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* This work was supported by a postdoctoral fellowship for physicians from the Howard Hughes Medical Institute (to D. E. K.), a predoctoral fellowship from the National Institute of Mental Health (to D. P.), a grant from the National Alliance for Research on Schizophrenia and Depression (to Y. L.), and grants from the National Institute of Mental Health and the National Institute on Drug Abuse (to R. H. E.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address: Rockefeller University, 1230 York Ave., New York, NY 10021. 1 The abbreviations used are: VMATsvesicular monoamine transportersCHOChinese hamster ovaryHAhemagglutininGSTglutathione S-transferaseCKIcasein kinase ICKIIcasein kinase II.
The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.
For classical neurotransmitters such as the monoamines, synaptic transmission involves two distinct transport activities. Transport across the plasma membrane removes neurotransmitter from the synaptic cleft, thereby terminating its action on the postsynaptic cell and recycling it for another round of exocytosis (
). Plasma membrane transport and vesicular neurotransmitter transport differ in their bioenergetic mechanism and sensitivity to drugs. Plasma membrane transport uses the sodium gradient across the plasma membrane and involves cotransport of sodium with the transmitter (
). Monoamine transport across the plasma membrane also differs from vesicular transport in terms of pharmacology. Whereas cocaine and antidepressants inhibit plasma membrane transport, the antihypertensives reserpine and tetrabenazine inhibit vesicular transport (
) then isolated the cDNA for a highly related protein expressed in the brain (VMAT2). Both of the VMATs have 12 predicted transmembrane domains and a large luminal loop between transmembrane domains 1 and 2 (
Considerable evidence indicates that the biosynthesis and transport of neurotransmitters undergo regulation by protein phosphorylation. In the case of catecholamines, tyrosine hydroxylase is the rate-limiting enzyme in transmitter biosynthesis and is regulated by several kinases, including cyclic AMP-dependent protein kinase and Ca2+- and calmodulindependent kinase II (
). Transport of monoamines across the plasma membrane also undergoes regulation by phosphorylation. Activation of Ca2+- and phospholipid-dependent kinase by phorbol esters inhibits plasma membrane serotonin transport (
). Although the phosphorylation of a plasma membrane transporter for monoamines has not yet been directly demonstrated, metabolic labeling of a plasma membrane transporter for glutamate has revealed phosphorylation-dependent changes in activity (
). The transport of neurotransmitter into synaptic vesicles may also be regulated by phosphorylation. Indeed, stimulation of protein kinases A and C in rat pheochromocytoma (PC12) cells has been suggested to inhibit vesicular transport activity (
). However, the mechanism of inhibition remains unclear, and the phosphorylation of a vesicular neurotransmitter transporter has not yet been demonstrated.
To assess the potential for regulation of the VMATs by phosphorylation, we have determined their phosphorylation state in cultured cells under a variety of conditions. Surprisingly, we observed phosphorylation of rat VMAT2 under all tested conditions, suggesting constitutive phosphorylation. Using mutational analysis, we have identified sites for constitutive phosphorylation at serines 512 and 514. In vitro assays using purified kinases as well as cellular homogenates suggest the involvement of casein kinase II and possibly casein kinase I. Interestingly, rat VMAT1 does not show this pattern of constitutive phosphorylation, suggesting that the two VMATs may be regulated differently.
MATERIALS AND METHODS
Chinese hamster ovary (CHO) fibroblast cells were maintained in Ham's F-12 medium containing 5% calf serum (Hyclone Laboratories), penicillin, and streptomycin. Rat pheochromocytoma PC12 cells were maintained in Dulbecco's modified Eagle's medium containing 5% calf serum, 10% equine serum, penicillin, and streptomycin. Monkey kidney COS cells were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum, penicillin, and streptomycin. For transient transfections, COS cells were electroporated with 15-30 μg of DNA using a Bio-Rad Gene Pulser apparatus as described previously (
), a fragment containing the mutagenized region was subcloned into the expression vector pcDNA1-Amp (Invitrogen) containing the wild-type VMAT2 cDNA. Glutathione S-transferase (GST)-VMAT2 fusion proteins (
) were constructed by subcloning EcoRV fragments encoding the carboxyl termini of wild-type and mutant rat VMAT2 cDNAs into the SmaI site of the pGEX-3X bacterial expression vector (Pharmacia Biotech Inc.). GST-VMAT1 was constructed by amplification of the region of rat VMAT1 cDNA encoding the carboxyl-terminal domain of the protein using the polymerase chain reaction (
) and the high-fidelity Pfu DNA polymerase (Stratagene). Oligonucleotides used for polymerase chain reaction amplification encoded in-frame EcoRI and XhoI cleavage sites, allowing insertion of the amplified fragment between the EcoRI and XhoI sites of the pGEX-5X-1 bacterial expression vector (Pharmacia Biotech Inc.). To facilitate cloning, the first amino acid of VMAT1 after the GST moiety was changed from isoleucine to phenylalanine.
For metabolic labeling with 32Pi, cells were washed three times in medium lacking phosphate and then incubated for 2 h at 37°C in the presence of 0.5-1.0 mCi/ml 32Pi (ICN). After labeling, cells were washed with ice-cold 10 mM HEPES-NaOH, pH 7.4, 140 mM NaCl (HEPES-buffered saline) and then frozen on dry ice. The frozen cells were harvested by scraping into HEPES-buffered saline; pelleted by centrifugation at 5000 × g for 5 min at 4°C; and then resuspended by trituration in 1 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 0.2 mM NaVO3, 10 mM EDTA, 5 mM EGTA, 10 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 1% (v/v) Nonidet P-40 detergent (homogenization buffer). After removal of the cell debris and nuclei by centrifugation at 14,000 × g for 5 min at 4°C, SDS was added to the supernatant to a final concentration of 0.2%. For immunoprecipitation, the mixture was incubated overnight at 4°C with protein A-Sepharose (Sigma) prebound either to affinity-purified polyclonal antibodies to VMAT1 or VMAT2 or to monoclonal antibody 12CA5 to HA (Boehringer Mannheim) (
), and the proteins were separated by electrophoresis through 10% polyacrylamide. The gels were then fixed in 10% acetic acid, 50% methanol; dried; and submitted to autoradiography.
For metabolic labeling with [35S]methionine/cysteine, cells were washed three times in medium lacking cysteine and methionine and then incubated for 2 h at 37°C in the presence of 0.05-0.1 mCi/ml [35S]Met/Cys (Tran35S-label, ICN). Cell harvesting, immunoprecipitation, and electrophoresis were performed as described above for labeling with 32Pi, except that the gels were incubated in 1 M sodium salicylate as a fluor prior to drying. Western analysis of HA-tagged protein was performed as described previously (
), with a primary rabbit polyclonal antibody to HA (Babco) diluted 1:1000, followed by a secondary antibody conjugated to horseradish peroxidase and visualization by enhanced chemiluminescence (Amersham Corp.).
Phosphoamino Acid Analysis
Phosphoamino acid analysis was performed as described (
). Briefly, extracts prepared from cells metabolically labeled with 32Pi were immunoprecipitated with antibody to HA or VMAT2, and the immunoprecipitates were separated by electrophoresis through polyacrylamide. Following autoradiography, the radiolabeled band was excised from the gel and rehydrated in 50 mM ammonium bicarbonate, and protein was eluted overnight in 0.2% SDS, 2%β-mercaptoethanol. The eluate was then precipitated with 20% trichloroacetic acid and partially hydrolyzed by boiling in 5.7 M HCl for 60 min. The hydrolysate was washed with distilled water and pH 1.9 buffer (7.8% acetic acid, 2.2% formic acid), resuspended in a minimal volume of pH 1.9 buffer containing phosphoamino acid standards, and spotted onto thin-layer cellulose plates. Electrophoresis was performed at 4°C using pH 1.9 buffer for the first dimension and pH 3.5 buffer (5% acetic acid, 0.5% pyridine) for the second dimension. The standards were then stained with ninhydrin, and the plates were submitted to autoradiography to visualize radiolabeled material.
Transport assays were performed essentially as described (
) with minor modifications. Briefly, transiently transfected COS cells were detached from the substrate with trypsin; resuspended in 320 mM sucrose, 10 mM HEPES-KOH, pH 7.4 (SH buffer), containing 2.5 mM MgSO4, 2.5 mM EGTA, 10 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 40 μg/ml bestatin, 2 μg/ml E-64, 50 mM NaF, and 0.2 mM NaVO3; and disrupted in the water-filled cup horn of an ultrasonic cell disrupter (Branson Ultrasonics Corp.) using 15 pulses at an intermediate setting. After removal of nuclei and cell debris by sedimentation at 1300 × g at 4°C for 5 min, 10-μl aliquots were added to SH buffer containing 2.5 mM MgSO4, 4 mM KCl, 100 mM ATP, 10 mM NaF, 0.2 mM NaVO3, and 20 nM [3H]serotonin with varying concentrations of unlabeled serotonin (60-300 nM). After incubation for 2 min at 29°C, reactions were terminated by dilution into ice-cold SH buffer and filtration through 0.2-μm Supor 200 membranes (Gelman Instrument Co.). Filters were air-dried and added to scintillation fluid, and bound radioactivity was determined by scintillation counting in Cytoscint (ICN). Transport reactions were done in duplicate and repeated four times. The protein concentration of each postnuclear supernatant was determined by the Bradford assay (
) (Bio-Rad), and the transport assay values were normalized by dividing by the amount of total protein (mg) added to the reaction. Km and Vmax values were determined by double-reciprocal plots of serotonin concentration and normalized transport activity.
In Vitro Phosphorylation
To purify GST fusion proteins, Escherichia coli cells were grown overnight in 1.6% Tryptone, 1% yeast extract, 0.5% NaCl and induced in 0.1 mM isopropyl-β-D-thiogalactoside for an additional 3-6 h at 37°C. Bacteria were then pelleted; resuspended in 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3, 140 mM NaCl, 2.7 mM KCl (phosphate-buffered saline); and disrupted by vigorous sonication for 1-2 min at O°C. The lysate was centrifuged at 14,000 × g to remove cell debris, and the resulting supernatant was used immediately or stored at −70°C. To partially purify the fusion protein, the cleared extract was bound to glutathione-Sepharose beads for 20 min at room temperature in phosphate-buffered saline and washed twice in phosphate-buffered saline and once in 20 mM Tris, pH 7.5, 50 mM KCl, 5 mM dithiothreitol, 10 mM MgCl (kinase buffer). Aliquots of fusion protein (0.2-8 μg) bound to glutathione-Sepharose (10-20-μl bed volume) were then incubated for 20 min at 30°C in kinase buffer containing 200 μM ATP and [γ-32P]ATP to a final specific activity of 500 μCi/μmol with 100-1000 units of purified CKI (New England Biolabs Inc.), 25-500 units of purified CKII (New England Biolabs Inc.), or 1 μl (∼10 μg of total protein) of a postnuclear supernatant from COS cells (see below). Reactions were stopped by washing with ice-cold phosphate-buffered saline containing 15 mM EDTA, and the phosphorylated proteins were eluted with 20 μl of 10 mM glutathione in 50 mM Tris-HCl, pH 8.0. Eluates were added to an equal volume of 2 × Laemmli sample buffer, and proteins were separated by electrophoresis through 12.5% polyacrylamide. Gels were fixed and stained with Coomassie Blue and then dried and submitted to autoradiography. For quantitation of incorporated radiolabel, gel slices containing the stained radiolabeled bands were excised and subjected to Cerenkov scintillation counting. To quantitate the amount of fusion protein in radiolabeled bands, comparable amounts of bovine serum albumin (Boehringer Mannheim) used as standards were separated by electrophoresis in parallel with the fusion proteins. After staining with Coomassie Blue, the fusion proteins and bovine serum albumin standards were excised from the gel and eluted as described above for phosphoamino acid analysis, and the amount of protein was determined by measuring the absorbance of the bound stain at 585 nm.
Preparation of a postnuclear supernatant was performed as described previously (
) with minor modifications. Briefly, COS or PC12 cells were detached from the substrate either with trypsin or with a cell scraper following one freeze-thaw cycle. Cells were resuspended in 10 mM HEPES-KOH, pH 7.4, 320 mM sucrose, 5 mM EGTA, 10 mM EDTA, 20 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 1 mM dithiothreitol (resuspension buffer) and disrupted in the water-filled cup horn of the ultrasonic cell disrupter using 15 pulses at an intermediate setting. Nuclei and cell debris were removed by sedimentation at 1300 × g at 4°C for 5 min, and the supernatant was decanted and stored at −70°C. Since CKI-7 (Seikagaku) and GTP both compete with ATP for kinase binding, the concentration of ATP was reduced to 2.5 μM in experiments using CKI-7 and to 5 μM with GTP, with final specific activities of [γ-32P]ATP of 40,000 and 16,600 μCi/μmol ATP, respectively. For experiments using CKI-7 to inhibit phosphorylation by the COS cell extract, the endogenous ATP concentration in the extract was reduced 10-fold by dilution in resuspension buffer, followed by reconcentration to the original volume using a Centricon 10 filtration device (Amicon, Inc.).
To investigate the regulation of vesicular transport, we have expressed rat VMAT cDNAs in CHO and monkey kidney (COS) cells (
). We have used these cell lines because they lack endogenous amines, eliminating the possibility of competition with radiolabeled neurotransmitter in transport assays. Although the cells also lack regulated secretory vesicles such as synaptic vesicles, the VMATs sort to an endocytic compartment in these fibroblast cells (
), and acidification of this compartment by the vacuolar H+-ATPase provides the driving force that supports transport activity.
Stable CHO transformants expressing VMAT1 and VMAT2 contain a prominent immunoprecipitable protein of ∼55 kDa after metabolic labeling with [35S]methionine/cysteine (data not shown). To assess phosphorylation of the VMATs, we labeled the CHO cells with 32Pi and detected phosphorylation of VMAT2 even in the absence of exogenous cell stimulation (Fig. 1A). In contrast, the ∼55-kDa form of VMAT1 showed no detectable phosphorylation under the same conditions (Fig. 1A). In addition to the ∼55-kDa forms, metabolic labeling of CHO cells with [35S]Met/Cys indicated less abundant forms of VMAT1 and VMAT2 migrating at ∼95 kDa (data not shown). Higher molecular mass forms of both VMAT1 and VMAT2 appeared to show weak phosphorylation (Fig. 1A); however, the presence of nonspecific bands complicates their analysis. To determine whether VMAT2 is also phosphorylated in neuroendocrine cells, we metabolically labeled rat pheochromocytoma (PC12) cells stably expressing rat VMAT2 with 32Pi. Both the ∼55-kDa form and a higher molecular mass form of VMAT2 were specifically phosphorylated (Fig. 1B). Neither the ∼55-kDa form nor higher molecular mass forms of endogenously expressed VMAT1 were detectably phosphorylated in the PC12 cells (data not shown). We have also detected phosphorylation of the ∼55-kDa form of VMAT2, but not VMAT1, in COS cells (Fig. 2B). These results indicate that VMAT2, but not VMAT1, undergoes constitutive phosphorylation in a variety of cell types.
To identify the site(s) of constitutive phosphorylation on VMAT2, we first used functional VMAT1-VMAT2 chimeras. We previously used these chimeras to identify regions responsible for differences in substrate affinity and inhibitor sensitivity between VMAT1 and VMAT2 (
). To identify the smallest region of VMAT2 sufficient for phosphorylation, chimeras containing the amino terminus of VMAT1 fused to progressively smaller regions from the carboxyl terminus of VMAT2 were metabolically labeled with 32Pi in transfected COS cells. Analysis of chimera C439S (Fig. 2) indicated that at least one phosphorylation site occurred in the carboxyl-terminal 81 amino acids of VMAT2. VMAT1-VMAT2 chimeras containing larger carboxyl-terminal regions of VMAT2 did not show increased levels of phosphorylation, suggesting that these regions do not contain additional constitutive phosphorylation sites (data not shown).
To determine the nature of the phosphorylated residue, we performed phosphoamino acid analysis of VMAT2 metabolically labeled with 32Pi in transfected COS cells. To reduce nonspecific background, we introduced an epitope tag from HA (
) into the large luminal loop between the first and second transmembrane domains of VMAT2. Expressed in COS cells, HA epitope-tagged VMAT2 (HA-VMAT2) showed protein expression, transport activity, and phosphorylation equivalent to wild-type VMAT2 (Fig. 3A and data not shown). Phosphoamino acid analysis of HA-VMAT2 metabolically labeled with 32Pi in COS cells and immunoprecipitated with anti-HA antibody showed phosphorylation on serine, but not threonine or tyrosine (Fig. 3B). CHO cells stably expressing wild-type VMAT2 yielded similar results (data not shown), confirming that the major constitutively phosphorylated sites in VMAT2 occur on one or more serines.
The cDNA sequence of VMAT2 predicts four carboxyl-terminal serines (Fig. 4A) (
). Epitope-tagged VMAT2 cDNAs containing these mutations were then expressed in COS cells, and the phosphorylation state of the protein was determined by metabolic labeling with 32Pi. Of these point mutations, only substitution of alanine at residue 512 (S512A) reduced phosphorylation (Fig. 4B). To determine whether phosphorylation of serine 514 might account for the residual labeling observed in the S512A mutant, we simultaneously mutagenized both serines 512 and 514 to alanine. This double mutant showed no phosphorylation (Fig. 4B), indicating that serine 514 also undergoes phosphorylation, albeit to a lesser extent than serine 512. To assess whether changes in expression of the mutant proteins might account for the reduced labeling, we performed both Western analysis (Fig. 4B) and metabolic labeling with [35S]Met/Cys (data not shown). These analyses showed that the mutations did not affect expression of VMAT2 and indicate a selective effect of these mutations on protein phosphorylation.
To assess the effect of these mutations on transport activity, we performed in vitro transport assays using extracts from transiently transfected COS cells. The Km and Vmax values for wild-type VMAT2 were similar to those for transporters containing alanine replacements at serine 512 or 514 or both serines 512 and 514 (Table I). These results suggest that phosphorylation of these residues is not required for base-line transport activity and that phosphorylation at these sites does not dramatically alter either the affinity of the transporter for substrate or its base-line rate of transport.
Table ITransport activity of VMAT2 with alanine replacements at serines 512 and 514
To identify the kinase(s) responsible for phosphorylation of serines 512 and 514, we compared the sequence surrounding these sites with consensus sequences for phosphorylation by previously characterized kinases (
). The proximity of multiple acidic residues suggested the involvement of the acidotropic kinases CKI and/or CKII. The consensus sequence for CKI is (S(Pi)/D/E)XX(S/T) (in which the phosphorylated site is shown in boldface and S(Pi) represents a phosphoserine specificity determinant), and that for CKII is (S/T)XX(D/E) (
). Thus, serine 512 occurs in a consensus sequence for phosphorylation by both CKI and CKII, and serine 514 in a consensus sequence for CKI (see Fig. 4A). To assess the role of CKI and CKII in VMAT2 phosphorylation, we incubated GST fusion proteins (
) containing the carboxyl-terminal 55 amino acids of VMAT2 (GST-VMAT2) with the purified kinases in vitro (see Fig. 2B). Both CKI and CKII (Fig. 5A), but not protein kinase A or C or glycogen synthetase kinase-3 (data not shown), phosphorylated GST-VMAT2. Neither GST alone (Fig. 5A) nor a GST fusion protein containing the amino terminus of VMAT2 (data not shown) was phosphorylated by CKI or CKII, indicating that the phosphorylation of the carboxyl terminus was specific (Fig. 5A and data not shown). These data suggest that CKI and/or CKII may also mediate the phosphorylation of VMAT2 in vivo
To determine whether purified CKI and CKII phosphorylate the same sites in GST-VMAT2 that are phosphorylated in intact cells, we introduced point mutations into GST-VMAT2. Simultaneous mutation of serines 512 and 514 eliminated phosphorylation by purified CKII and greatly reduced phosphorylation by CKI (Fig. 5B). Mutation of serine 512 alone decreased the phosphorylation of GST-VMAT2 by CKI and, to a lesser extent, by CKII (Fig. 5B). These results suggest that CKI and CKII phosphorylate both serines 512 and 514 and support the relevance of phosphorylation in vitro to events observed in intact cells.
To assess further the relationship between phosphorylation of GST-VMAT2 in vitro and phosphorylation of the transporter in intact cells, we examined the phosphorylation of the fusion protein by COS and PC12 cell extracts. Concomitant replacement of both serines 512 and 514 by alanine or of serine 512 alone decreased phosphorylation of GST-VMAT2 by both extracts (Fig. 5C), similar to the effect of these mutations in intact cells. To determine whether the kinase(s) in the extracts responsible for phosphorylating GST-VMAT2 are CKI and/or CKII, we first used the protein kinase inhibitor CKI-7. CKI-7 inhibits CKI and CKII with Ki values of 9.5 and 90 μM, respectively (
). Using purified CKI and CKII to test the effects of this inhibitor in our in vitro system, 5-10 μM CKI-7 inhibited phosphorylation of GST-VMAT2 by purified CKI, and 50-100 μM CKI-7 inhibited phosphorylation by CKII (Fig. 6). Using the COS cell extract, 10-50 μM CKI-7 inhibited phosphorylation of GST-VMAT2, consistent with a role for CKI and/or CKII in the phosphorylation of VMAT2. Furthermore, at 50 μM, the extract was inhibited at an intermediate level between CKI and CKII, suggesting that both kinases may be involved.
To examine further the involvement of CKII in VMAT2 phosphorylation, we have determined the sensitivity of the in vitro phosphorylation reaction to GTP. Unlike CKI and other known kinases, CKII uses GTP as a phosphate donor almost as well as ATP, with Km values for ATP and GTP of ∼10 and ∼20 μM, respectively (
). Indeed, GTP potently inhibited the labeling of GST-VMAT2 with [γ-32P]ATP by purified CKII, but not CKI (Fig. 7). Using the COS cell extract, we found that GTP partially inhibited phosphorylation of GST-VMAT2 by [γ-32P]ATP, indicating that CKII contributes to the phosphorylation of VMAT2 (Fig. 7). Similar results were obtained using the PC12 cell extract (data not shown). However, GTP inhibited the extracts less potently than it inhibited purified CKII, suggesting the involvement of one or more additional kinases such as CKI.
Although the presence of consensus sites for both CKI and CKII in VMAT1 suggests the potential for phosphorylation by these enzymes (see Fig. 4A), the phosphorylation of VMAT2 greatly exceeds that of VMAT1 in intact cells (see Fig. 1). We therefore determined whether purified CKII could phosphorylate a GST-VMAT1 carboxyl-terminal fusion protein similar to the GST-VMAT2 fusion protein described above (see Fig. 4A). Although GST-VMAT1 is a substrate for CKII, it was phosphorylated 10-fold less than comparable amounts of GST-VMAT2 at each substrate concentration tested (Fig. 8). In vitro phosphorylation using purified CKI yielded similar results (data not shown). These observations indicate that CKII and possibly CKI are responsible for the selective constitutive phosphorylation of VMAT2 and not VMAT1.
To assess the potential for regulation of vesicular monoamine transport, we have studied the phosphorylation state of the transport proteins and determined that the ∼55-kDa form of the brain monoamine transporter VMAT2, but not the adrenal gland transporter VMAT1, undergoes constitutive phosphorylation in intact cells. Phosphoamino acid analysis of VMAT2 indicates that phosphorylation occurs on serine residues, and metabolic labeling of VMAT1-VMAT2 chimeras and VMAT2 point mutants maps the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of the protein, with serine 512 phosphorylated to a greater extent than serine 514.
Serines 512 and 514 occur in consensus sequences for phosphorylation by the acidotropic kinases CKI and CKII (
). We have found that both purified CKI and CKII specifically phosphorylate serine 512 and, to a lesser extent, serine 514 of a bacterial fusion protein containing the carboxyl terminus of VMAT2. More important, the VMAT2 fusion protein is a much better substrate for CKI and CKII than an equivalent VMAT1 fusion protein, consistent with the prominent phosphorylation of VMAT2 relative to VMAT1 in intact cells. To assess the relative role of CKI and CKII in intact cells, we used cell extracts to phosphorylate the VMAT2 fusion protein in vitro The inhibitor CKI-7 blocks phosphorylation by the extract at concentrations intermediate between CKI and CKII, consistent with a role for one or both of these kinases in the phosphorylation of VMAT2. Furthermore, unlabeled GTP inhibits phosphorylation of the fusion protein by the extract. Since CKII can use GTP as a phosphate donor, this supports a role for CKII in the constitutive phosphorylation of VMAT2, although CKI may also contribute.
CKII occurs ubiquitously in vertebrate tissues, with particularly high levels in the brain (
). Thus, CKII occurs in the same tissue as VMAT2 and could be responsible for constitutive phosphorylation of VMAT2 in the brain. Neuronal processes previously linked to CKII activity include neuritogenesis in neuroblastoma cells (
). Phosphorylation of VMAT2 by CKII (or CKI) may regulate its activity directly or indirectly by facilitating additional phosphorylation events. Eliminating the constitutive phosphorylation of VMAT2 does not appear to alter base-line transport activity, suggesting that phosphorylation may not regulate activity directly. However, we cannot exclude this possibility entirely since we do not know the proportion of wild-type VMAT2 constitutively phosphorylated in vivo. If only a small fraction of total steady-state VMAT2 is phosphorylated, mutation of the acceptor sites might not show a difference in activity even if phosphorylation significantly affected transport function.
In addition to regulating intrinsic protein activity, phosphorylation by CKII may influence subcellular localization. Phosphorylation of both cation-dependent and -independent mannose 6-phosphate receptors by CKII may activate binding to the clathrin adaptor protein AP-1 and thereby influence membrane trafficking (
). These results raise the possibility that phosphorylation influences the localization of the VMATs. Indeed, differential phosphorylation of VMAT1 and VMAT2 may contribute to differences in the localization of the two transporters that we have observed in PC12 cells.2
In summary, we have found that VMAT2, but not VMAT1, undergoes constitutive phosphorylation by casein kinase II and possibly casein kinase I on two carboxyl-terminal serine residues, providing the first direct evidence for phosphorylation of a vesicular neurotransmitter transporter or any monoamine transporter. The identification of this phosphorylation event will now enable us to assess its role in the membrane trafficking and regulation of VMAT2 function, with the attendant implications for modulating the release of monoamine transmitters.
We thank Tom Kornberg, Ed Cooper, Edward Fon, and Pam Swedlow for critically reading the manuscript.