERT by a specific kinase. Studies of 32PO4 incorporation in SERT after kinase stimulation or inhibition have demonstrated changes in phosphorylation levels for pathways involving PKC,5,6,12,20 p38MAPK,6 Src,8 PKA,5,23 and PKG.5,7 However, evidence for the identity of specific SERT phosphorylation sites for specific kinases and their role in SERT regulation is very limited. For PKG, increase in SERT phosphorylation following kinase stimulation was abolished by mutation of Thr276 to Ala, providing the first indication of a specific phosphorylation site in SERT.7 However, recent evidence suggests that PKG does not directly phosphorylate SERT.24 Thus, for the majority of kinase-dependent SERT regulatory processes, it remains unknown whether regulation is attributable to direct SERT phosphorylation. A developing and highly advantageous strategy for identification of specific phosphorylation sites is analysis of tryptic peptides from the protein of interest by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).25,26 However, such analysis has not yet proven Received: September 18, 2013 Accepted: January 22, 2014 Published: January 22, 2014 935

dx.doi.org/10.1021/cb4007198 | ACS Chem. Biol. 2014, 9, 935−944

ACS Chemical Biology

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Figure 1. Overview of peptides investigated for in vitro phosphorylation. (Left) Membrane topology of hSERT with transmembrane segments shown as cylinders connected by intracellular and extracellular loops. Stipulated lines demarcate boundaries of the intracellular segments that were produced by solid-phase peptide synthesis for use in in vitro phosphorylation assays. Potential phosphoacceptor residues are shown as gray (Ser/Thr) and black (Tyr) spheres. (Right) Amino acid sequence of hSERT peptides. Peptides were designed to cover all intracellular Ser, Thr and Tyr residues (highlighted in bold) flanked by at least four residues on either side.

Figure 2. In vitro phosphorylation of hSERT peptides. (A) Peptide samples were analyzed for phosphorylation using analytical HPLC and LC-MS. Shown are representative HPLC traces of the C2 peptide (left) and the PKC control peptide (QKRPSQRSKYL) (right) following 0, 1, and 5 h incubation with PKC. Phosphorylation is detected as the occurrence of an additional peptide peak in the HPLC trace after incubation with the kinase and the detection of an ion in the mass spectrum (not shown) corresponding to the phosphorylated peptide. (B) Summary of kinase activity at hSERT peptides. Fields in the matrix are color coded according to % phosphorylation detected at different time points following kinase incubation. Dark green, >30% at 1 h and >90% at 5 h; medium green, >5% at 1 h and >20% at 5 h; light green, >5% at 24 h; gray, no detectable phosphorylation at 24 h; NT, not tested, Tyr not present in peptide. (C) Summary of the time course of kinase activity of all observed phosphorylation events.

individual domains. For example, in vitro phosphorylation of the isolated N-terminal segment from the dopamine transporter (DAT), a close paralogue to SERT, was used to identify a

successful for native or recombinant SERT. An alternative approach is the use of in vitro phosphorylation strategies using purified protein preparations, either of full-length protein or 936



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Figure 3. MS or MS/MS spectra of phosphorylated hSERT peptides. (A) Full-scan mass spectrum of the CaMKII-phosphorylated N1 fragment after tryptic digest with the phosphorylated residue contained within the Gln11-Gly18 (QLSACEDG) fragment, containing a single phosphoacceptor residue (Ser13). (B) Full scan mass spectrum of the PKC-phosphorylated IL1 peptide (Ala138-Ile157, ALGQYHRNGCISIWRKICPI), containing a single phosphoacceptor residue (Ser149). (C−E) Product ion spectra from the MS/MS analyses showing ions resulting from fragmentation of the phosphorylated precursor. The b and y ions are labeled. (C) PKC-phosphorylated IL2 (W271−V281, WKGVKTSGKVV) with phosphorylation located to Ser277 (nonphosphorylated b6 ion). (D) PKC-phosphorylated C2 after tryptic digest with the phosphorylated residue contained within the Leu597−Lys605 (LIITPGTFK) fragment. Phosphorylation is located to Thr603 (nonphosphorylated b4 ion and phosphorylated y3 and y5 ions). (E) p38MAPK-phosphorylated C2 after tryptic digest with the phosphorylated residue contained within the Ser611-Arg626 (SITPETPTEIPCGDIR) fragment. Phosphorylation is located to Thr616 (nonphosphorylated y10 ion and phosphorylated y11 and y13 ions). 937



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PKC phosphorylated IL1, IL2, and C2 to a degree of ∼95%, 20%, and 90%, respectively, within 5 h. Phosphorylation of IL3 by PKC was also detected, but with lower activity (∼10% in 24 h). p38MAPK phosphorylated C2 and displayed the highest catalytic activity among any of the phosphorylated peptides, with phosphorylation level reaching ∼90% in 1 h. CaMKII displayed robust phosphorylation of N1 (∼25% in 1 h) in addition to weak activity at IL1 (20% phosphorylation after 5 h and their surrounding amino acid sequence. The N-terminus harbors a phosphorylation site for CaMKII and the C-terminus two sites for PKC and p38MAPK. Additionally, two sites for PKC are found in the first and second intracellular loop (IL1 and IL2).

Km (μM) 0.98 1.03 0.76 0.38 0.18 0.59 0.20 1.26 1.02 1.25 1.18 0.30

± ± ± ± ± ± ± ± ± ± ± ±

0.08 0.19 0.13 0.09c 0.06c 0.10 0.09c,d 0.13 0.08 0.15 0.15 0.05c

0.31 ± 0.06c

n

Vmaxb (% of WT)

27 7 7 5 6 6 4 5 5 5 5 4

100 77 ± 6c 55 ± 5c 26 ± 1c 10 ± 1c,d 41 ± 5c 6 ± 2c,d 66 ± 11c 51 ± 8c 73 ± 5c 64 ± 9c 36 ± 4c

4 4 4 4 4 4 4 4 4 4 4

100 66 ± 51 ± 34 ± 10 ± 43 ± NB 62 ± 48 ± 71 ± 63 ± ND

3

63 ± 10c

4

ND

n

Bmaxb (% of WT) 12 5c 5c 4c,d 7c 21 7c 9 16

n 3 3 3 3 3 3 3 3 3 3

Values are mean ± SEM; n is the number of experiments. bExpressed as % of WT determined in parallel. For WT, mean Vmax = 48 ± 4 pmol/min·mg (n = 30) and mean Bmax = 2.4 ± 0.2 pmol/mg (n = 20). c p < 0.05, significantly different from WT (ANOVA with Dunnet’s hoc test). dp < 0.05, significantly different from corresponding Ala mutant (Student’s t-test). NB: no measurable binding. ND: not determined. a

ylation of SERT by PKC can occur during PKC-mediated regulatory mechanisms. We found phosphorylation of the C-terminal C2 peptide by p38MAPK to occur at Thr616 (Figure 3E) within a consensus motif for proline-directed kinases of the MAP kinase family36,37,40 (Figure 4). p38MAPK activity has been found to regulate SERT activity in heterologous and native cells,6,10,13,14 and p38MAPK inhibition leads to a reduction of basal SERT phosphorylation in synaptosomes.6 However, the role of direct SERT phosphorylation by p38MAPK is unknown, and Thr616 within the SERT C-terminus thus represent the first candidate for a p38MAPK phosphorylation site in SERT. As the sequence containing Thr616 constitutes a consensus motif for proline-directed kinases, we decided to test the ability of the C2 peptide to serve as substrate for three additional MAP kinases: ERK1, ERK2, and JNK2. We found all three kinases to phosphorylate Thr616 to a similar or even higher degree than the control peptide (Supporting Information Figure 1), thus establishing Thr616 as a general phosphoacceptor site for proline-directed kinases. To our knowledge, this is the first report of potential SERT phosphorylation by ERK1, ERK2, and JNK2, and the role of these kinases in SERT regulation remains to be elucidated. Effect of Ala and Asp Mutations on hSERT Function and Expression. As a first approach to examine functional consequences of potential phosphorylation at the five identified sites, we generated Ala (phosphodeficient) and Asp (phosphomimicking) mutants of each site. We expressed the mutants in COS7 cells and determined transport kinetics (Km and Vmax for cellular uptake of [3H]5-HT) in parallel with determination of cell surface Bmax by whole cell binding of [125I]-labeled (−)-2βcarbomethoxy-3β-(4-iodophenyl)tropane ([ 125 I]RTI-55) (Table 1). We furthermore assessed total and cell surface expression of all mutants by surface biotinylation and immunoblotting (Supporting Information Figure 2). Total expression levels of mutants were not significantly different from wild-type (WT) hSERT except for S149D where expression was reduced by approximately 45% (Supporting Information Figure 2). All Ala and Asp mutants of the potential CaMKII site (S13A and S13D) and p38MAPK site (T616A and T616D) had Km

identical to WT hSERT, but significantly decreased Vmax in the range from 51 to 71% (Table 1) compared to WT. The decrease in Vmax was paralleled by decrease in Bmax, suggesting that decreased transport capacity is caused by lower cell surface expression levels and not decreased turnover rate. (The Kd for binding of RTI-55 was not significantly different from WT for any of the Ala or Asp mutants, Supporting Information Table 2.) However, surface biotinylation analysis did not detect these decreases in cell surface expression (Supporting Information Figure 2). This discrepancy may be caused by the limited quantitative resolution of the biotinylation analysis but needs to be taken into account in relation to the suggestion that lower transport capacity of the mutants is mainly caused by lower surface levels of active transporter. In summary, with the caution that mutation to Asp potentially cannot sufficiently mimic the properties of a phosphoryl group at Ser13 and Thr616, these data suggest that potential phosphorylation of Ser13 and Thr616 does not influence transport kinetics. Furthermore, with regard to the potential involvement of these sites in control of cell surface expression as indicated by the lower Bmax for the mutants compared to WT, the observation of similar Bmax for Ala and Asp mutants suggest that phosphorylation state does not play a role in this effect. Mutation of the identified PKC phosphoacceptor residues (Ser149, Ser277, and Thr603) promoted substantially decreased Vmax, which was paralleled by decreased Bmax (Table 1), thus suggesting that decrease in transport capacity is caused by lower cell surface expression levels of the mutants. Surface biotinylation also revealed lower expression of these mutants, although only significant for S149D and S277D (Supporting Information Figure 2). For S149D, however, the decrease in Vmax and Bmax was mainly ascribed to a lower total expression level of this mutant. Interestingly, the Asp mutant of Ser277 displayed 17- and 7-fold decreased Vmax compared to WT and the Ala mutant, respectively, and 5-fold decreased Km compared to WT (Table 1). Furthermore, RTI-55 binding of 939



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Figure 5. 5-HT uptake kinetics and surface expression of hSERT WT coexpressed with constitutively active kinases. (A, B) Representative saturation [3H]5-HT uptake (A) and [125I]RTI-55 surface binding (B) in COS7 cells expressing hSERT WT alone (■) or with a constitutively active mutant of PKC (Δ), CaMKII (□), or p38MAPK (○). Data points represent mean ± SEM from triplicate determinations.

Table 2. Effect of Co-expression of Constitutively Active Kinases on WT and Mutant hSERT Transport Kinetics and Surface Expressiona kinase

hSERT mutation

CaMKII p38MAPK PKC PKC PKC PKC PKC PKC

WT WT WT S149A S277A T603A T276A/S277A S149A/S277A/T603A

Km (μM)

n

Vmaxb (% of control)

n

Bmaxb (% of control)

n

± ± ± ± ± ± ± ±

6 6 6 4 4 3 4 3

106 ± 5 96 ± 4 40 ± 2c 40 ± 4c 45 ± 2c 45 ± 2c 38 ± 8c 37 ± 8c

15 15 7 4 4 4 4 4

111 ± 4 106 ± 5 28 ± 6c ND ND ND ND ND

6 5 4

0.84 0.93 0.72 0.54 0.53 0.99 0.36 0.35

0.08 0.10 0.02 0.14 0.11 0.08 0.13 0.07

Values are mean ± SEM; n is the number of experiments. bExpressed as % of control (WT or respective mutant in the absence of coexpressed kinase) determined in parallel. cp < 0.05, significantly different from control (paired t-test). ND: not determined. None of the Km values for WT and mutant hSERT in the presence of kinase are significantly different from the corresponding values in the absence of kinase (Student’s t-test); listed in Table 1. a

regulation. Pharmacological induction of p38MAPK in systems with native expression of SERT such as platelets and synaptosomes has been found to increase Vmax for 5-HT transport.10,13 However, absence of effect on 5-HT transport of p38MAPK stimulation in synaptosomes and LCC-PK cells has also been reported.44 The lack of effect of p38MAPK overexpression in COS7 cells might indicate that this model cell line lacks the cellular factors required for regulation of SERT expression. Future studies of the role of Thr616 as a potential p38MAPK phosphorylation site could be conducted in other cell systems where modulation of SERT by p38MAPK activity is well-established. In contrast to CaMKII and p38MAPK, coexpression of hSERT with PKC led to a dramatic decrease in transport capacity to 40 ± 2% of the Vmax for hSERT in the absence of PKC (Table 2). This decrease in Vmax was paralleled by a reduction in cell surface Bmax, demonstrating that decreased transport capacity is due to decreased cell surface expression levels of hSERT without a significant change in 5-HT turnover rate (0.33 ± 0.05 s−1 vs 0.49 ± 0.12 s−1, for WT in absence or presence of PKC coexpression). To test if the observed effects could be blocked by Ala substitutions at the three potential PKC sites, we coexpressed single (S149A, S277A, T603A) and triple (S149A/S277A/T603A) mutants together with constitutively active PKC. The T276A/S277A mutant was also included as we speculated whether the adjacent Thr residue could assume a compensatory role upon Ala mutation of Ser277. For all mutants, PKC coexpression decreased Vmax to ∼40%, similarly to the effect observed for WT (Table 2). These findings indicate that the mechanism by which persistent, constitutive PKC activity down-regulates hSERT cell surface

S277D was decreased to a level were specific binding could not be detected (Table 1). However, cell surface level assessed by biotinylation showed only a 40% reduction compared to WT (Supporting Information Figure 2). Together, these results show that introduction of negative charge at Ser277 substantially influences transport kinetics and surface expression. First, a lower level of transporter is present at the cell surface for S277D compared to WT, and second, the transporter that reaches the surface has a dramatically decreased transport capacity, altered Km, and is incapable of binding RTI55. These changes in functional phenotype are in agreement with the generally observed decrease in SERT uptake capacity and surface expression upon acute activation of PKC.12,20,21 Coexpression with Constitutively Active Kinases. As a second approach to address possible functions of phosphorylation at the identified sites, we coexpressed hSERT bicistronically with constitutively active variants of CaMKII,41 p38MAPK,42 and PKC43 in COS7 cells and determined transport function and cell surface expression (Figure 5 and Table 2). To ensure that kinase coexpression yielded constitutive activity in COS7 cells, we performed control expressions with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1, which harbors a wellestablished CaMKII and PKC phosphorylation site, and found CaMKII and PKC coexpression to induce increased phosphorylation of GluA1 (Supporting Information Figure 3). We found no difference in Km, Vmax or Bmax for hSERT with or without coexpression of CaMKII or p38MAPK (Table 2). The absence of effects of CaMKII and p38MAPK coexpression on hSERT in COS7 cells prevents us from addressing the ability of Ala mutants to block potential CaMKII or p38MAPK 940



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Figure 6. β-PMA-mediated down-regulation of transport activity in HEK293 or COS7 cells expressing hSERT WT and Ala mutants of Ser149, Thr276, Ser277, and Thr603. (A) Representative saturation [3H]5-HT uptake for hSERT WT following 30 min incubation with β-PMA (1 μM; ○), β-PMA + staurosporine (both 1 μM, □) or vehicle (■). Data points represent mean ± SEM from triplicate determinations. (B) Graphical summary of the decrease in Vmax for WT and mutant hSERT induced by 5 min (gray) and 30 min (black) β-PMA treatment. Bars represent mean ± S.E.M; n = 3 to 5 experiments. ** p < 0.01, significantly different from WT hSERT (ANOVA with Dunnett’s post hoc test).

Table 3. Km Values for Vehicle and β-PMA Treated WT and Mutant hSERT Km (μM), 5 mina hSERT mutation WT S149A T276A S277A T603A T276A/S277A S149A/S277A/T603A

vehicle 2.25 1.07 1.73 0.96 2.16 1.10 0.60

± ± ± ± ± ± ±

0.17 0.03 0.06 0.08 0.18 0.13 0.04

Km (μM), 30 mina β-PMA

n

± ± ± ± ± ± ±

5 3 3 3 3 5 7

2.03 0.90 1.60 0.88 1.68 1.10 0.55

0.22 0.11 0.05 0.09 0.04 0.11 0.03

vehicle 2.69 1.49 2.34 1.59 3.11 1.09 0.83

± ± ± ± ± ± ±

0.09 0.12 0.15 0.17 0.31 0.05 0.06

β-PMA

n

± ± ± ± ± ± ±

7 5 3 3 3 7 6

2.30 1.21 1.88 1.51 2.85 1.02 0.74

0.08 0.14 0.15 0.13 0.15 0.04 0.05

Values are mean ± SEM; n is the number of experiments. All experiments involving β-PMA were performed at 37 °C, which underlies the higher Km values compared to those obtained from experiments listed in Tables 1 and 2, which all were performed at RT (∼20 to 22 °C). a

following 30 min β-PMA treatment (Figure 6). Thus, potential direct PKC phosphorylation of hSERT at these sites is not involved in down-regulation by prolonged β-PMA treatment. Following short-term (5 min) β-PMA treatment, the single Ala (S149A, S277A and T603A) and the triple Ala (S149A/S277A/ T603A) mutants behaved like WT with ∼25% reduction of Vmax (Figure 6). However, interestingly, T276A/S277A displayed significantly less reduction of Vmax (12 ± 2%) (Figure 6). We tested whether the effect could be ascribed to the T276A mutation alone; however, this mutant maintained ∼25% reduction in Vmax (Figure 6). The observation that substitution of Thr276 and Ser277 to Ala inhibits short-term β-PMAmediated down-regulation of hSERT suggests that direct phosphorylation by PKC of the Thr276-Ser277 motif in IL2 could be involved in the short term phase of β-PMA-mediated down-regulation of hSERT. Interestingly, the IL2 sequence that contains the Thr276−Ser277 motif is conserved in both the norepinephrine transporter (NET) and DAT. In NET, the equivalent T258A/S259A mutation inhibits PKC-mediated NET internalization, where Ser259 was proposed as the de facto site of kinase action.46 In contrast, the observed hSERT down-regulation during persistent activation of PKC activity (30 min β-PMA treatment or constitutive PKC activity) seems independent of direct phosphorylation of hSERT by PKC, at least at any of the sites that PKC was able to phosphorylate in vitro. Similarly, for PKC-mediated down-regulation of DAT, it has been demonstrated that removal of the N-terminal Ser cluster abolished β-PMA-mediated DAT phosphorylation without affecting the β-PMA-induced internalization of the

expression in COS7 cells is independent of direct transporter phosphorylation by PKC, at least at the residues identified in vitro. Ala Mutants of Thr276-Ser277 Inhibit Short-Term hSERT Down-Regulation. Activation of endogenous PKC by the PKC activator β-phorbol 12-myristate 13-acetate (β−PMA) triggers acute down-regulation of 5-HT uptake capacity in native and heterologous SERT expressing cells, which correlates with an increase in SERT phosphorylation.5,20 This regulation proceeds in a biphasic manner with short-term (5 min) β-PMA exposure leading to changes in SERT transport kinetics (increased Km and decreased Vmax), whereas prolonged (≥30 min) treatment promotes SERT endocytosis.12 To study the role of the candidate PKC sites in β−PMA-mediated regulation of SERT, we determined Vmax and Km for WT and Ala mutants following 5 or 30 min of β-PMA treatment (Figure 6 and Table 3). Previous studies have suggested that β-PMA treatment does not promote SERT phosphorylation in COS7 cells,45 and we therefore also performed the β-PMA experiments in HEK293 cells. The results from the two cell lines, however, were indifferent and data therefore pooled. Consistent with previous studies, we found that 5 and 30 min β-PMA treatment induced a 25 ± 1% and 28 ± 2% decrease in Vmax, respectively, which was blocked by the PKC inhibitor staurosporine (Figure 6). There was no significant effect on Km for WT following either 5 or 30 min treatment (Table 3). All single Ala mutants of Ser149, Ser277, and Thr603 as well as the triple S149A/S277A/T603A and double T276A/S277A mutant were found to exhibit similar decreases in Vmax as WT 941



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triisopropylsilane (90:5:5), followed by precipitation with cold ether. Crude peptides were analyzed by LC-MS using an Agilent 6410 Triple Quadrupole Mass Spectrometer instrument with electron spray coupled to an Agilent 1200 HPLC system with autosampler and diode-array detector. A linear gradient of the binary solvent system of H2O/acetonitrile/TFA (A, 95/5/0.1; B, 5/95/0.086) with a flow rate of 1 mL min−1 was used with a C18 reverse phase (RP) column (Zorbax, 4.6 × 50 mm). Crude peptides were purified to >98% purity by preparative HPLC on an Agilent 1100 system using a C18 RP column (Zorbax 300 SB-C18, 21.2 × 250 mm) with the same solvent system as for LC-MS, a flow rate of 20 mL min−1 and UV detection at 230 nm. Purified peptides were lyophilized and analyzed by LC-MS. In Vitro Phosphorylation Assays. Phosphorylation reactions were carried out in a reaction volume of 50 μL in thin-walled PCR tubes containing 12.5 nmol peptide. Reaction buffers contained 25 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 10 mM NaF, 0.5 mM Na3VO4 and 0.3 mM adenosine triphosphate (ATP). Additional buffer components were added for certain kinases (Supporting Information Table 3). Seventeen units of kinase were used per nmol peptide in all reactions except for JNK2, where 2 units were used, due to low specific activity of the enzyme. For peptides with low solubility, dimethyl sulfoxide (DMSO) was added (≤0.5%). Reactions were incubated at 30 °C and 5 μL samples were collected at 0 min, 30 min, 1 h, 2 h, 5 h, and 24 h. Kinase activity was terminated by addition of an equal volume of HPLC solvent A and stored at −80 °C until analysis. Enzymatic activity of the included kinases under assay conditions was assessed using control peptide substrates (Supporting Information Table 1). Quantification of Peptide Phosphorylation and Phosphorylation Site Assignment. Peptide phosphorylation was quantified using LC-MS and analytical HPLC. Briefly, the samples from the in vitro phosphorylation assay were analyzed for the presence of a phosphorylated peptide species by LC-MS. The degree of phosphorylation was assessed by peak integration of the phosphorylated and nonphosphorylated species using analytical HPLC with the same system as described above for preparative HPLC; using a C18 RP column (Zorbax 300 SB-C18, 4.6 × 150 mm). In case of multiple possible phosphoacceptor residues within a peptide, identification of the phosphorylated residue was performed by LC-MS/MS using the same system as for single LC-MS. Where applicable, purified phosphorylated peptide was treated with trypsin (peptide:trypsin 1:50 w/w in 50 μL 0.1 M Tris-HCl, pH 7.5, 37 °C, 1 h) prior to LCMS or LC-MS/MS analysis.

transporter, indicating that the down-regulation is independent of direct transporter phosphorylation.47 Conclusions. Understanding the molecular mechanisms underlying SERT regulation by phosphorylation has so far been hampered by lack of knowledge of specific phosphorylation sites. In the present study, we show that segments from the intracellular region of hSERT are readily phosphorylated in vitro by PKC, CaMKII, and p38MAPK. We furthermore localize the specific phosphoacceptor residues, leading to identification of five potential in vivo phosphorylation sites: Ser149, Ser277, and Thr603 for PKC, Ser13 for CaMKII, and Thr616 for p38MAPK. Among these, our analysis in a heterologous expression system suggests that direct phosphorylation of Ser277 is involved in the rapid phase of PKCmediated down-regulation of hSERT, whereas a prolonged phase is possibly independent of direct phosphorylation of the transporter by PKC. The five proposed hSERT phosphorylation sites expand the knowledge base for future studies of the correlation between SERT phosphorylation and transporter regulatory processes.

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METHODS

Materials. Information regarding cell culturing materials, enzymes, pharmacological agents and chemicals are provided in Supporting Information. Molecular Biology and Functional Assays. Creation of cDNA constructs, [3H]5-HT transport measurements and [125I]RTI-55 radioligand binding assays were performed as described previously48,49 and are described in detail in Supporting Information. Total hSERT Expression and Cell Surface Biotinylation Assays. COS7 cells were transfected as described in Supporting Information and seeded in poly-D-lysine coated 6-well plates 48 h prior to experiments. Cells were washed three times with cold phosphatebuffered saline (PBS) containing 0.5 mM MgCl2 and 0.1 mM CaCl2 (PBSCM) and incubated with the membrane-impermeable biotinylating reagent sulfosuccinimidyl-2[biotinamido]ethyl-1,3-di-thiopropionate (sulfo-NHS-SS-biotin, 1 mg mL−1 in PBSCM, 1 h, 4 °C). The reaction was quenched by washing three times and subsequently 30 min incubation with 100 mM glycine in PBSCM and cells were solubilized in solubilization buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche). After centrifugation (18,000 × g, 30 min, 4 °C), solubilized biotinylated protein was isolated by incubation with NeutrAvidin beads (Pierce) for 1 h at RT. Beads were washed three times in solubilization buffer and protein was eluted by incubating beads with SDS loading buffer containing 100 mM DTT for 30 min at RT. Samples of total and biotinylated hSERT were separated by SDS-PAGE and immunoblotted with anti-hSERT (1:2000, mAb Technologies) followed by HRP-conjugated antimouse (1:20 000, Promega). Immunoreactive bands were visualized with ECL prime detection reagent (GE Healthcare) using a DNR MicroChemi System and quantified with GelQuant software (both DNR BioImaging Systems Ltd.). The presence of calnexin in total cell lysate and bead eluate was assessed by immunoblotting with anticalnexin (1:2000, Enzo Life Sciences) followed by HRP-conjugated antirabbit (1:20 000, Promega). Solid Phase Peptide Synthesis. Peptides were synthesized by Fmoc-based solid-phase peptide synthesis on a 0.25 mmol scale either manually or using a Liberty microwave peptide synthesizer (CEM). All peptides were synthesized from preloaded Wang resins (Novabiochem). Fmoc deprotection was performed with 20% piperidine in DMF (2 × 10 min) and coupling was carried out with O(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) (resin/ amino acid/HBTU/DIPEA 1:4:4:4). The final peptide was cleaved from the resin by 2 h treatment with trifluoroacetic acid (TFA)/H2O/

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ASSOCIATED CONTENT

S Supporting Information *

In vitro phosphorylation of C2 by ERK1, ERK2, and JNK2; total and cell surface expression of WT and mutant hSERT; activity of PKC and CaMKII expressed in COS7 cells; amino acid sequence of control peptides; Kd values for RTI-55 at WT and mutant hSERT; buffer composition for in vitro phosphorylation assays; supplementary methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J. Andersen for excellent technical assistance and for fruitful discussions. This work was supported by the School of Pharmaceutical Sciences, University of Copenhagen (PhD stipend to L.S.), the Brødrene Hartmanns Foundation, and the Torben Frimodt and Alice Frimodt Foundation. 942



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Characterization of Intracellular Regions in the Human Serotonin

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