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Vol. 19, Issue 7, 2818-2829, July 2008
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*Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; and Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912
Submitted September 28, 2007;
Revised April 3, 2008;
Accepted April 16, 2008
Monitoring Editor: Jean Gruenberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Therapeutic drugs, such as antidepressants, and addictive drugs such as cocaine, act by inhibiting monoamine transporters and provide a compelling reason for understanding the regulation of transport during normal neurotransmission and in pathological states.
Many studies have shown that the activity of monoamine transporters can be regulated acutely by intracellular second-messenger systems, and some of these have implicated direct protein–protein interactions in the regulation of carrier function. One of the best studied examples of acute transporter regulation is the activation of protein kinase C (PKC) by phorbol esters that results in a decrease in dopamine transport in cellular systems and rat striatal synaptosomes (Mortensen and Amara, 2003). The down-regulation of activity occurs through the internalization of transporter molecules from the cell surface through a dynamin-dependent process (Daniels and Amara, 1999; Sorkina et al., 2005). Although one recent study has implied that the direct phosphorylation of the dopamine transporter (DAT) can regulate its intrinsic activity (Khoshbouei et al., 2004), others have ruled out the premise that the direct phosphorylation of the dopamine transporters after PKC activation triggers the internalization of the DAT (Granas et al., 2003). In the latter study the deletion of all potential phosphorylation sites in the N-terminus of DAT eliminates PKC-mediated phosphorylation, but did not eliminate PKC-mediated down-regulation. Thus, other proteins, particularly those linked to endocytosis and trafficking of membrane proteins, are more likely to be the direct substrates for PKC-mediated phosphorylation.
Recent studies have identified several specific regions within DAT that could mediate a direct interaction between DAT and regulatory proteins linked to membrane protein internalization. For example, several amino acids in the DAT C-terminus have been found to be important for both the constitutive and the PKC-regulated internalization of the transporter (Holton et al., 2005; Sorkina et al., 2005). Other work has shown that PKC activation causes DAT to become ubiquitylated in a process that requires the ubiquitin ligase Nedd4-2 (Sorkina et al., 2006), indicating that the addition of ubiquitin moieties could serve to trigger the internalization and degradation of DAT (Miranda et al., 2005, 2007). It should be noted that the degradation of DAT after PKC activation has not been examined in dopaminergic neurons. Other proteins linked to the process of DAT internalization include dynamin, clathrin heavy chain, rab GTPases, epsins, and eps15 (Daniels and Amara, 1999; Sorkina et al., 2005, 2006), and the identification of additional proteins involved in PKC-regulated endocytosis continues to be a central focus for understanding plasma membrane monoamine transporter regulation and physiology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular Biology
The coding sequences of all phosphatases were cloned, and all transporters were subcloned into pOTV using normal primer-based RT-PCR or PCR cloning. The resulting plasmids were linearized and used to prepare cRNA for injection into oocytes. MKP3 was subcloned into pFLAG-CMV-2 to N-terminally tag the protein with FLAG. For production of stable Madin-Darby canine kidney (MDCK) cell lines both MKP1 (a kind gift from Prof. S. M. Keyse, University of Dundee, United Kingdom; Alessi et al., 1993) and MKP3 were subcloned into an IRES vector expressing the phosphatase together with the blasticidin resistance gene. Point mutations were generated using the QuikChange Site-Directed Mutagenesis Kit according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA).
Primary Neuronal Cultures
Primary cultures from substantia nigra and ventral tegmental areas were prepared from 2- to 4-d-old Sprague Dawley rat pups as described previously (Prasad and Amara, 2001).
RNA Interference in MN9D Cells
MN9D cells were obtained from Drs. Alfred Heller and Lisa Won (University of Chicago) and propagated as described (Choi et al., 1992). The cells were transiently cotransfected with DAT cDNA and one of the following MISSION short hairpin RNA (shRNA) plasmids: TRCN0000055040 (shRNA-B); TRCN0000055041 (shRNA-C), which produces shRNAs that targets the coding region of MKP3; or the SHC002 MISSION Non-Target shRNA Control Vector (shRNA-nontarget; Sigma Aldrich, St. Louis, MO). To produce an MKP3 knockdown cell line, MN9D cells were transfected with the MISSION shRNA plasmid TRCN0000055041 (shRNA-C). A pool of shRNA-expressing cells was selected using 5 µg/ml puromycin. These cells were transfected with cDNAs for transporters using Fugene HD transfection reagent (Roche Applied Science, Indianapolis, IN) according to manufacturer's protocol. Functional uptake assays were performed 72–96 h after transfection.
Uptake Assays
Defollicated Xenopus oocytes were injected with 50 ng of cRNA. Uptake of radiolabeled [3H]dopamine (60 Ci/mmol; Perkin Elmer-Cetus, Wellesley, MA) was assayed in oocytes for 10 min in a frog Ringer's buffer with 10 µM RO41-0960 (COMT inhibitor) at room temperature. Nonspecific accumulation of substrate was determined with water-injected oocytes for each condition. Uptake assays in all mammalian cells were carried out in PBS containing 1 mM MgCl2, 0.1 mM CaCl2, and 10 µM RO41-0960. Uptake assays were performed at room temperature for 10 min using radiolabeled [3H]dopamine (60 Ci/mmol; Perkin Elmer-Cetus) at concentrations between 50 and 100 nM. Nonspecific accumulation of substrate was determined with either naïve or mock-transfected cells.
Immunofluorescence Imaging
After either vehicle or 1 µM PMA treatment, MDCK cells were fixed in freshly prepared 4% paraformaldehyde in PBS. Paraformaldehyde-fixed cells were washed in PBS and incubated overnight at 4°C with the primary anti-FLAG antibody (Sigma-Aldrich). After incubation with primary antibody, the cells were washed with PBS and incubated for 1 h in blocking buffer containing secondary antibody conjugated to rhodamine red-X (Jackson Immuno Research Laboratories, West Grove, PA). After incubation with secondary antibody, the cells were washed in PBS and mounted on glass slides with ProLong (Invitrogen) antifade reagent. Images were produced using confocal microscopy (MRC 1024 system; Bio-Rad, Hercules, CA). Image analysis and quantitation of intracellular fluorescence was carried out using NIH ImageJ software (http://rsb.info.nih.gov/ij/).
Cell Surface Biotinylation Assay
Cell surface expression of DAT was assayed with modifications of the method described previously (Daniels and Amara, 1999). After drug treatment cells were washed and incubated with 2 mg/ml sulfo-NHS-SS-biotin (Pierce, Rockford, IL). The cells were quenched with 100 mM glycine buffer, washed with PBS, and lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100 and protease inhibitor cocktail; Roche Applied Science). The cell lysate was incubated on ice and centrifuged at 14,000 x g before incubation with NetrAvidin Resin (Pierce) overnight at 4°C. The beads were separated from the supernatant by centrifugation at 5000 x g for 15 min, washed three times with lysis buffer, twice with a high-salt wash buffer, and once with a no-salt wash buffer. Proteins were separated on SDS-PAGE gels and immunoblotted. Expression of DAT was probed with a rabbit polyclonal antiserum against DAT. Antibodies against the transferrin receptor were obtained from Zymed/Invitrogen, and the antibody against the Na+/K+ ATPase was from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City). EAAT3 was detected using a rabbit polyclonal antibody against the C-terminus. Surface DAT degradation was monitored using the same biotinylation protocol as above with the modification that biotinylation was carried out before drug treatments. After drug treatments cells were lysed and biotinylated proteins were isolated and analyzed as above.
Ubiquitylation of DAT
To examine the ubiquitylation of DAT, cells were lysed in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM N-ethyl-maleimide, and protease inhibitor cocktail). After drug treatments, cell lysates were immunoprecipitated with a DAT-specific polyclonal rabbit antibody, and samples were separated on SDS-PAGE gels and immunoblotted. Ubiquitylated DAT was detected using a mAb to ubiquitin (P4D1) from Santa Cruz Biotechnology (Santa Cruz, CA). Total DAT was detected with a green fluorescent protein (GFP) antibody from Clontech (Mountain View, CA).
Immunoblotting to Detect Phosphorylation State of ERK
For immunoblotting experiments oocytes were lysed in ice cold lysis buffer (100 mM NaCl, 50 mM β-glycerophosphate, pH 7.4, 10 mM EDTA, 2 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail; Roche Applied Science) and centrifuged at 700 x g for 2 min, and the resulting supernatant was mixed with 2x sample buffer. MDCK protein samples were produced by washing of cells directly in their well using PBS and lysed in 2x sample buffer. All samples were separated by SDS-PAGE and immunoblotted. Antibodies from Cell Signaling Technology (Danvers, MA) were used to detect the level of and the activation state of ERK1/2. The mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD184352 was a kind gift from Prof. Philip Cohen (University of Dundee, Scotland).
Statistical Analysis
GraphPad prism software (San Diego, CA) was used to determine statistical significance using Student's t test or one-way ANOVA followed by Bonferroni's multiple comparison test.
Post Hoc Microarray Analyses
To confirm the expression of MKP3 in dopaminergic neurons, we performed post hoc analyses of data from two publicly available sets of microarray data. One dataset produced by Greene et al. (2005) was obtained from the NIH Neuroscience microarray consortium (http://arrayconsortium.tgen.org/np2/home.do). The raw data from this dataset was analyzed using the Affymetrix (Santa Clara, CA) GCOS software statistical expression algorithm. We also obtained expression data for MKP3 (Gene ID: 93285_at) from the supplemental dataset produced by Miller et al. (2004).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Granas et al., 2003). In the experiments presented here cells were incubated with PMA for 30 min at room temperature and not at 37°C as in previous studies. When cells were incubated at 37°C, we also found small, but significant down-regulation (data not shown).
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; Kondoh and Nishida, 2006). When expressed along with DAT in oocytes, MKP3 prevented the PMA-induced down-regulation. PMA still induced a down-regulation with an IC50 of 8 nM, similar to the IC50 of 4 nM found in oocytes injected with the hDAT cRNA alone, but in the presence of MKP3 the magnitude of down-regulation was dramatically reduced from nearly 100% to 52 ± 6%.
Although no specific inhibitors of MKP3 exist, we used the vanadate analogue bpv(phen), a general in vitro inhibitor of cysteine-based tyrosine and dual specificity phosphatases (Wiland et al., 1996), to test the involvement of this superfamily of tyrosine phosphatases in the PKC-mediated regulation of neurotransmitter transporters. SK-N-SH cells that express both NET and MKP3 endogenously were pretreated with 100 µM bpv(phen) before activation of PKC with PMA. Although vehicle-treated SK-N-SH cells displayed no significant change in uptake activity in response to PMA, cells pretreated with bpv(phen) displayed a 54% reduction in activity after PMA (data not shown), confirming the involvement of tyrosine phosphatases in PKC-mediated regulation of NET activity.
Only Catalytically Active MAP Kinase Phosphatase Can Prevent the PMA-induced Down-Regulation of the Dopamine Transporter
Because bpv(phen) can inhibit other phosphatases in addition to MKP3, we next examined whether other tyrosine phosphatases or even serine/threonine phosphatases could modulate the PKC induced down-regulation of DAT activity in Xenopus oocytes. We chose to investigate PP2C
a prototypic serine/threonine phosphatase expressed in the brain (Price and Mumby, 1999) and PTP-1B a prototypic tyrosine phosphatase involved in the dephosphorylation of several receptor tyrosine kinases (Tonks, 2003; Figure 2B). Coexpressing either PP2C or PTP-1B with DAT in Xenopus oocytes had no effect on the PMA-induced down-regulation of DAT, suggesting the effect observed above in SK-N-SH cells is the effect of bpv(phen) inhibiting the endogenous MKP3 in these cells.
To further explore the mechanism of action of MKP3 in regulating DAT activity, we used a mutant of MKP3 in which the catalytic activity was removed by mutating a single cysteine in the catalytic site to a serine (C293S). This mutant phosphatase binds and traps MAP kinases, but because the dephosphorylating activity of the mutant is highly reduced, it does not inactivate its MAP kinase substrates (Brunet et al., 1999). Thus, although the C293S mutant does not prevent the MAP kinase from activating cytosolic targets, it disrupts the nuclear translocation of the activated MAP kinase by sequestering it in the cytosol, preventing activation of nuclear targets of the MAP kinases. In contrast to the effects observed with wild-type MKP3, the MKP3-C293S mutant did not prevent PKC-mediated down-regulation of DAT (Figure 2B), indicating that a catalytically active phosphatase is required for blocking the inhibition of DAT activity. Because the MKP3 mutant sequesters its MAP kinase substrates within the cytoplasm, this experiment also addresses whether translocation of MAP kinase to the nucleus and activation of downstream nuclear targets is required for PKC-mediated regulation of DAT. The mutant was unable to prevent PKC-mediated down-regulation and thus, these results also suggest that nuclear translocation of MAP kinases is not required for the effect of PKC on transporter activity.
PKC Regulation of All Plasma Membrane Monoamine Transporters Is Modulated by MKP3
To determine whether the effect of MKP3 expression was specific for dopamine transporters or whether MKP3 could also modulate PMA-induced down-regulation of other monoamine transporters, we coexpressed three monoamine transporters in Xenopus oocytes alone or with MKP3 (Figure 2C). The effect of PMA on transport activity varied greatly between the different transporters. DAT and NET were the most sensitive, with almost complete inhibition of transport activity, 98 ± 1 and 91 ± 7%, respectively. The serotonin transporter (SERT) was moderately affected with only 47 ± 5% of the uptake activity remaining after PMA treatment. Although differences in expression of the three transporters could contribute to the variation in sensitivity to PMA, coexpression of MKP3 with either DAT, NET, or SERT consistently reduced the PMA-mediated decrease in activity. With the SERT and the NET we found modest effects of MKP3 expression, with 17 and 25% increases in serotonin and norepinephrine uptake, respectively. However, DAT showed the most dramatic effect with a recovery of about 48% of its maximal uptake activity.
Overexpression of MKP3 Prevents DAT Internalization in MDCK Cells
We next transiently expressed FLAG-tagged MKP3 into MDCK cells stably expressing GFP-tagged DAT to examine how MKP3 influences the trafficking of DAT in the system we have used previously to study PKC regulation of DAT (Daniels and Amara, 1999). MDCK cells can be grown to form a polarized epithelium with tight junctions in a very distinct honeycomb pattern, making them extremely useful as a model for membrane protein trafficking and sorting. Representative confocal images of GFP-DAT in MDCK cells are shown in Figure 3, A and C. We have previously shown in MDCK cells that after PKC activation dopamine transporters are rapidly endocytosed through a dynamin-dependent and clathrin-mediated process resulting in a very distinctive punctate intracellular pattern (Daniels and Amara, 1999). The internalized DAT molecules present within these puncta initially colocalize with internalized transferrin receptors, but subsequently transit through an endosomal pathway into lysosomes where they are ultimately degraded (Daniels and Amara, 1999). The dramatic increase in intracellular puncta reflecting internalized GFP-DAT occurs within 30 min of treatment with 1 µM PMA (Figure 3C) and precisely parallels the decrease in transporter surface expression. Vehicle-treated cells showed virtually no intracellular puncta (Figure 3A). The intracellular accumulation of DAT in response to PMA treatment was not apparent in cells transiently transfected with FLAG-tagged MKP3, shown in red on Figure 3C. Quantitative analysis of the amount of intracellular fluorescence using ImageJ (NIH; Figure 3D) shows that after PMA treatment there was a significantly lower level of intracellular fluorescence in MKP3-expressing cells with almost double the amount of intracellular fluorescence in cells that did not express MKP3. In control cells that were not treated with PMA, we did not find a significant difference in intracellular fluorescence between MKP3- expressing cells and naïve cells (Figure 3, A and B).
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; Miranda et al., 2005). To test if MKP3 regulates internalization or recycling, we carried out a conventional biotinylation assay in which surface proteins were biotinylated after drug treatments, purified using streptavidin beads, and visualized using immunoblotting (Figure 4A). The PMA treatment was carried out on cells pretreated either with vehicle or with 25 µM monensin, which blocks the recycling of vesicles back to the surface (Miranda et al., 2004). We found that the down-regulation of surface DAT density after PMA treatment (
65% remaining) was dramatically reduced in cells expressing MKP3 (90% remaining; Figure 4C). Pretreatment of the cells with monensin had no effect on the ability of MKP3 to attenuate the removal of DAT from the surface, suggesting that MKP3 regulates internalization without altering the process of vesicle recycling (Figure 4C).
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90% of surface DAT remains even after 2 h of PMA treatment. Taken together these results suggest that MKP3 attenuates the internalization of DAT, thereby delaying the degradation of the protein. This is also in agreement with the time course of DAT trafficking imaged with confocal microscopy in Figure 3E. We next examined whether the effects of MKP3 were selective for the DAT by assessing the effects of PMA and MKP3 expression on the surface distribution of several endogenously expressed integral membrane proteins. Figure 4E shows that the steady-state surface levels of both the transferrin receptor (TfR) and the sodium potassium ATPase (Na+/K+) remain unchanged after incubation with PMA and, as expected, MKP3 expression had no effect on the surface expression of the two proteins. Intriguingly, the glutamate transporter EAAT3, a member of a distinct neurotransmitter transporter family, showed a decrease in surface expression in response to PMA, and this decrease also could be prevented by overexpression of MKP3. Thus, the effects of MKP3 appear selective for membrane proteins that are modulated by PKC and may reflect a more general regulatory mechanism that is not limited to the DAT.
PKC-induced Ubiquitylation of DAT Is Not Affected by MKP3
Recently it has been established that DAT is ubiquitylated after PMA treatment and that this ubiquitylation is necessary for the internalization of DAT (Miranda et al., 2005, 2007), and thus we hypothesized that MKP3 could be regulating the ubiquitylation of DAT. However, we found no difference in the level of DAT ubiquitylation in cells expressing MKP3 compared with cells expressing DAT alone (Figure 4F). We did find that PMA would increase the ubiquitylation of DAT, but only in a very small subfraction of the DAT population, because the GFP antibody detecting all GFP-tagged DAT detects a band with a different and smaller size constituting nonubiquitylated DAT. This suggests that the ubiquitylation is a dynamic process in which ubiquitinylated DAT exists only transiently and is readily deubiquitinylated by enzymes involved in ubiquitin turnover. To confirm that ubiquitylation is required for DAT internalization in the MDCK cell line, we stably expressed a DAT mutant (
Ub-DAT) in which all three previously reported N-terminal lysines (positions 19, 27, and 35) that are responsible for the ubiquitin-mediated down-regulation of DAT (Miranda et al., 2007) have been mutated to arginine. As in the previous study, we found that the ubiquitylation of this mutant was reduced and that the PMA-induced down-regulation, internalization and degradation was attenuated, but not completely prevented. Moreover, cells expressing this DAT mutant displayed a very similar behavior to the cells expressing MKP3 and wild type DAT (Figures 3E and 4, A–F). These results are not likely due to differences in the amount of DAT, as comparable expression was observed in DAT, DAT+MKP3, and Ub-DAT cell lines (data not shown).
The Effects of MKP3 on DAT Internalization Is Independent of Classical MAP Kinases
MAP kinases are thought to be the primary target of MKP3, and thus we anticipated that a MAP kinase might be the downstream target of the signaling cascade activated by PMA. Previous work has demonstrated that PKC can activate MAP kinases through activation of raf-1 and its MAP kinase kinase substrates (MEKs), although the precise mechanism and PKC substrates required remain controversial. We therefore investigated the activation state of various MAP kinases after PMA treatment either in the absence or presence of MKP3 in MDCK cells and Xenopus oocytes expressing DAT (Figure 5). The phosphorylation state of ERK1/2 was elevated in untreated MDCK cells when compared with oocytes. PMA treatment of MDCK cells did induce an increase in the phosphorylation and activation of ERK1/2 (Figure 5A), but did not induce activation of the stress-induced JNKs or p38 MAPKs (data not shown). We observed that untransfected cells had levels of activated ERK very similar to cells stably expressing either MKP1 or MKP3. The PMA-induced increase in phosphorylation and activation of ERK1/2 was abolished when MKP1 or MKP3 was stably expressed in PMA-treated MDCK cells, even though MKP1 was unable to prevent PKC-mediated down-regulation of DAT activity (see below). Pharmacological inhibition of the upstream MAP kinase kinase MEK1 using the compound PD98059 also resulted in inhibition of PMA-induced activation of ERK.
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). Because our results suggested that blocking ERK activation did not have the same effect as expressing MKP3, we tested the involvement of other members of the MAP kinase family, even those that do not appear to be activated by PKC. In these experiments, inhibition of MAP kinases such as JNKs with SP600125 (50 µM) or p38 MAPK with SB203580 (10 µM) or MEK with U0126 (20 µM) could not prevent the PMA-induced down-regulation of DAT activity (data not shown). As was found in MDCK cells, there was no correlation with the activation state of MAP kinases and the down-regulation of DAT in Xenopus oocytes (Figure 5B). The MEK1 inhibitor PD184352 had no effect on the down-regulation of transporter activity, but did remove the very limited activation of ERK1/2 after PMA treatment. None of the other MAP kinases were activated by PMA, nor could the p38 MAP kinase inhibitor SB203580 (10 µM) or the SAP/JNK MAP kinase inhibitor SP600125 (50 µM) inhibit the PMA-induced down-regulation (data not shown). Table 1 lists all the compounds tested for their effects on the PMA-mediated down-regulation of DAT in oocytes. The only compounds within this list that can prevent the down-regulation are the two PKC inhibitors bisindolylmaleimide I (GF109203X; 10 µM) and staurosporine (10 µM). One interpretation of these results is that the expression of MKP3 leads to the inhibition of PKC itself. To test this, we assayed the activity of PKC after PMA treatment in oocytes expressing MKP3 or not. We found no difference in basal and PMA-induced PKC activity between the two in a crude assay using an antibody that detects phosphorylated PKC substrates to estimate PKC activity (data not shown).
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MKP3 Effects on DAT Down-Regulation in a Dopaminergic Cell Line
To examine whether the effects of MKP3 on monoamine transport activity can be observed in dopaminergic cells, we used MN9D cells, a dopaminergic neuronal hybrid cell line (Choi et al., 1992). These cells contain dopamine and tyrosine hydroxylase and express DAT at very low levels (Chen et al., 2005). As shown in Figure 6, A, C, and D, MN9D cells transiently transfected with the DAT or the SERT exhibited no down-regulation of transport activity after acute PMA treatment. Interestingly, MKP3 is expressed endogenously at readily detectable levels in these cells (Figure 6B), a finding consistent with the idea that expression of MKP3 disrupts PKC-mediated regulation. To test this hypothesis, we transiently cotransfected MN9D cells with DAT and either a control shRNA directed against no known mouse gene targets (shRNA-Non-Target) or two different shRNAs directed against the MKP3 coding region (shRNA-B and shRNA-C). When MN9D cells are transfected with either MKP3-targeted shRNA, dopamine transport activity is decreased (20%) by PMA application (Figure 6A). We also established a pool of MN9D cells (MN9D shRNA-C) stably expressing the shRNA-C. This cell pool expressed significantly lower levels of MKP3 protein as demonstrated by immunoblotting (Figure 6B). In these stably transfected cell pools, PMA induced a decrease in transport activity of the DAT (Figure 6C) and the SERT (Figure 6D) confirming that when MKP3 is reduced the transporters are no longer refractory to down-regulation by PKC in MN9D cells. In cell surface biotinylation experiments we also obtained data consistent with the results of the uptake data present in Figure 6. However, unlike the robust effects we present in Figure 4 for MDCK cells, the changes in the MN9D cells were modest and not always significant within the variability of the biochemical assay (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mortensen and Amara, 2003). By coexpressing DAT with a cRNA library from the human SK-N-SH neuroblastoma cell line, we obtained evidence for a factor expressed in SK-N-SH cells that could prevent or reverse the dramatic internalization of DAT observed in response to PKC activation in Xenopus oocytes. Using the oocyte system, we were able to establish a complementation assay to screen pools of clones from the SK-N-SH cRNA library, which led to the isolation of a MAP kinase phosphatase MKP3 (DUSP6 or PYST1) with an activity that attenuates the PKC-mediated down-regulation of DAT function.
MAP kinase phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate MAP kinases at both threonine and tyrosine residues and thereby inactivate them (Dickinson and Keyse, 2006; Kondoh and Nishida, 2006). Within the family of MKPs, the individual phosphatases show some selectivity between the three conventional families of MAP kinases. For example, MKP3 has a preference for the ERK kinases (Muda et al., 1996), and MKP1 has a preference for the stress activated kinases p38 and JNK/SAPK (Chu et al., 1996). Most of this work is from studies in transfected mammalian cells, and only recently studies have begun to examine the physiological role of MKP3 in vivo. These studies have focused on the involvement of the phosphatase in signaling pathways during development. One study described the effects of targeted disruption of the MKP3 gene in mice (Li et al., 2007). These mice displayed increased ERK activation, and their phenotype led to dominant postnatal lethality and included serious developmental defects such as skeletal dwarfism, coronal craniosynostosis, and hearing loss. Other genetic and mutational approaches to examine the developmental role of MKP3 have been carried out in zebrafish (Tsang et al., 2004), chick embryos (Eblaghie et al., 2003; Smith et al., 2005), and Drosophila (Rintelen et al., 2003).
To understand how MKP3 regulates DAT trafficking in mammalian cells, we used canine kidney MDCK cells as a model system. We had demonstrated previously in these cells that PKC activation stimulates clathrin-mediated endocytosis and trafficking of DAT to lysosomes where it is ultimately degraded (Daniels and Amara, 1999). Expression of MKP3 did indeed result in less intracellular DAT (Figure 4) and using biochemical assays, we showed that the step MKP3 inhibits is the PKC-activated dynamin-dependent endocytosis of DAT (Figures 5 and 7). In mammalian cells clathrin-mediated endocytosis is a well-studied process that involves a variety of proteins including epsins, dynamin, adaptor proteins, and clathrin, as well as several accessory proteins that work as scaffolding proteins (Conner and Schmid, 2003). Not surprisingly, several of these are also required for internalization of DAT (Sorkina et al., 2006). Various steps in clathrin-mediated endocytosis have been shown to be regulated by phosphorylation and/or dephosphorylation of proteins present in the endocytic complex (Cousin and Robinson, 2001). A number of studies have identified phosphoproteins required for dynamin-dependent endocytosis (AP-2, dynamin), as well as associated kinases (PKC, Casein kinase II, cyclin-G–associated kinase, cdk5, and adaptor-associated kinase 1), and phosphatases (Calcineurin and Synaptojanin; reviewed in Conner and Schmid 2003).
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), other modifications such as ubiquitinylation, have been considered as potential signals for the internalization of DAT. DAT becomes ubiquitinylated after activation of PKC (Miranda et al., 2005) in a process that appears to require the E3 ubiquitin ligase Nedd4-2 (Sorkina et al., 2006). However, intriguingly, we do not find any effect of MKP3 on PMA-induced ubiquitylation of DAT and thus conclude that the process regulated by MKP3 must be downstream from ubiquitylation events that take place while the transporter sits on the plasma membrane (Figure 7). Whether only the initial ubiquitylation of DAT is regulated by PKC activation or whether other steps in internalization are regulated by PKC cannot be established from our data. One possibility is that the machinery for internalization is primed and awaits the ubiquitylated DAT, which is sufficient for activation of internalization process, but that other proteins involved in the internalization of ubiquitylated cargo are regulated by MKP3. It could also be imagined that PKC-dependent phosphorylation of several key proteins occurs simultaneously and multiple processes are activated to produce the internalization of DAT. Thus, one required PKC-dependent step would be turned on by the ubiquitylation of DAT and another step would be turned off by dephosphorylation by MKP3.
To date two different MAP kinase families, p38 and ERK MAP kinases, have been implicated in the regulation of plasma membrane transporters. The p38 MAP kinase was found to inhibit insulin mediated up-regulation of NET in SK-N-SH cells (Apparsundaram et al., 2001), and it was in one study also found to increase membrane insertion of the serotonin transporter (SERT) in a PKC-independent manner in both synaptosomes and HEK-293 cells (Samuvel et al., 2005). In several studies another group found that the activation of the p38 MAP kinase would result in increased intrinsic activity of SERT (Zhu et al., 2004, 2005). The inhibition of the ERK MAP kinases was found in a study to result in a small inhibition of DAT believed to be a result of internalization (Moron et al., 2003), and recently it was similarly found that effects of dopamine receptor activation on DAT regulation were mediated through the MEK/ERK pathway (Bolan et al., 2007). The results of these studies were obtained using inhibitors of MAP kinases that have no effect on the PKC-dependent internalization of DAT in our studies and are likely to involve different mechanisms.
Our results suggest that the three best characterized MAP kinase families are not directly involved in PKC-mediated regulation of the DAT, because the activation state of these kinases does not correlate with the effect of MKP3 on DAT down-regulation in either oocytes or MDCK cells (Figure 6). We did find, as has been found in many cell systems, that ERK is activated by PMA treatment, but using pharmacological inhibitors of the upstream MAP kinase kinase, MEK1 to reduce ERK activation, did not effect the PMA-induced down-regulation of DAT. Moreover, in oocytes, when we used progesterone to activate ERK through a non-PKC–mediated pathway no change in transport activity was observed.
Thus, it seems likely that PKC activates a protein target of MKP related to MAP kinases such as ERK3, ERK7, MOK (DYF-5), MAK, ICK, and DYRK1A (Chen et al., 2001), a hypothesis that is further supported by the fact that only MKP3 and not the closely related MAP kinase phosphatase, MKP1, can prevent the internalization of DAT. Little is known about the signaling roles of these orphan MAP kinases, and even less is known about their involvement in endocytosis and trafficking. One study in Caenorhabditis elegans has examined the orphan MAP kinase DYF-5 and implicated it directly in the docking and undocking of kinesin motors (Burghoorn et al., 2007). Obviously, the identification of the unknown substrate of MKP3 would further our understanding of the mechanism mediating DAT trafficking. It has been found that replacement of the cysteine in the active site of MKPs with serine abolishes catalytic activity, but dramatically stabilizes the otherwise transient interaction between substrate and phosphatase, creating a "substrate-trap" to enable the isolation of substrates of tyrosine-directed phosphatases (Blanchetot et al., 2005).
An issue critical to the physiological relevance of MKP3 to DAT regulation is whether it is expressed in the same cell type. Post hoc analyses (see Materials and Methods) of data on gene expression in laser-captured tyrosine hydroxylase–positive neurons from substantia nigra (SN) and ventral tegmental area (Greene et al., 2005) demonstrate that the DAT and MKP3 are expressed in the same cells. In addition, others have shown that MKP3 expression decreases significantly when dopamine neurons in SN are selectively lesioned by the neurotoxin, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP; Miller et al., 2004).
Our studies in MN9D cells, which exhibit a dopaminergic phenotype (Choi et al., 1992; Chen et al., 2005), also demonstrate that MKP3 is expressed in dopamine neurons; however, in most studies to date, MKP3 displays relatively low basal expression in the brain. Most work on the regulation of intracellular signal transduction has focused on the activity of kinases. However, it has been proposed that the acute activation of phosphatases could be a more effective way of controlling the activity of signaling cascades (Bhalla et al., 2002). This might explain why MKP3 and other MAP kinase phosphatases are relatively nonabundant in the brain, but can be turned on by activity (Boschert et al., 1997). MKP3 is also strongly regulated posttranslationally and displays an enhanced sensitivity to proteasomal degradation that is phosphorylation-dependent (Marchetti et al., 2005). Midbrain dopamine neuron cultures display robust down-regulation (Figure 1) consistent with the idea that MKP3 is turned off in these cells, whereas cells lines such as MN9D or SK-N-SH, which express MKP3, are more refractory to the effects of PKC activation. Because MKP1 and MKP3 mRNAs are up-regulated by acute and chronic treatment of rats with the DAT substrate methamphetamine (Takaki et al., 2001), we hypothesize that MKP3 plays a regulatory homeostatic role in maintaining and stabilizing neurotransmitter transporters on the surface to limit the actions of neurotransmitter during periods of increased neuronal activity.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Ole Valente Mortensen (mortense{at}pitt.Edu)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apparsundaram, S., Schroeter, S., Giovanetti, E., and Blakely, R. D. (1998). Acute regulation of norepinephrine transport: II. PKC-modulated surface expression of human norepinephrine transporter proteins. J. Pharmacol. Exp. Ther 287, 744–751.
Apparsundaram, S., Sung, U., Price, R. D., and Blakely, R. D. (2001). Trafficking-dependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transport in SK-N-SH cells. J. Pharmacol. Exp. Ther 299, 666–677.
Bhalla, U. S., Ram, P. T., and Iyengar, R. (2002). MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network. Science 297, 1018–1023.
Blanchetot, C., Chagnon, M., Dube, N., Halle, M., and Tremblay, M. L. (2005). Substrate-trapping techniques in the identification of cellular PTP targets. Methods 35, 44–53.[CrossRef][Medline]
Bolan, E. A. et al. (2007). D2 receptors regulate dopamine transporter function via an ERK 1/2-dependent and PI3 kinase-independent mechanism. Mol. Pharmacol 71, 1222–1232.
Boschert, U., Muda, M., Camps, M., Dickinson, R., and Arkinstall, S. (1997). Induction of the dual specificity phosphatase PAC1 in rat brain following seizure activity. Neuroreport 8, 3077–3080.[Medline]
Brondello, J. M., McKenzie, F. R., Sun, H., Tonks, N. K., and Pouyssegur, J. (1995). Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibroblasts. Oncogene 10, 1895–1904.[Medline]
Brunet, A., Roux, D., Lenormand, P., Dowd, S., Keyse, S., and Pouyssegur, J. (1999). Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 18, 664–674.[CrossRef][Medline]
Burghoorn, J., Dekkers, M. P., Rademakers, S., de Jong, T., Willemsen, R., and Jansen, G. (2007). Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 104, 7157–7162.
Chen, C. X., Huang, S. Y., Zhang, L., and Liu, Y. J. (2005). Synaptophysin enhances the neuroprotection of VMAT2 in MPP+-induced toxicity in MN9D cells. Neurobiol. Dis 19, 419–426.[CrossRef][Medline]
Chen, Z., Gibson, T. B., Robinson, F., Silvestro, L., Pearson, G., Xu, B., Wright, A., Vanderbilt, C., and Cobb, M. H. (2001). MAP kinases. Chem. Rev 101, 2449–2476.[CrossRef][Medline]
Choi, H. K., Won, L., Roback, J. D., Wainer, B. H., and Heller, A. (1992). Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions. Proc. Natl. Acad. Sci. USA 89, 8943–8947.
Chu, Y., Solski, P. A., Khosravi-Far, R., Der, C. J., and Kelly, K. (1996). The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J Biol. Chem 271, 6497–6501.
Conner, S. D., and Schmid, S. L. (2003). Regulated portals of entry into the cell. Nature 422, 37–44.[CrossRef][Medline]
Cousin, M. A., and Robinson, P. J. (2001). The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci 24, 659–665.[CrossRef][Medline]
Daniels, G. M., and Amara, S. G. (1999). Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters. J Biol. Chem 274, 35794–35801.
Dickinson, R. J., and Keyse, S. M. (2006). Diverse physiological functions for dual-specificity MAP kinase phosphatases. J. Cell Sci 119, 4607–4615.
Eblaghie, M. C., Lunn, J. S., Dickinson, R. J., Munsterberg, A. E., Sanz-Ezquerro, J. J., Farrell, E. R., Mathers, J., Keyse, S. M., Storey, K., and Tickle, C. (2003). Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos. Curr. Biol 13, 1009–1018.[CrossRef][Medline]
Granas, C., Ferrer, J., Loland, C. J., Javitch, J. A., and Gether, U. (2003). N-terminal truncation of the dopamine transporter abolishes phorbol ester- and substance P receptor-stimulated phosphorylation without impairing transporter internalization. J Biol. Chem 278, 4990–5000.
Greene, J. G., Dingledine, R., and Greenamyre, J. T. (2005). Gene expression profiling of rat midbrain dopamine neurons: implications for selective vulnerability in parkinsonism. Neurobiol. Dis 18, 19–31.[CrossRef][Medline]
Holton, K. L., Loder, M. K., and Melikian, H. E. (2005). Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization. Nat. Neurosci 8, 881–888.[Medline]
Khoshbouei, H., Sen, N., Guptaroy, B., Johnson, L., Lund, D., Gnegy, M. E., Galli, A., and Javitch, J. A. (2004). N-terminal phosphorylation of the dopamine transporter is required for amphetamine-induced efflux. PLoS. Biol 2, E78.[CrossRef][Medline]
Kondoh, K., and Nishida, E. (2007). Regulation of MAP kinases by MAP kinase phosphatases. Biochim. Biophys. Acta 1773, 1227–1237.[Medline]
Li, C., Scott, D. A., Hatch, E., Tian, X., and Mansour, S. L. (2007). Dusp6 (Mkp3) is a negative feedback regulator of FGF-stimulated ERK signaling during mouse development. Development 134, 167–176.
Marchetti, S., Gimond, C., Chambard, J. C., Touboul, T., Roux, D., Pouyssegur, J., and Pages, G. (2005). Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation. Mol. Cell. Biol 25, 854–864.
Miller, R. M. et al. (2004). Dysregulation of gene expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse substantia nigra. J. Neurosci 24, 7445–7454.
Miranda, M., Dionne, K. R., Sorkina, T., and Sorkin, A. (2007). Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter. Mol. Biol. Cell 18, 313–323.
Miranda, M., Sorkina, T., Grammatopoulos, T. N., Zawada, W. M., and Sorkin, A. (2004). Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum. J Biol. Chem 279, 30760–30770.
Miranda, M., Wu, C. C., Sorkina, T., Korstjens, D. R., and Sorkin, A. (2005). Enhanced ubiquitylation and accelerated degradation of the dopamine transporter mediated by protein kinase C. J. Biol. Chem 280, 35617–35624.
Moron, J. A. et al. (2003). Mitogen-activated protein kinase regulates dopamine transporter surface expression and dopamine transport capacity. J. Neurosci 23, 8480–8488.
Mortensen, O. V., and Amara, S. G. (2003). Dynamic regulation of the dopamine transporter. Eur. J. Pharmacol 479, 159–170.[CrossRef][Medline]
Muda, M., Boschert, U., Dickinson, R., Martinou, J. C., Martinou, I., Camps, M., Schlegel, W., and Arkinstall, S. (1996). MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. J Biol. Chem 271, 4319–4326.
Prasad, B. M., and Amara, S. G. (2001). The dopamine transporter in mesencephalic cultures is refractory to physiological changes in membrane voltage. J. Neurosci 21, 7561–7567.
Price, N. E., and Mumby, M. C. (1999). Brain protein serine/threonine phosphatases. Curr. Opin. Neurobiol 9, 336–342.[CrossRef][Medline]
Rintelen, F., Hafen, E., and Nairz, K. (2003). The Drosophila dual-specificity ERK phosphatase DMKP3 cooperates with the ERK tyrosine phosphatase PTP-ER. Development 130, 3479–3490.
Samuvel, D. J., Jayanthi, L. D., Bhat, N. R., and Ramamoorthy, S. (2005). A role for p38 mitogen-activated protein kinase in the regulation of the serotonin transporter: evidence for distinct cellular mechanisms involved in transporter surface expression. J. Neurosci 25, 29–41.
Smith, T. G., Sweetman, D., Patterson, M., Keyse, S. M., and Munsterberg, A. (2005). Feedback interactions between MKP3 and ERK MAP kinase control scleraxis expression and the specification of rib progenitors in the developing chick somite. Development 132, 1305–1314.
Sorkina, T., Hoover, B. R., Zahniser, N. R., and Sorkin, A. (2005). Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism. Traffic 6, 157–170.[CrossRef][Medline]
Sorkina, T., Miranda, M., Dionne, K. R., Hoover, B. R., Zahniser, N. R., and Sorkin, A. (2006). RNA interference screen reveals an essential role of Nedd4–2 in dopamine transporter ubiquitination and endocytosis. J. Neurosci 26, 8195–8205.
Takaki, M., Ujike, H., Kodama, M., Takehisa, Y., Nakata, K., and Kuroda, S. (2001). Two kinds of mitogen-activated protein kinase phosphatases, MKP-1 and MKP-3, are differentially activated by acute and chronic methamphetamine treatment in the rat brain. J. Neurochem 79, 679–688.[CrossRef][Medline]
Tonks, N. K. (2003). PTP1B: from the sidelines to the front lines! FEBS Lett 546, 140–148.[CrossRef][Medline]
Torres, G. E., Gainetdinov, R. R., and Caron, M. G. (2003). Plasma membrane monoamine transporters: structure, regulation and function. Nat. Rev. Neurosci 4, 13–25.[CrossRef][Medline]
Tsang, M., Maegawa, S., Kiang, A., Habas, R., Weinberg, E., and Dawid, I. B. (2004). A role for MKP3 in axial patterning of the zebrafish embryo. Development 131, 2769–2779.
Wiland, A. M., Denu, J. M., Mourey, R. J., and Dixon, J. E. (1996). Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6. J Biol. Chem 271, 33486–33492.
Zahniser, N. R., and Doolen, S. (2001). Chronic and acute regulation of Na+/Cl–-dependent neurotransmitter transporters: drugs, substrates, presynaptic receptors, and signaling systems. Pharmacol. Ther 92, 21–55.[CrossRef][Medline]
Zhu, C. B., Carneiro, A. M., Dostmann, W. R., Hewlett, W. A., and Blakely, R. D. (2005). p38 MAPK activation elevates serotonin transport activity via a trafficking-independent, protein phosphatase 2A-dependent process. J Biol. Chem 280, 15649–15658.
Zhu, C. B., Hewlett, W. A., Feoktistov, I., Biaggioni, I., and Blakely, R. D. (2004). Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation. Mol. Pharmacol 65, 1462–1474.
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