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Originally published In Press as doi:10.1074/jbc.M310734200 on October 21, 2003

J. Biol. Chem., Vol. 279, Issue 3, 2182-2191, January 16, 2004
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Enhancement of Tyrosine Hydroxylase Phosphorylation and Activity by Glial Cell Line-derived Neurotrophic Factor*

Nobuhide Kobori{ddagger}, Jack C. Waymire§, John W. Haycock¶, Guy L. Clifton{ddagger}, and Pramod K. Dash{ddagger}§||

From the {ddagger}The Vivian L. Smith Center for Neurological Research, §Departments of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77225 and the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70119

Received for publication, September 29, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although glial cell-line derived neurotrophic factor (GDNF) acts as a potent survival factor for dopaminergic neurons, it is not known whether GDNF can directly alter dopamine synthesis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis, and its activity is regulated by phosphorylation on three seryl residues: Ser-19, Ser-31, and Ser-40. Using a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuron cultures, the present study examined whether GDNF alters the phosphorylation of TH and whether these changes are accompanied by increased enzymatic activity. Exposure to GDNF did not alter the TH protein level in either neuroblastoma cells or in primary neurons. However, significant increases in the phosphorylation of Ser-31 and Ser-40 were detected within minutes of GDNF application in both cell types. Enhanced Ser-31 and Ser-40 phosphorylation was associated with increased TH activity but not dopamine synthesis in neuroblastoma cells, possibly because of the absence of L-aromatic amino acid decarboxylase activity in these cells. In contrast, increased phosphorylation of Ser-31 and Ser-40 was found to enhance dopamine synthesis in primary neurons. Pharmacological experiments show that Erk and protein kinase A phosphorylate Ser-31 and Ser-40, respectively, and that their inhibition blocked both TH phosphorylation and activity. Our results indicate that, in addition to its role as a survival factor for dopaminergic neurons, GDNF can directly increase dopamine synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dopamine, a neurotransmitter of the mesostriatal, mesolimbic, and mesocortical neural projections, regulates various neurological functions including memory, attention, motivation, reward, and motor control. Alterations in the levels of this neurotransmitter have been linked to pathological conditions such as Parkinson's disease, schizophrenia, psychosis, drug dependence, dementia, and attention deficit. Glial cell line-derived neurotrophic factor (GDNF)1 is a potent survival factor for dopamine neurons and is necessary for differentiation and maintenance of this phenotype. GDNF administration protects dopaminergic neurons from neurotoxin- and axotomy-induced death. These beneficial effects of GDNF have led to the suggestion that this trophic factor could be used as a therapeutic agent for the treatment of Parkinson's disease (1).

In addition to preventing the death of dopaminergic neurons, several studies have reported that GDNF can enhance dopamine levels and increase the quantal size of small synaptic vesicles in dopaminergic neurons (2, 3). One possible mechanism for the increases in dopamine levels and quantal size is stimulation of tyrosine hydroxylase (EC 1.14.16.2 [EC] , tyrosine 3-monooxygenase; L-tyrosine tetrahydropteridine:oxidoreductase (3-hydroxylating); TH) activity. TH is the rate-limiting enzyme in the biosynthesis of dopamine, and therefore, the activity of this enzyme is likely to be a key determinant of dopamine levels. Although there have been reports of higher TH levels and more TH-positive neurons in physically or chemically lesioned animals treated with GDNF, others (4-6) indicate that GDNF cannot reverse injury-induced decreases in TH levels. These apparently contradictory results are likely to be caused by the following two factors: 1) difficulty in distinguishing the effect of GDNF as a survival factor from its ability to modulate dopamine synthesis in these in vivo studies; and 2) TH protein levels may not directly reflect its activity and dopamine biosynthesis.

Although enhanced transcription and translation can increase TH protein levels, the enzymatic activity is regulated by phosphorylation of the protein (7). Phosphorylation of seryl residues (Ser-19, Ser-31, and Ser-40) has been observed both in vitro and in situ, and protein kinases that phosphorylate each of these sites have been identified in part (8). These studies report that phosphorylation at Ser-31 and Ser-40 correlates with stimulation of dopamine synthesis (9). However, the effect of GDNF on phosphorylation of TH and its enzymatic activity have not been examined. The present study uses a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuronal cultures in order to examine the effect of GDNF on TH phosphorylation. TH enzymatic activity and dopamine synthesis were measured to examine whether GDNF-mediated alterations in TH phosphorylation are accompanied by changes in enzymatic activity and dopamine synthesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—A BE(2)-C human neuroblastoma cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA). Human recombinant GDNF and rat recombinant GDNF were purchased from R&D Systems (Minneapolis, MN). Anti-synaptophysin polyclonal antibody and Alexa dye-conjugated secondary antibody were purchased from DakoCytomation (Carpinteria, CA) and Molecular Probes (Eugene, OR), respectively. Pan-specific anti-TH polyclonal antibody, phospho-specific antibody for rat TH (Ser-31 and Ser-40), and anti-NeuN monoclonal antibody were from Chemicon (Temecula, CA). Anti-phospho-Erk1/2 polyclonal antibody and LY294002 were from Cell Signaling Technology (Beverly, MA). Okadaic acid and PD098059 were from Calbiochem. 3,5-[3H]L-Tyrosine and [1-14C]L-tyrosine were obtained from Amersham Biosciences and Moravek Biochemicals (Brea, CA), respectively. (Rp)-cAMP was from Biomol%20Research%20Laboratories">Biomol Research Laboratories (Plymouth Meeting, PA). U0126, D, L-6-Met-5,6,7,8-tetrahydropterine, L-tyrosine, ascorbic acid, and catalase were purchased from Sigma. Culture media and fetal bovine serum were from Invitrogen.

BE(2)-C Cell Culture—Cells in culture were maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum, non-essential amino acids, and an antibiotics/antimycotics mixture (100 units/ml penicillin G, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B) in Falcon culture flasks (BD Biosciences). It has been reported that high serum concentration induces dopamine phenotype in these cells (10). After four to six passages in 10% serum, the serum concentration was raised to 20% for an additional two to four passages. Cells were then re-plated either in 35-mm Falcon culture dishes or in 24-well culture plates (BD Biosciences) at a density of 1 x 105/cm2 and treated with 10 µM all-trans-retinoic acid (RA) plus 20% fetal bovine serum for 6 days. Culture medium and RA were renewed every other day. Exposure to retinoic acid for 6 days resulted in differentiation of cells to a dopamine neuronal phenotype as determined by morphology and immunoreactivity for synaptophysin and TH. Differentiated cells were used for the TH phosphorylation and activity experiments.

Serum Deprivation and GDNF Treatment for BE(2)-C Cells—Human recombinant GDNF was reconstituted in 0.1% bovine serum albumin (BSA) in PBS (Invitrogen) at a concentration of 50 µg/ml as recommended by the manufacturer and stored at -80 °C until used. Cells were washed with DMEM/F-12 culture medium two times followed by incubation in DMEM/F-12 supplemented with non-essential amino acids and antibiotics/antimycotics mixture with or without 50 ng/ml GDNF. Control cells received an equivalent amount of BSA. GDNF was maintained throughout the incubation period. Cells were harvested either immediately, 10, 30, or 60 min, or 3 or 6 h following treatment and lysed in a buffer containing 10 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.5 µM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.1 µM okadaic acid. Extracts were snap-frozen and stored at -80 °C until needed. For the inhibition studies using the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (5 µM) or PD098059 (25 µM) or the phosphatidylinositol 3-kinase inhibitor LY294002 (20 µM), cells were preincubated in the culture medium containing an inhibitor at the indicated concentration for an hour before serum deprivation. Following the incubation, cells were washed with DMEM/F-12 followed by incubation in serum-deprived culture medium including the inhibitor in the presence or absence of GDNF for 60 min. Cells were lysed, and extracts were prepared as described above. Prior to use, the cell lysate was sonicated, and the amount of protein in each sample was measured by a Bradford assay using BSA as the standard.

Primary Mesencephalic Neuronal Culture—Primary neurons were cultured from E15 Sprague-Dawley rat embryos. The ventral mesencephalic tissue was dissected out in an ice-cold DMEM/F-12 culture medium followed by treatment with phosphate buffered-saline (PBS, pH 7.4) containing 0.05% trypsin and 0.53 mM EDTA (Invitrogen) at 37 °C for 15 min. The treatment was terminated by adding an equal volume of the culture medium supplemented with 10% fetal bovine serum. The tissue was resuspended in the culture medium and triturated using a 1-ml pipette tip 10 times followed by incubation on ice for 10 min. Suspended cells were removed, and clumped materials at the bottom of the tube were again triturated using a 200-µl pipette tip 10 times and kept on ice for additional 10 min. Suspended cells were collected and combined, followed by centrifugation at 250 x g. Cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum in poly-L-lysine-coated 35-mm culture dish or 24-well plate (Falcon) at a density of 3 x 105 cells/cm2 for 36 h. The cells were treated with 5 µM cytosine 1-{beta}-D-arabinofuranoside (Sigma) for 24 h and switched to the serum-free culture medium containing neurobasal medium supplemented with B27 component, 2 mM GlutaMax-1, and the antibiotics/antimycotics mixture (Invitrogen). Cells were maintained in the same medium for an additional 10 days prior to use. Rat GDNF was added to the culture medium at a concentration of 50 ng/ml, and the cells were incubated for 30 min unless otherwise described. Addition of protein kinase A inhibitor (Rp)-cAMP (50 µM) or U0126 (5 or 50 µM) to cultured neurons, cell lysis, and sample preparations were carried out as described above for BE(2)-C cells.

Immunohistochemical Staining—For the immunohistochemical staining, cells were washed briefly in PBS and fixed with 4% paraformaldehyde in PBS for an hour at 4 °C. Following fixation, cells were washed in 10 mM Tris-buffered saline, pH 7.4 (TBS), and permeabilized with ice-cold methanol for 5 min followed by blocking with 5% normal goat serum for an hour at room temperature. Cells were incubated with Pan-specific anti-TH polyclonal antibody (1:5000 dilution), anti-synaptophysin polyclonal antibody (1:5000 dilution), or anti-NeuN monoclonal antibody (1:2000 dilution) in TBS at 4 °C. Following overnight incubations, cells were washed three times in TBS and incubated with an Alexa 488-conjugated anti-rabbit IgG or Alexa568-conjugated anti-mouse IgG as suggested by the vendor. Immunoreactivity was detected using a Leica DMIRB microscope, and images were adjusted for size and labeled using Adobe Photoshop 6.0.

Production of Phosphorylation-specific Antibodies and Western Blotting—The following phosphorylated peptides (corresponding to the bovine TH sequence) were synthesized and used for immunizing rabbits: phospho-Ser-31, QAEAIMpSPRF (identical to human TH-1 isoform, where pS is phosphoserine); phospho-Ser-40, GRRQpSLIQDAR (human TH-1 sequence GRRQSLIEDAR). Antibodies were sequentially purified first by using phosphopeptide affinity columns, followed by removal of any residual non-phosphorylation-specific antibodies by using non-phosphopeptide columns. Non-phosphorylated specific antibodies for Ser-31 and Ser-40 were produced by immunizing rabbits with the appropriate peptides and then purified using non-phosphopeptide affinity chromatography followed by adsorption against a phosphopeptide column to remove cross-reacting antibodies. The affinity-purified antibodies were characterized by Western blotting. Samples were resolved in SDS-PAGE and transferred to an Immobilon-P (Millipore, Bedford, MA) membrane by using a semi-dry transfer apparatus (Millipore). Membranes were blocked overnight in TBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) plus 5% BSA and incubated with a primary antibody (1:1000-2500 dilution for phospho- and non-phospho-TH antibodies, 1:50,000 for Pan-specific TH antibody, or 1:2000 for phospho-Erk1/2 antibody) for 3 h at room temperature. The migration of TH was confirmed using a Pan-specific polyclonal antibody, which recognized both the phosphorylated and unphosphorylated forms of TH (Chemicon). In addition, commercially available phosphorylation-specific antibodies for Ser-31 or Ser-40 (Chemicon), which recognize rat TH, were also used to corroborate immunoreactivity in rat neuronal culture. Following incubation with the primary antibody, membranes were washed three times for 20 min each in TBST. Immunoreactivity was assessed by an alkaline phosphatase-conjugated secondary antibody and a CDP-star chemiluminescent substrate (Cell Signaling Technology). The optical density of the immunoreactive bands was measured utilizing ImageQuant band-analysis software (Amersham Biosciences). Prior to reprobing, blots were stripped by two 10-min washes in 50 mM NaOH at room temperature. The membrane was then washed extensively with TBST and reblocked for an hour in 2% BSA prior to immunodetection.

TH Activity and Dopamine Synthesis Assays—TH catalyzes the hydroxylation of tyrosine to generate 3,4-dihydroxy-L-phenylalanine (L-DOPA) and water using DL-6-Met-5,6,7,8-tetrahydropterine as a cofactor. TH activity was measured by quantifying tritiated water production from 3,5-[3H]L-tyrosine (water assay), as described previously by Levine et al. (11), with minor modifications. 35 µl of cell lysates was added to an equal volume of assay mixtures to yield a final reaction mixture containing 150 mM Tris malate buffer, pH 6.4, 0.35 µCi of 3,5-[3H]L-tyrosine, 50 µM L-tyrosine, 5 mM ascorbic acid, 3 mM DL-6-Met-5,6,7,8-tetrahydropterine, and 1500 units of catalase. After an incubation of 10 min at 37 °C, the reaction was stopped by cooling the samples on ice, followed by addition of 700 µl of 7.5% activated charcoal in 1 N HCl. The samples were then centrifuged, and the aqueous phase was recovered and mixed with 4 ml of Universol (ICN Pharmaceuticals, Costa Mesa, CA) liquid scintillation fluid. Radioactivity was counted in a liquid scintillation analyzer. Blank values were obtained from identically prepared samples that did not contain cell lysate. The assays were performed in duplicate.

An assay to measure dopamine synthesis, which monitors 14CO2 production following the conversion of [1-14C]-L-tyrosine to dopamine, was performed as described previously by Salvatore et al. (12) (CO2 assay). BE(2)-C cells were cultured in 24-well plates. Following treatment, cells in each well were equilibrated in 200 µl of HEPES-buffered, pH 7.4 (15 mM HEPES, 150 mM NaCl, 1.5 mM CaCl2, 0.5 mM EGTA, 0.5 mM ascorbic acid, 5.6 mM glucose, 1 mM MgCl2, and 1.9 mM K2HPO4), for 3 min at 37 °C. Each well was fitted with a section of Tygon tubing to enable collection of 14CO2 generated during the enzymatic reaction. Ten µl of HEPES-buffered saline containing 0.1 µCi of [1-14C]L-tyrosine was added in each well and incubated for 10 min at 37 °C. At the end of the incubation, 200 µl of 20% trichloroacetic acid was added to terminate the reaction, and a rubber stopper fitted with a suspended plastic well containing Whatman-3 filter paper saturated with Soluen-350 (Packard, Meriden, CT) was placed into the tubing. After allowing absorption of the generated 14CO2 for 2 h, the filter paper was transferred to 4 ml of Universol liquid scintillation fluid. Radioactivity was counted as described above. Blank values were obtained from identically prepared samples that did not contain cells. The assays were performed in duplicate.

Statistical Analysis—Statistical significance was determined by a repeated measures analysis of variance followed by post-hoc analysis. Data were considered significant at p <= 0.05. Statistical analysis was performed using either the integrated optical densities (Western blot) or scintillation counts (enzyme activity assay).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Retinoic Acid (RA) Treatment Induces Neuronal Morphology and Increases the Expression of Synaptophysin and TH in BE(2)-C Human Neuroblastoma Cells—RA exposure has been shown to cause differentiation of BE(2)-C cells (10, 13), resulting in dopamine-like neurons. BE(2)-C cells were treated with 10 µM all-trans-retinoic acid, and morphological changes were examined using phase-contrast microscopy as well as by immunohistochemical staining for TH and synaptophysin, a synaptic-vesicle protein (Fig. 1a). Cells not exposed to RA possess short neurites and show weak synaptophysin and TH immunoreactivity (Fig. 1a). Differentiation by RA is associated with cessation of proliferation and extensive branching of the neuronal processes. By 6 days post-RA exposure, the expression of synaptophysin and TH are markedly increased (Fig. 1a). We next examined if this increase in TH immunoreactivity is accompanied by enhanced TH activity and dopamine synthesis. TH activity using cell extracts and dopamine synthesis in intact cells were measured as described under "Experimental Procedures." Fig. 1b shows that RA treatment significantly increases TH activity as compared with the untreated cells. In contrast, measurement of dopamine synthesis by monitoring 14CO2 production did not show any detectable synthesis in either untreated or RA-treated cells (data not shown). This could be due to the absence of L-aromatic amino acid decarboxylase activity, which catalyzes the conversion of L-DOPA to dopamine in these cells. It has been reported that other neuroblastoma cell lines also lack this enzyme and do not synthesize dopamine (14, 35). Dopamine inhibits its synthesis by directly binding to TH, and this binding of dopamine is blocked when TH is phosphorylated on Ser-40. Thus a lack of dopamine synthesis in BE(2)-C cells minimizes the involvement of Ser-40 phosphorylation in regulating TH activity. These cells are therefore useful in isolating the contribution of Ser-31 phosphorylation to TH activity.



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  		FIG. 1.
Differentiation of BE(2)-C human neuroblastoma cells and characterization of TH antibodies. a, morphology of the cells before and after the treatment with all-trans-retinoic acid (RA) for 6 days as seen by using a phase-contrast microscope. RA treatment increased the number and length of processes and the immunoreactivities for Pan-specific TH and synaptophysin. b, RA treatment for 6 days (n = 3) increased TH activity as assessed by using the water assay. *, p <= 0.05. c, immunoblotting of bacterially expressed bovine TH protein using Pan-, phosphorylation-, and non-phosphorylation-specific TH antibodies. The phosphorylation-specific antibodies did not bind to bacterially expressed TH. d, immunoblotting for Pan-specific and phosphorylation-specific TH antibodies. 1st lane, bovine adrenal gland; 2nd lane, BE(2)-C; 3rd lane, Jurkat T-cell; and 4th lane, pre-absorbed phosphorylation-specific antibody.

 
Characterization of TH Antibodies—Phosphorylation- and non-phosphorylation-specific antibodies were generated to examine phosphorylation changes in response to treatments. The specificities of the phospho-specific antibodies were evaluated using bacterially expressed bovine TH and by Western blot analysis. Bacterially expressed proteins are not phosphorylated as bacteria lack protein kinases required to phosphorylate TH (15). Fig. 1c shows that the Pan-specific TH antibody detects bacterially expressed TH. As anticipated, the phosphorylation-specific antibodies for Ser-40 or Ser-31 did not detect bacterially expressed TH. In contrast, the non-phosphorylation-specific antibodies for Ser-40 and Ser-31 reacted with the TH protein. When BE(2)-C cell extracts were used, both phospho-specific antibodies detected a band slightly larger than the band detected in bovine adrenal gland protein extracts (Fig. 1d). This observation is consistent with the reported size difference between human and bovine TH (16, 17). The identity of this band was confirmed by reprobing the membranes with a Pan-specific TH antibody (Fig. 1d, right). Neither the Pan-specific nor the phospho-specific antibodies cross-reacted with extracts from Jurkat T-cells, which do not express TH. Furthermore, preincubation of each of the phospho-specific antibodies with 20-fold molar excess of the phosphopeptide used for immunization blocked the corresponding immunoreactivity (Fig. 1d, 4th lane). The linear range for TH immunoreactivity in Western blots was determined for all antibodies by using increasing amounts of protein samples. The immunoreactivity was found to increase in a linear manner with amounts of total protein ranging from 5 to 62 µg (data not shown). Subsequent experiments were performed using 10-30 µg of total proteins.

GDNF Treatment Does Not Alter TH Levels—Although previous studies have shown that GDNF is a survival factor for dopamine neurons (4, 18), it is not clear if GDNF alters TH protein levels. GDNF binds to two receptors (GFR{alpha}1 and GFR{alpha}2) that recruit the Ret tyrosine kinase to the lipid raft (19). This results in the phosphorylation of Ret at multiple tyrosine residues that serve as docking sites for intracellular signaling molecules (20, 21). By using mRNA prepared from both undifferentiated and differentiated cells, we tested if these cells express GDNF receptors by PCR. The mRNA for ret and gfr{alpha}2, but not for gfr{alpha}1, are present in both differentiated and undifferentiated BE(2)-C cells (data not shown).

To examine whether exposure to GDNF changes TH protein levels, BE(2)-C cell extracts were prepared at different time points following serum deprivation and GDNF exposure, and were analyzed by Western blotting using the Pan-specific TH antibody. The optical density of the immunoreactive band at each time point was normalized with respect to the zero time point and expressed as fold change. A representative Western blot and the summary data compiled from three independent experiments are shown in Fig. 2a. The figure shows that Pan-TH immunoreactivity does not significantly change as a result of serum deprivation or GDNF treatment at any of the time points examined.



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  		FIG. 2.
GDNF exposure alters TH phosphorylation in BE(2)-C cells. Optical density of immunoreactive bands at each time point is plotted as fold change (mean ± S.E.) compared with the density at zero time point (n = 3). Pictures of representative Western blots are shown. a, Pan-specific (total) TH; b, TH phospho-Ser-40; c, TH nonphospho-Ser-40; d, TH phospho-Ser-31; and e, TH nonphospho-Ser-31. *, p <= 0.05 between GDNF-treated and -untreated groups; +, p <= 0.05 compared with zero time point controls.

 
GDNF Treatment Increases Ser-31 and Ser-40 Phosphorylation—The protein samples used to examine Pan-TH immunoreactivity in Fig. 2a were analyzed for changes in TH phosphorylation. Fig. 2b shows representative Western blots and summary data indicating that serum deprivation did not alter phospho-Ser-40 immunoreactivity at any of the time points examined. In contrast, GDNF treatment significantly increased the phosphorylation of Ser-40 as early as 30 min compared with both the serum-deprived group (*) and the zero time point samples (+) used as controls. This increase in phospho-Ser-40 immunoreactivity was detected for up to 3 h post-GDNF application (Fig. 2b). Changes in Ser-40 phosphorylation were further examined by re-probing the membranes with Ser-40 non-phosphorylation-specific antibodies. The immunoreactivity of non-phospho-Ser-40 did not change as a result of serum deprivation but was significantly decreased as a result of GDNF treatment for up to 60 min (Fig. 2c). Consistent with the observed increases in immunoreactivity by using the phospho-Ser-40 antibody, the immunoreactivity using the non-phospho-Ser-40 shows a corresponding decrease at the time points examined.

Fig. 2d shows that serum deprivation increased phospho-Ser-31 immunoreactivity at the 30- and 60-min time points. GDNF treatment significantly augmented phospho-Ser-31 immunoreactivity with changes detected as early as 10 min post-GDNF application and lasting for up to 3 h. A corresponding decrease in non-phospho-Ser-31 immunoreactivity was detected as a result of GDNF exposure, beginning as early as 10 min post-treatment and returning to control values by 3 h (Fig. 2e).

Erk Activity Is Required for Ser-31, but Not Ser-40, Phosphorylation—Previous studies have shown that phosphorylation of TH on Ser-31 enhances enzymatic activity by increasing the Vmax of the enzyme. In contrast, phosphorylation of Ser-40 increases TH activity by increasing the rate of dissociation of inhibitory catecholamines from the enzyme (12, 30). Furthermore, in the absence of catecholamines, Ser-40 phosphorylation does not increase TH activity (23). It has been reported (12, 22) that Erk can phosphorylate Ser-31. Therefore, we examined if the increases in Ser-31 phosphorylation are because of GDNF-mediated Erk activation. A representative Western blot and the summary data compiled from three independent experiments showing the temporal change in phospho-Erk immunoreactivity are shown in Fig. 3a. The phosphorylation of Erk reaches a maximum within 10 min and returns to control values by 6 h following both serum deprivation and/or GDNF exposure. The temporal profile of Erk activation is consistent with it being the Ser-31 phosphorylating kinase. To examine if Ser-31 and Erk phosphorylation co-vary at different concentrations of GDNF, a dose-response study was carried out. Phospho-Ser-31 and phospho-Erk immunoreactivities showed parallel increases (subtracting changes caused by serum deprivation) in a dose-dependent manner up to 100 ng/ml GDNF (Fig. 3b).



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  		FIG. 3.
Increases in Ser-31 phosphorylation in response to GDNF exposure is caused by enhanced Erk activity in BE(2)-C cells. a, GDNF treatment significantly increases the immunoreactivity for phospho-Erk1/2 (n = 3). b, phospho-Ser-31 and phospho-Erk immunoreactivities are enhanced with increasing concentrations of GDNF (n = 2). Immunoreactivity signals obtained from untreated samples were used to calculate the fold-changes at each GDNF concentration. c, effect of MEK inhibitors on phospho-Ser-31, phospho-Erk, Pan-TH, and phospho-Ser-40 immunoreactivities (n = 3). d, effect of LY294002 on phospho-Ser-31 and phospho-Erk immunoreactivities (n = 3). Control samples were obtained from cells not treated with either GDNF or MEK inhibitors. *, p <= 0.05 between GDNF-treated and -untreated groups; +, p <= 0.05 compared with zero time point controls.

 
To evaluate if a causal link exists between Erk activity and Ser-31 phosphorylation, the MEK inhibitors U0126 and PD098059 were used. Fig. 3c shows that both U0126 and PD098059 blocked the serum-deprived and GDNF-induced increases in phospho-Erk immunoreactivity. This was accompanied by a corresponding decrease in phospho-Ser-31 immunoreactivity (Fig. 3c). Re-probing of these membranes with the Pan-specific TH antibody indicated that the decrease in phospho-Ser-31 immunoreactivity was not because of an overall change in the protein levels. To determine whether PD098059 decreases Ser-40 phosphorylation, membranes were re-probed with antibodies for phospho-Ser-40. PD098059 did not alter the immunoreactivity of phospho-Ser-40 (Fig. 3c). In addition to stimulation of the Erk cascade, the binding of GDNF to its receptors activates phosphoinositide 3,4,5-kinase (24-27). To examine if the phosphoinositide 3,4,5-kinase cascade contributes to Ser-31 phosphorylation, LY294002 was utilized. Fig. 3d shows that LY294002 did not alter phospho-Erk or phospho-Ser-31 immunoreactivities in either serum-deprived or GDNF-treated cells.

GDNF Treatment Increases TH Activity—To examine if GDNF-induced changes in TH phosphorylation in BE(2)-C cells resulted in altered enzymatic activity, in vitro assays were performed by using cell lysates. Fig. 4 shows activity measurements using the water assay. A linear increase in TH activity was observed with increasing amounts of total protein for up to 35 µg (Fig. 4a). Twenty micrograms of total protein was used in the subsequent experiments. Serum deprivation modestly increased TH activity at the 60-min time point (Fig. 4b). Consistent with the effect of GDNF on Ser-31 phosphorylation, TH activity was significantly augmented at the 60-min time point compared with the serum-deprived control. To examine if the inhibition of Ser-31 phosphorylation by the MEK inhibitor blocks this increase in TH activity, cells were treated with GDNF for 30 min in the presence or absence of 5 µM U0126. GDNF-mediated increases in TH activity were completely blocked in the presence of U0126 (Fig. 4c).



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  		FIG. 4.
Measurements of TH enzymatic activity in BE(2)-C cells by using the water assay. a, radioactive water production increases in a linear fashion with respect to protein amount in the assay mixture (n = 2). b, TH activity increases with GDNF treatment (n = 3). TH activity was plotted as fold changes compared with the zero time point controls. *, p < 0.05 between GDNF-treated and -untreated groups; +, p < 0.05 compared with the zero time point controls. c, GDNF-mediated increases in TH activity are blocked by treatment with the Erk inhibitor U0126 (n = 3). Control samples were obtained from cells untreated with U0126 or GDNF. *, p < 0.05 between U0126-treated and -untreated groups; +, p < 0.05 between GDNF-treated and -untreated groups.

 
GDNF Increases TH Phosphorylation, but Not TH Protein Levels, in Primary Dopaminergic Cells—Because BE(2)-C cells do not synthesize dopamine, we used primary rat mesencephalic cell cultures to examine if the increased phosphorylation of TH in response to GDNF exposure leads to enhanced dopamine synthesis. To characterize the cell population, double immunostainings using a neuron-specific marker, NeuN, and Pan-TH antibodies were performed. Fig. 5a shows that ~95% of the cells in these cultures were NeuN-positive and that 2.43 ± 0.25% (mean ± S.E.) of neurons are TH-positive in these primary cultures. These cell cultures were exposed to GDNF, and cell extracts were prepared at different time points. The extracts were analyzed by Western blotting using the Pan-specific TH antibody. A representative Western blot and the summary data compiled from five independent experiments in Fig. 5b show that GDNF treatment did not change TH immunoreactivity at any of the time points examined.



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  		FIG. 5.
GDNF exposure increases TH phosphorylation, but not TH protein levels, in mesencephalic dopaminergic cells. a, pictures of cultured mesencephalic dopaminergic cells stained with the neuron-specific marker NeuN and NeuN + Pan-specific TH antibodies. GDNF treatment did not alter the immunoreactivity of Pan-specific TH (b), but significantly increased the immunoreactivities for TH phospho-Ser-31 (c) and TH phospho-Ser-40 (d). Optical density of immunoreactive bands at each time point is plotted as fold change (mean ± S.E.) compared with the zero time point (n = 5). Pictures of representative Western blots are shown. +, p <= 0.05 compared with zero time point controls. e, phospho-Ser-31 and phospho-Ser-40 immunoreactivities increase with increasing GDNF concentrations. The cells were treated with GDNF for 30 min (n = 2). The graph was plotted as fold changes of immunoreactivity compared with samples obtained from the untreated cells.

 
The protein samples used to examine Pan-specific TH immunoreactivity in Fig. 5b were further analyzed for TH phosphorylation. Fig. 5, c and d, shows representative Western blots and summary data for phospho-Ser-31 and phospho-Ser-40 immunoreactivity in response to GDNF application. The immunoreactivities for both phosphorylation sites were significantly increased within minutes and remained elevated at the 3-h time point compared with the GDNF-untreated controls (the 0 time points). The temporal change in phospho-Ser-31 and phospho-Ser-40 immunoreactivity was qualitatively similar for primary neurons when compared with GDNF-treated BE(2)-C cells. Fig. 5e shows dose-response graphs for phospho-Ser-31 and phospho-Ser-40 immunoreactivities in response to a 30-min GDNF exposure. Both phospho-Ser-31 and phospho-Ser-40 immunoreactivities showed comparable increases with increasing GDNF concentrations up to 50 ng/ml.

Erk Activity Is Required for Ser-31 and PKA for Ser-40 Phosphorylation in Primary Neuronal Cells—As shown in Fig. 3, the increases in Ser-31 phosphorylation are because of GDNF-mediated Erk activation in BE(2)-C cells. To examine if GDNF-mediated Ser-31 phosphorylation depends on Erk activation in dopaminergic cells, the cells were pretreated with MEK inhibitor U0126 for an hour, followed by the treatment with GDNF for 30 min. A representative Western blot and the summary data compiled from three independent experiments are shown in Fig. 6a. GDNF significantly enhanced the phospho-Ser-31 immunoreactivity in the absence of the inhibitor. The GDNF-mediated increases in both Ser-31 and Erk phosphorylations were inhibited by 5 µM U0126 (Fig. 6a, left and center). Re-probing of the membrane with the phospho-Ser-40 antibody followed by the Pan-specific TH antibody indicated that U0126 did not affect Ser-40 phosphorylation or TH protein levels (Fig. 6a, right). As previous studies have shown that protein kinase A (PKA) can phosphorylate Ser-40, we examined if GDNF-mediated Ser-40 phosphorylation is altered by the PKA inhibitor (Rp)-cAMP. Phospho-Ser-40 immunoreactivity was significantly decreased by 50 µM (Rp)-cAMP (Fig. 6b, left). The membranes used for the Western blotting of phospho-Ser-40 were reprobed for phospho-Ser-31, showing that phospho-Ser-31 level was not affected by (Rp)-cAMP (Fig. 6b, right).



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  		FIG. 6.
Increases in Ser-31 and Ser-40 phosphorylation in response to GDNF exposure is mediated via Erk and PKA, respectively, in dopaminergic neurons. Optical density of immunoreactive bands in each time point is plotted as fold change (mean ± S.E.) compared with the zero time point (n = 5). Pictures of representative blots are shown in the upper rows. a, U0126 treatment blocked the GDNF-mediated increases in phospho-Ser-31 and phospho-Erk, but not phospho-Ser-40, immunoreactivities. b, (Rp)-cAMP treatment reduced the GDNF-mediated increase in phospho-Ser-40, but not phospho-Ser-31, immunoreactivity. *, p <= 0.05 between inhibitor-treated and -untreated groups; +, p <= 0.05 between GDNF-treated and -untreated groups.

 
GDNF Increases Dopamine Synthesis in Primary Neuronal Cells—Dopamine synthesis in mesencephalic neurons was measured using the CO2 assay. Fig. 7a shows a dose-dependent increase in CO2 release in response to exposure with different concentrations of GDNF for 30 min. To examine the effect of MEK and PKA inhibitors on GDNF-mediated increases in dopamine synthesis, cells were preincubated with U0126 and/or (Rp)-cAMP for an hour followed by treatment with 50 ng/ml GDNF for 30 min, and CO2 assay was performed. Fig. 7b shows that GDNF increases dopamine synthesis by ~3-fold. (Rp)-cAMP treatment modestly decreased this GDNF-mediated enhanced dopamine synthesis. In contrast, U0126 significantly inhibited dopamine synthesis at two different concentrations. However, the degree of inhibition observed was similar at these two concentrations. Interestingly, treatment of cells with both U0126 and (Rp)-cAMP completely blocked GDNF-stimulated dopamine synthesis.



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  		FIG. 7.
Measurement of dopamine synthesis in mesencephalic cell cultures using the CO2 assay. Results are plotted as the mean ± S.E. a, dopamine synthesis, as measured by radioactive CO2 production, is enhanced with increasing concentrations of GDNF. Cells were treated with GDNF for 30 min (n = 3 per concentration). b, inhibition of Erk activity by UO126 significantly reduced the GDNF-mediated increase in dopamine synthesis. The combination of (Rp)-cAMP and U0126 blocked the GDNF effect on dopamine synthesis, resulting in CO2 production comparable with untreated controls (n = 3). *, p <= 0.05 between inhibitor-treated and -untreated groups; +, p <= 0.05 between GDNF-treated and -untreated groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The study of the effect of GDNF on dopaminergic cells has included the investigation of its ability to maintain the survival of these neurons, and the usefulness of this factor as a potential therapy for diseases of the central nervous system (28). In addition to effects directly attributable to the survival of dopaminergic cells, results that appear to be related to altered dopamine biosynthesis (e.g. increases in quantal size) have been reported (2). It has been hypothesized that increases in the level of TH, the rate-limiting enzyme in the synthesis of dopamine, may underlie these effects, although experiments investigating the levels of TH following GDNF exposure have yielded contradicting results (6). As TH activity is modulated by changes in its phosphorylation state (7), we investigated the effect of GDNF on the phosphorylation of two key seryl residues. In this report, we have examined the effect of GDNF on TH phosphorylation and activity by using a human neuroblastoma cell line and rat primary mesencephalic neuronal cultures. Exposure of these cells to GDNF increased TH activity and in the primary neurons dopamine synthesis. This enhancement of activity was primarily caused by increased phosphorylation of TH on Ser-31.

Although previous studies have reported increased phosphorylation of TH on three seryl residues (Ser-19, Ser-31 and Ser-40) in response to treatments of cells, contribution of phosphorylation at each of these sites to TH activity remains unclear. Ser-19 phosphorylation does not appear to influence TH activity in vitro (29). Phosphorylation of Ser-31 and Ser-40 has been reported to increase TH activity via different mechanisms. For example, Ser-31 phosphorylation enhances enzymatic activity by increasing the Vmax of the enzyme (22), whereas phosphorylation of Ser-40 is thought to enhance the rate of dissociation of catecholamines that suppresses TH activity (30). Erk1/2 are the only protein kinases, to date, that have been shown to phosphorylate Ser-31 in situ both in response to depolarization and nerve growth factor treatment (12, 22, 36). In this report, we present data to show that exposure of BE(2)-C cells to GDNF caused a rapid increase in Erk phosphorylation that lasted for up to 3 h. The phosphorylation of both Erk and Ser-31 was significantly reduced by pretreatment with two different MEK inhibitors, suggesting that GDNF-induced Erk activity results in enhanced Ser-31 phosphorylation leading to increased TH activity.

As observed by using BE(2)-C cells, GDNF increased Ser-31 phosphorylation in primary neuron cultures with comparable temporal changes (Fig. 5c). GDNF treatment also increased Erk phosphorylation in these cells. The increased phosphorylation of both Erk and Ser-31 was blocked by the MEK inhibitor U0126. In contrast, the PKA inhibitor (Rp)-cAMP did not block GDNF-stimulated Ser-31 phosphorylation but decreased Ser-40 phosphorylation. These results are consistent with previous studies indicating that Ser-31 is primarily phosphorylated by Erk in situ.

Phosphorylation of Ser-40 increases TH activity, possibly via a catecholamine dissociation. Consistent with this, mutation of Ser-40 to either leucine or tyrosine decreases the affinity of TH for dopamine (31). Several protein kinases including PKA, protein kinase C, cyclic GMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase II, and mitogen-activated protein kinase-activated protein kinase 2 have been shown to phosphorylate this site. Our results show that GDNF exposure increases the phosphorylation of Ser-40 at time points when increased TH activity was detected in both BE(2)-C cells and primary neurons. Dopamine synthesis assay in BE(2)-C cells did not yield any detectable activity at any of the time points or conditions examined. As many neuroblastoma cell lines are known to lack L-aromatic amino acid decarboxylase activity (14), which catalyzes the conversion of L-DOPA to dopamine, this would result in an overall failure of the assay. In the absence of dopamine production, the feedback inhibition of TH would not occur in BE(2)-C cells. Thus the increase in Ser-40 phosphorylation we observed is unlikely to have contributed to the increases in TH enzymatic activity detected, leaving us to conclude that the increases in TH activity in response to GDNF application is most likely because of the increases in Ser-31 phosphorylation in BE(2)-C cells. Consistent with this, inhibition of Ser-31 phosphorylation by a MEK inhibitor completely blocked GDNF-induced increase in TH activity measured in the water assay.

In mesencephalic neuronal cells, Ser-40 is also increased in response to GDNF exposure. The PKA inhibitor (Rp)-cAMP decreased the phosphorylation of Ser-40 without changing the Ser-31 phosphorylation. This indicates that GDNF-mediated activation of PKA is primarily responsible for Ser-40 phosphorylation. At present, it is not known how GDNF can stimulate synthesis of cAMP and activity of PKA. One possibility is that binding of GDNF to GFR{alpha}-Ret receptor stimulates phospholipase C{gamma} activity leading to increases in intracellular calcium (32). Increases in intracellular calcium can stimulate the activity of calcium-sensitive adenylyl cyclase and cAMP levels (33).

Unlike the BE(2)-C cells, mesencephalic neurons show increased dopamine synthesis in response to GDNF exposure. A 30-min exposure to GDNF increased dopamine synthesis by ~3-fold. Both 5 and 50 µM U0126 inhibited dopamine synthesis to the same degree. When a combination of U0126 and (Rp)-cAMP was utilized, a complete blockade of GDNF-mediated enhanced dopamine synthesis was observed, returning the activity to the basal levels. In addition to a direct activation of TH via increased phosphorylation, GDNF can indirectly increase TH activity. For example, it has been reported that GDNF increases GTP-cyclohydrolase I activity (a key enzyme in BH4 synthesis) and BH4 levels in primary dopamine neurons (34). GTP-cyclohydrolase I activation was observed at the 24 and 48 h, but not at 6 h, following GDNF treatment. This suggests that increases in GTP-cyclohydrolase I and BH4 levels can contribute to TH activity at longer time points and may not have contributed to the changes in TH activity observed in the present study.

Can chronic GDNF administration elevate dopamine levels? It has been reported that chronic GDNF treatment decreases TH protein levels (6). In our findings, TH activity is initially increased followed by a steady decline over time. The mechanism(s) for this decline in activity over time in the continued presence of GDNF is not known at present. Several mechanisms are plausible including receptor desensitization and activation of a protein phosphatase. Our preliminary examination shows that chronic treatment with GDNF causes a decrease in Ser-19 phosphorylation (data not shown), consistent with a phosphatase activation. In addition to the effect of GDNF on TH phosphorylation and dopamine synthesis, it also acts as a potent survival factor and causes collateral sprouting of dopamine cells. Even if chronic GDNF treatments may not increase TH activity, its effect on dopamine cell survival and collateral sprouting may result in an overall increase in dopamine levels. This, however, remains to be demonstrated in animal models.

In conclusion, the present study shows that in addition to its survival effects, GDNF can increase the activity of the rate-limiting enzyme of dopamine biosynthesis and suggests that impaired dopaminergic function can be acutely altered by GDNF treatment. Future studies will examine whether administration of GDNF can alter the phosphorylation and activity of TH in vivo and offer beneficial effects for neurological dysfunctions associated with decreased dopamine levels.


   FOOTNOTES
 
* This work was supported by Grant NS35457 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Neurobiology and Anatomy, University of Texas Medical School, P. O. Box 20708, Houston, TX 77255. Tel.: 713-500-5575; Fax: 713-500-0621; E-mail: p.dash{at}uth.tmc.edu.

1 The abbreviations used are: GDNF, glial cell line-derived neurotrophic factor; TH, tyrosine hydroxylase; Erk, extracellular signal-regulated kinase; PKA, protein kinase A; MEK, mitogen-activated protein kinase/Erk kinase; L-DOPA, 3,4-dihydroxy-L-phenylalanine; RA, all-trans-retinoic acid; TBS, Tris-buffered saline; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Pan, pantothenate. Back


   ACKNOWLEDGMENTS
 
We thank Anthony Moore and John Redell for their invaluable assistance and comments on the manuscript.



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 DISCUSSION
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