Enhancement of Tyrosine Hydroxylase Phosphorylation and Activity by Glial Cell Line-derived Neurotrophic Factor
2004; Elsevier BV; Volume: 279; Issue: 3 Linguagem: Inglês
10.1074/jbc.m310734200
ISSN1083-351X
AutoresNobuhide Kobori, Jack C. Waymire, John W. Haycock, Guy L. Clifton, Pramod K. Dash,
Tópico(s)Nuclear Receptors and Signaling
ResumoAlthough 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. 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. 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) 1The 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. 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.Nutt J.G. Burchiel K.J. Comella C.L. Jankovic J. Lang A.E. Laws Jr., E.R. Lozano A.M. Penn R.D. Simpson Jr., R.K. Stacy M. Wooten G.F. Neurology. 2003; 60: 69-73Crossref PubMed Scopus (726) Google Scholar). 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.Beck K.D. Irwin I. Valverde J. Brennan T.J. Langston J.W. Hefti F. Neuron. 1996; 16: 665-673Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 3.Pothos E.N. Davila V. Sulzer D. J. Neurosci. 1998; 18: 4106-4118Crossref PubMed Google Scholar). One possible mechanism for the increases in dopamine levels and quantal size is stimulation of tyrosine hydroxylase (EC 1.14.16.2, 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.Winkler C. Sauer H. Lee C.S. Bjorklund A. J. Neurosci. 1996; 16: 7206-7215Crossref PubMed Google Scholar, 5.Kearns C.M. Cass W.A. Smoot K. Kryscio R. Gash D.M. J. Neurosci. 1997; 17: 7111-7118Crossref PubMed Google Scholar, 6.Lu X. Hagg T. J. Comp. Neurol. 1997; 388: 484-494Crossref PubMed Scopus (78) Google Scholar) 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.Lew J.Y. Garcia-Espana A. Lee K.Y. Carr K.D. Goldstein M. Haycock J.W. Meller E. Mol. Pharmacol. 1999; 55: 202-209Crossref PubMed Scopus (44) Google Scholar). 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.Fitzpatrick P.F. Annu. Rev. Biochem. 1999; 68: 355-381Crossref PubMed Scopus (433) Google Scholar). These studies report that phosphorylation at Ser-31 and Ser-40 correlates with stimulation of dopamine synthesis (9.Lindgren N. Xu Z.Q. Herrera-Marschitz M. Haycock J. Hokfelt T. Fisone G. Eur. J. Neurosci. 2001; 13: 773-780Crossref PubMed Google Scholar). 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. 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 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.Ross R.A. Biedler J.L. Spengler B.A. Reis D.J. Cell. Mol. Neurobiol. 1981; 1: 301-311Crossref PubMed Scopus (66) Google Scholar). 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 × 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 × 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 × 105 cells/cm2 for 36 h. The cells were treated with 5 μm cytosine 1-β-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.Levine R.A. Pollard H.B. Kuhn D.M. Anal. Biochem. 1984; 143: 205-208Crossref PubMed Scopus (22) Google Scholar), 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.Salvatore M.F. Waymire J.C. Haycock J.W. J. Neurochem. 2001; 79: 349-360Crossref PubMed Scopus (91) Google Scholar) (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). 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.Ross R.A. Biedler J.L. Spengler B.A. Reis D.J. Cell. Mol. Neurobiol. 1981; 1: 301-311Crossref PubMed Scopus (66) Google Scholar, 13.Bunone G. Borrello M.G. Picetti R. Bongarzone I. Peverali F.A. de Franciscis V. Della Valle G. Pierotti M.A. Exp. Cell Res. 1995; 217: 92-99Crossref PubMed Scopus (51) Google Scholar), 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.Ikeda H. Pastuszko A. Ikegaki N. Kennett R.H. Wilson D.F. Neurochem. Res. 1994; 19: 1487-1494Crossref PubMed Scopus (12) Google Scholar, 35.Waymire J.C. Gilmer-Waymire K. J. Neurochem. 1978; 31: 693-699Crossref PubMed Scopus (25) Google Scholar). 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. 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.Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar). 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.Grima B. Lamouroux A. Boni C. Julien J.F. Javoy-Agid F. Mallet J. Nature. 1987; 326: 707-711Crossref PubMed Scopus (308) Google Scholar, 17.Saadat S. Stehle A.D. Lamouroux A. Mallet J. Thoenen H. J. Neurochem. 1988; 51: 572-578Crossref PubMed Scopus (18) Google Scholar). 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.Winkler C. Sauer H. Lee C.S. Bjorklund A. J. Neurosci. 1996; 16: 7206-7215Crossref PubMed Google Scholar, 18.Beck K.D. Valverde J. Alexi T. Poulsen K. Moffat B. Vandlen R.A. Rosenthal A. Hefti F. Nature. 1995; 373: 339-341Crossref PubMed Scopus (619) Google Scholar), it is not clear if GDNF alters TH protein levels. GDNF binds to two receptors (GFRα1 and GFRα2) that recruit the Ret tyrosine kinase to the lipid raft (19.Airaksinen M.S. Titievsky A. Saarma M. Mol. Cell. Neurosci. 1999; 13: 313-325Crossref PubMed Scopus (388) Google Scholar). This results in the phosphorylation of Ret at multiple tyrosine residues that serve as docking sites for intracellular signaling molecules (20.Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 21.Saarma M. Trends Neurosci. 2001; 24: 427-429Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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 gfrα2, but not for gfrα1, 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. 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.Salvatore M.F. Waymire J.C. Haycock J.W. J. Neurochem. 2001; 79: 349-360Crossref PubMed Scopus (91) Google Scholar, 30.McCulloch R.I. Daubner S.C. Fitzpatrick P.F. Biochemistry. 2001; 40: 7273-7278Crossref PubMed Scopus (33) Google Scholar). Furthermore, in the absence of catecholamines, Ser-40 phosphorylation does not increase TH activity (23.Harada W.J. Haycock J.W. Goldstein M. J. Neurochem. (Tokyo). 1996; 67: 629-635Crossref PubMed Scopus (68) Google Scholar). It has been reported (12.Salvatore M.F. Waymire J.C. Haycock J.W. J. Neurochem. 2001; 79: 349-360Crossref PubMed Scopus (91) Google Scholar, 22.Haycock J.W. J. Neurosci. Methods. 2002; 116: 29-34Crossref PubMed Scopus (16) Google Scholar) 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 immunoreacti
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