From the Laboratoire de Génétique
Moleculaire de la Neurotransmission et des Processus
Neurodégénératifs, Hôpital de la
Pitié Salpêtrière, 75013 Paris, France,
¶ Department of Biochemistry and Molecular Biology, University of
Bergen, 5009 Bergen, Norway, and the
Laboratoire de Biologie
de l'Ecole Supérieure de Physique et Chimie Industrielles,
75231 Paris, France
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ABSTRACT |
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Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.
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INTRODUCTION |
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Tyrosine hydroxylase (TH,1 tyrosine 3-monooxygenase, EC 1.14.16.2) catalyzes the formation of L-3,4-dihydroxyphenylalanine (L-dopa) from L-tyrosine in central and peripheral neurons and in adrenal medulla chromaffin cells (1). This reaction is the rate-limiting step in the synthesis of catecholamines which are both neurotransmitters and hormones. Full-length cDNA for TH has been obtained for various animal species as follows: rat (2), mouse (3), cow (4, 5), Drosophila (6), macaque monkey (7), and human (8, 9). In human, four types of mRNA are produced in different tissues by alternative splicing of a single primary transcript (8-10). hTH1 mRNA is similar to rat TH. hTH2 and hTH3 species differ from hTH1 by the insertion of 12 and 81 nucleotides, respectively, between the 90th and 91st residue. Both the 12 and 81 nucleotide insertions are found in hTH4. The four isoforms of hTH are differently distributed among tissues. Brain and adrenal medulla contain mainly the hTH1 and hTH2 isoforms. Detectable amounts of hTH3 and hTH4 are found in human pheochromocytoma tumors.
The active site of TH lies in the C-terminal domain where the physiological co-substrate (6R)-tetrahydrobiopterin (BH4) binds. The enzyme requires Fe(II) and O2 for the hydroxylation reaction. The molecular weight of the purified active TH indicates that it is a tetramer. A leucine zipper motif in the C terminus of TH might contribute to tetramerization (11). The N terminus acts as a regulatory domain. It contains four serines that can be phosphorylated by several kinases including cyclic AMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase (CaM-PKII), and protein kinase C (12-18). The different phosphorylation states of TH have different catalytic behaviors.
The four isoforms of hTH have been produced in frog oocytes (19) and appear to have different and specific enzymatic properties. To delineate their characteristics, TH isoforms have been produced in bacteria and purified to homogeneity. The activities and effects of phosphorylation by PKA and CaM-PKII have been described for hTH1 and hTH2 (20). Sutherland et al. (21) recently studied the activities of the four human TH following phosphorylation by mitogen-activated protein kinases. We here report further studies of the hTH3 isoform in its unphosphorylated state and following phosphorylation by PKA and CaM-PKII. We determined the initial velocity conditions of the enzyme and established that the co-substrate BH4 exerts an inhibitory effect and that the L-dopa synthesis activity of hTH3 is not regulated by phosphorylation. The particular properties of hTH3 may be relevant to diseases affecting dopaminergic cells.
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EXPERIMENTAL PROCEDURES |
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Construction of a Tyrosine Hydroxylase Type 3 Expression Vector-- To construct the hTH3 pET expression vector, a DNA fragment containing the nucleotide sequence of the exons 1a and 2 and 84 base pairs of the exon 3 of human TH gene, was synthesized by polymerase chain reaction. hTH3 cDNA inserted into pSPT18 (19) was used as the template. The amplified fragment was inserted between the NdeI and NotI restriction sites of hTH1 cDNA which was in turn inserted into the expression vector pET-3a (20). The pET-hTH3 construction was sequenced before its introduction into Escherichia coli BL21(DE3) (22, 23).
Large Scale Purification of Recombinant Human Tyrosine Hydroxylase Type 3-- To ensure that the recombinant hTH3 isoform is correctly expressed in E. coli BL21 (DE3), a Western blot experiment was performed using a monoclonal anti-TH antibody (Boehringer Mannheim) at a final dilution of 1:1,000. After washing in 0.2% Tween 20 in phosphate-buffered saline, bound antibody was detected by labeling with 125I-protein A and autoradiography (data not shown).
hTH3 was then purified on a large scale from E. coli by a slight modification of a previously published procedure (24). In brief, the following modifications were used. The bacterial lysate from 36 liters of culture was applied to a DEAE-Sepharose column. After washing the column with 20 mM Tris maleate, pH 7.0, the protein was eluted onto a heparin-Sepharose column using 0.3 M Tris-HCl in 20 mM Tris maleate, pH 7.0. The protein was eluted from the heparin-Sepharose column with 0.3 M KCl in 20 mM Tris maleate and, after concentration, was subjected to Sephacryl S-300 chromatography. The protein preparations had an estimated purity of 80% as assessed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined according to the method of Bradford (25) using bovine serum albumin as the standard. The molecular weight of the purified hTH3 was determined by electrospray mass spectrometry. Five micrograms of protein in 10 µl of a solution of acetonitrile/water/formic acid (50/50/0.2%) was injected at 2 µl/min into a Platform mass spectrometer (Fisons Instruments, Manchester, UK) previously calibrated with myoglobin.Tyrosine Hydroxylase Enzymatic Activity-- TH activity was assayed as described previously (26) with some modifications. We determined the initial velocity conditions by working with different quantities of purified recombinant hTH3, as given in Fig. 3. Kinetic assays were carried out at 30 °C in a total volume of 100 µl containing 100 mM Na-Hepes buffer, pH 6.9, 0.2 µCi of [3H]tyrosine, 20 µM L-tyrosine (Sigma), 0.5 mg/ml catalase (Sigma), 0.1 mM FeSO4, and BH4 (Schircks Laboratory, Jona, Switzerland) as specified in the figure legends.
Phosphorylation of Human Tyrosine Hydroxylase--
Purified TH3
and TH1 (1 µg) were phosphorylated by either PKA or CaM-PKII. The
conditions for the phosphorylation reaction (total volume 10 µl) were
as follows: 20 mM Na-Hepes, pH 6.9, 10 mM
MgCl2, 5 mM 2-mercaptoethanol, 0.1 mg/ml
ovalbumin, 0.1 mM ATP, and [-32P]ATP (1300 cpm/pmol). TH was phosphorylated by CaM-PKII in the same assay mix,
except that 1 mM CaCl2 and 30 µg/ml
calmodulin were also included. Purified protein kinases were used in
the following amounts: 120 units of the catalytic subunit of PKA (from bovine heart, Sigma) and 45 units of the CaM-PKII (from rat brain, generously provided by Dr. J. A. Girault, CNRS-Collège de
France, Paris). To evaluate the ratio of phosphate incorporation, the reactions were stopped after 10 min at 30 °C by the addition of 1.0 ml of 25% (w/v) trichloroacetic acid and left to stand for 10 min. The
suspensions were then filtered through GF/C glass filters (Whatman),
and the filters were washed with 10% (w/v) trichloroacetic acid to
remove ATP, and radioactivity was measured. Phosphorylation
stoichiometries were calculated using a subunit mass of 60 kDa.
Isolation and Analysis of Tryptic Peptides-- The PKA or CaM-PKII phosphorylation reaction medium containing hTH (180 µg) was desalted by reverse phase high pressure liquid chromatography using an RP300 C8 column. After phosphorylation by CaM-PKII, the eluted peak containing radioactive material was composed of labeled phosphorylated hTH and of calmodulin-protein kinase II which co-elutes because the molecular mass of its subunit is similar to that of the hTH (58-60 kDa). The eluted phosphorylated protein was digested at 30 °C for 20 h with 3 µg of trypsin in a total volume of 200 µl, containing 0.1 M Tris, pH 8, 0.2 M guanidinium chloride, and 2 mM calcium chloride. Tryptic peptides were purified by high pressure liquid chromatography on a Vydac C18 column (2.1 mm internal diameter, 15 cm length). Phosphorylated hTH tryptic peptides were localized by monitoring the 32P radioactivity in Tcherenkov mode, using an on-line radiomatic flow beta detector (the cell detection volume was 25 µl) coupled with an UV 441 Waters detector at 214 nm. Peptides were eluted with a 0.32% per min linear gradient of acetonitrile in buffer A (0.1% trifluoroacetic acid in water) and buffer B (0.9% trifluoroacetic acid in 80% acetonitrile in water), at a flow rate of 200 µl/min. The labeled phosphorylated tryptic fragments of TH were identified by comparison of their elution time with the elution time of synthetic phosphorylated peptides used as markers. The synthetic peptides (Altergen) used as standards had the same amino acid sequences as three tryptic fragments of hTH as follows: peptide 1, AVS(PO3H2)ELDAK; peptide 2, RQS(PO3H2)LIEDAR; peptide 3, GQS(PO3H2)PR. Peptides 1-3 contain the unlabeled phosphorylated serine residues 19, 40, and 31, respectively (numbers correspond to positions in the hTH1 sequence).
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RESULTS |
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Purification Analysis of hTH3 Recombinant
hTH3 was produced in the E. coli Bl 21(DE3) expression system. The recombinant protein was purified to homogeneity by chromatography on DEAE-Sepharose, heparin-Sepharose, and Sephacryl S-300. The fraction recovered contained an active enzyme with an apparent molecular mass of 240 kDa, consistent with the eluted protein being in tetrameric form. The apparent molecular mass as determined by SDS-polyacrylamide gel electrophoresis was 60 kDa (Fig. 1). The precise mass, determined by mass spectroscopy, was 58082.4 Da. The theoretical mass of TH is 58212.9 Da. The mass difference is equivalent to the average mass of a methionine residue (131.2 Da). Thus, the purified recombinant hTH3 contains the entire deduced amino acid sequence other than, presumably, the N-terminal methionine.
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PKA and CaM-PKII Phosphorylation Sites in hTH3
The bacterial expression system produces an almost iron-free (24) and nonphosphorylated enzyme. The effect of phosphorylation on the enzymatic activity of the protein can therefore be easily tested in vitro. We first determined the stoichiometry of the phosphorylation reaction. Ratios of 0.8 and 1.7 mol of phosphate per mol of hTH3 phosphorylated subunit were obtained with PKA and CaM-PKII, respectively. Thus, hTH3 is phosphorylated at one site by PKA and two sites by CaM-PKII.
To localize the phosphorylation sites, labeled hTH3 was digested with trypsin, and the tryptic fragments were analyzed by high pressure liquid chromatography. Synthetic phosphorylated tryptic peptides of hTH were used as markers. These peptides corresponded to regions containing either Ser19, Ser31, or Ser40 as described under "Experimental Procedures." The radioactivity was recovered in a single phosphopeptide fraction which had a retention time identical to that of synthetic phosphopeptide containing the Ser40 (Fig. 2A). PKA thus phosphorylates the Ser40 of hTH3 as in hTH1. Phosphorylation of hTH3 and hTH1 by CaM-PKII was also investigated. Both hTH1 (data not shown) and hTH3 were found to be phosphorylated on Ser19 and Ser40 (Fig. 2B), as previously demonstrated for hTH1 (20). In conclusion, hTH3 has the same phosphorylation sites as hTH1 for PKA and CaM-PKII, and therefore the 27 amino acid insertion between Met30 and Ser31 does not create a new phosphorylation site for either PKA or CaM-PKII.
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Kinetic Parameters of hTH3 and hTH1 with the BH4 Co-substrate
We determined the kinetic properties of nonphosphorylated hTH3 and hTH3 phosphorylated by PKA and CaM-PKII and compared them to those of hTH1.
Determination of Initial Velocity Conditions-- Various experimental conditions were tested to determine the initial velocity of the hydroxylation reaction for hTH3 (Fig. 3A). The same determination was done for hTH1. For both enzymes the following conditions were deduced to be optimal whether or not the enzymes were phosphorylated by PKA or CaM-PKII (Fig. 3B): 10 ng of hTH, 20 µM L-tyrosine, pH 6.9, incubation time 15 min, 30 °C. These initial velocity conditions were valid over the range of BH4 concentrations from 5 to 500 µM, independent of the phosphorylation state of the enzyme.
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Enzymatic Activity of Phosphorylated and Unphosphorylated hTH3-- The activity of hTH3 was measured under the initial velocity conditions with various BH4 concentrations from 5 to 80 µM. The resulting Lineweaver-Burk plots (Fig. 4A) are typical of a substrate excess inhibition as described by Dixon and Webb (27). The curve can be subdivided into two parts: at low co-substrate concentrations the curve is similar to the classic Michaelis-Menten curve; at higher BH4 concentrations the curve reflects inhibition by excess of substrate. According to the typical analysis of substrate excess inhibition kinetics described by Axelrod (28) and Dixon and Webb (27), the inhibition constant (Ki) was assessed by plotting 1/V versus BH4 concentrations (Fig. 4B). The experimental results were fitted to the following equation: V = Vmax × [BH4]/(Km + [BH4] + ([BH4]2/Ki)) to determine the apparent Michaelis constant (Km) and the maximal velocity (Vmax). Kinetic data were then analyzed by nonlinear regression using the Grafit computer package.
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Catalytic Constant of hTH3-- Experimental determination of kcat implies maximal velocity conditions. Due to substrate excess inhibition we have determined the BH4 concentration yielding to the experimental maximum of velocity. The corresponding BH4 concentrations for the enzyme were found to be 40 and 30 µM BH4 for PKA-phosphorylated and unphosphorylated hTH3, respectively. The kinetics of dopa production by hTH3 were studied under these two conditions, and the corresponding velocities were used to compute the kcat (Table II). The kcat values were similar for PKA-phosphorylated and unphosphorylated hTH3. Consequently, phosphorylation did not modify the enzyme activity, and the increase in Ki is presumably compensated exactly by the increase in Km.
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Enzymatic Activity of hTH1 Phosphorylated or Not by PKA-- Under initial velocity conditions, hTH1 was subject to co-substrate excess inhibition (Fig. 4 and Table I), but this inhibition was less pronounced for hTH1 than for hTH3 (higher Ki). The Km of BH4 for hTH1 was 3.6-fold higher than that of hTH3. PKA phosphorylation of hTH1, unlike that of hTH3, did not modify its Ki value for the co-substrate but, in contrast, decreased its Km by 2.7-fold without changing the Vmax.
Thus, the phosphorylation of hTH1 by PKA activates the enzyme by decreasing its Km of BH4, whereas the other kinetic parameters remain constant (Ki and Vmax values). To compare the activities of hTH3 and hTH1, it was necessary to determine the hTH1 kcat, since Km, Vmax, and Ki differ between the two isoforms and have opposite effects on the activity. The kcat values for PKA-phosphorylated and unphosphorylated hTH1 (Table II) confirm the activation of the enzyme following phosphorylation and indicate that hTH3 is enzymatically more active than hTH1. ![]() |
DISCUSSION |
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In human, alternative splicing of exons 1 and 2 of the TH primary transcript results in the production of four isoenzymes. A further hTH mRNA species resulting from the skipping of exon 3 has been observed recently (29). We compared the enzymatic characteristics of the hTH3 isoform with those of hTH1, before and following phosphorylation by PKA and CaM-PKII. hTH3 differs from hTH1 by an additional 27 amino acids in the N terminus. The enzymatic analyses were rigorously performed with enzymes purified from recombinant bacteria. The purified enzymes were thus devoid of phosphate moieties and free of co-factor and catecholamines, which affect enzymatic characteristic determinations. To determine the genuine kinetic parameters of the enzymes, experiments were performed under initial velocity conditions.
One major finding of this work is that the co-substrate BH4 exerts an inhibitory effect on the activity of both hTH3 and hTH1 when in excess (Fig. 4). Earlier studies have shown that TH activity is inhibited by its tyrosine substrate (30) and by its catecholamine end products such as dopamine (31). Inhibition by BH4 had not previously been detected (32), most likely because kinetic studies were not performed under initial velocity conditions. The Ki values for BH4 were 24.7 and 53.0 µM for hTH3 and hTH1, respectively. The physiological concentration of BH4 has been estimated to be in the range of 1 to 13 µM in catecholamine tissues of various species (33-35). These data suggest that in vivo the activity of hTH3 and hTH1 is optimal. Given this inhibition effect of BH4, the activity of the enzymes is best represented by the kcat value (see Table II). We show that the activity of hTH3 is over 5 times that of hTH1. This represents the first determination of the true kinetic characteristics of the activity of TH enzyme variants.
Another important finding was that the phosphorylation of hTH3 by PKA does not modify its kinetics (Tables I and II). The hTH variants contain four serines, at positions 8, 19, 31, and 40 in their N-terminal regulatory domain. In hTH3 only Ser40 is phosphorylated by PKA, whereas Ser19 and Ser40 are phosphorylated by CaM-PKII. Ser40 in hTH1 has been previously found to be phosphorylated by these two kinases (20). Although the same site is phosphorylated by PKA and CaM-PKII, the effects of these kinases on hTH3 and hTH1 enzyme activity are quite different. Consistent with previous findings, hTH1 activity was doubled under initial velocity conditions by phosphorylation at Ser40 by PKA (Table II). In sharp contrast, the phosphorylation at Ser40 by PKA had no effect on the enzymatic activity of hTH3 for BH4. The activity of hTH3 is nevertheless twice that of the phosphorylated form of hTH1. Several studies on the regulation of the activity of purified native rat TH (which is the TH most similar to hTH1) suggest that when the enzyme is tightly bound in a dopamine-iron complex, it may be in a state of low basal activity. The major effect of phosphorylation may thus be the activation of TH by favoring dopamine release, which reduces the inhibitory effects of catecholamines (36-38). Recombinant hTH3 bound in a complex with dopamine and Fe(III) may be regulated by phosphorylation by PKA. Although it would be of interest to investigate this issue, the main point of our study is that it demonstrates a major difference between the effects of phosphorylation on hTH3 and hTH1. In the absence of catecholamine, hTH3 but not hTH1 escapes regulation by PKA phosphorylation.
The phosphorylation by CaM-PKII had no effect on the activity of hTH3 (Table I). An absence of regulation by CaM-PKII has previously been reported for rat TH. Furthermore, CaM-PKII phosphorylation appears to have no direct effect on the properties of the TH enzyme unless a 14.3.3-activator protein is present to activate the phosphorylated TH (39, 40). This activator protein, which has been purified and its gene isolated (41, 42), is a very abundant protein found throughout the brain (43). Although the role of the 14.3.3-activator protein is uncertain, it is possible that it does not activate TH directly but rather stabilizes the Ser19-phosphorylated enzyme (21).
The behavior of hTH3 may be due to a specific conformation associated with the presence of an addition of 27 amino acids in the N terminus of the enzyme. Martinez et al. (44) have shown that the activation of hTH1 by the phosphorylation on Ser40 was associated with a change in the conformation of the enzyme. Moreover, in its activated state, the enzyme appears to be less prone to bind dopamine, explaining why the activity is higher. It could be that the presence in hTH3 of the 27 amino acid fragment with hydrophobic residues could cause the enzyme to adopt a different conformational structure, similar to that generated by the addition of a phosphate group at position Ser40 in hTH1. This induced conformation would then be insensitive to activity regulation.
The effect of an insertion on the enzymatic characteristics of TH has also been observed in the Drosophila tyrosine hydroxylase (DTH). Birman et al. (6) have shown that two isoforms of DTH are produced following alternative splicing of a single DTH primary transcript. The DTH type I is an isoform predominantly produced in the central nervous system. The DTH type II, which contains an acidic extension of 71 amino acids at the N terminus not present in the DTH type I, is mostly produced in the hypoderm, where the dopamine synthesized is not a neurotransmitter. The two DTH isoenzymes have different properties possibly due to differences in activity regulation. The authors suggest that the group of negative charges at the N terminus of DTH type II could have the same effects as certain polyanions such as heparin. Thus, an acidic regulatory domain seems to be a characteristic of the regulation of TH activity.
It is striking that both in human and Drosophila, the same mechanism of alternative RNA splicing leads to insertion in the N-terminal regulatory domain of TH. The resulting modification of enzymatic properties is likely to be due to specific conformational changes, although hydrophobic segment is added in human TH and an acidic one in Drosophila TH. Further studies are required to elucidate the details of the mechanism of the activity changes in hTH3 and DTH types I and II. Secondary structure analysis as described by Martinez et al. (44) would be valuable.
In summary, the two main enzymatic characteristics we report are first that hTH3 is a very active enzyme. The dopa synthesis activity, in absence of end product, is not regulated by phosphorylation by PKA. Second, hTH3 activity is inhibited by excess BH4 (the Ki of BH4 is 24 µM). These particular enzymatic properties may be relevant to pathological situations. Although the hTH3 isoform seems not to be abundant in catecholamine cells (45), its high enzymatic activity even at low BH4 concentrations may safeguard a minimum level of dopamine synthesis in catecholaminergic neurons and could alleviate some diseases.
There are also therapeutic implications arising from this study of hTH3. One way to restore the level of dopamine in the striatum would be to generate ectopic synthesis of L-dopa and dopamine using gene therapy approaches. We have recently shown that dopamine can be produced by direct gene transfer to the brain using a recombinant adenovirus encoding tyrosine hydroxylase (46). The local expression of an hTH1 transgene in the striatum leads to a functional recovery in an animal model of Parkinson's disease. The production of the hTH3 isoform would be more appropriate because it is the most active isoform and would therefore require least virus to be introduced into the brain.
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ACKNOWLEDGEMENTS |
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We are grateful to Jean Antoine Girault for providing purified calmodulin-dependent protein kinase. We also thank Annie Lamouroux and Bernard Guibert for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by grants from the CNRS, the INSERM, the EEC Science Programme, and Rhône-Poulenc Rorer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Received a fellowship from l'Institut de Recherche sur la Moelle Epinière.
** To whom correspondence should be addressed: L.G.N.-Bâtiment CERVI, Hôpital de la Pitié Salpêtrière, 83-Bd de l'Hôpital, 75013 Paris, France.
1 The abbreviations used are: TH, tyrosine hydroxylase; hTH, human TH; PKA, cAMP-dependent protein kinase; CaM-PKII, calmodulin-dependent protein kinase II; BH4, (6R)-tetrahydrobiopterin; DTH, Drosophila tyrosine hydroxylase.
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REFERENCES |
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