Complex molecular regulation of tyrosine hydroxylase

Izel Tekin, Robert Roskoski, Nurgul Carkaci-Salli & Kent E. Vrana

Journal of Neural Transmission Translational Neuroscience, Neurology and Preclinical Neurological Studies, Psychiatry and Preclinical Psychiatric Studies

ISSN 0300-9564 Volume 121 Number 12

J Neural Transm (2014) 121:1451-1481 DOI 10.1007/s00702-014-1238-7

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1 23 Author's personal copy

J Neural Transm (2014) 121:1451–1481 DOI 10.1007/s00702-014-1238-7

TRANSLATIONAL NEUROSCIENCES - REVIEW ARTICLE

Complex molecular regulation of tyrosine hydroxylase

Izel Tekin • Robert Roskoski Jr. • Nurgul Carkaci-Salli • Kent E. Vrana

Received: 3 April 2014 / Accepted: 4 May 2014 / Published online: 28 May 2014 Ó Springer-Verlag Wien 2014

Abstract Tyrosine hydroxylase, the rate-limiting enzyme Introduction in catecholamine biosynthesis, is strictly controlled by several interrelated regulatory mechanisms. Enzyme syn- Tyrosine hydroxylase (TH) is an important component of thesis is controlled by epigenetic factors, transcription the central and the peripheral nervous systems. It is factors, and mRNA levels. Enzyme activity is regulated by essential for the production of three catecholamines end-product feedback inhibition. Phosphorylation of the (dopamine, norepinephrine, and epinephrine) whose func- enzyme is catalyzed by several protein kinases and tions include modulating autonomic reflexes, behavior, dephosphorylation is mediated by two protein phosphatases circulatory control, cognition, endocrine function, move- that establish a sensitive process for regulating enzyme ment, pain, reward, and vigilance. The catecholamines activity on a minute-to-minute basis. Interactions between function as neurotransmitters while norepinephrine and tyrosine hydroxylase and other proteins introduce addi- epinephrine, which are released from adrenal medullary tional layers to the already tightly controlled production of chromaffin cells, function as hormones. If one performs a catecholamines. Tyrosine hydroxylase degradation by the PubMed search using ‘‘tyrosine hydroxylase,’’ 19,972 ubiquitin–proteasome coupled pathway represents yet citations (as of 4/4/2014) are retrieved. These citations another mechanism of regulation. Here, we revisit the encompass disparate subjects including enzyme properties, myriad mechanisms that regulate tyrosine hydroxylase oxidative stress in neurodegenerative disorders, and the use expression and activity and highlight their physiological of this enzyme as a marker of catecholaminergic cells. As importance in the control of catecholamine biosynthesis. would be expected from its functional importance, TH is under strict regulation at several distinct and overlapping Keywords Alternative splicing Catecholamine levels. We have previously reviewed these mechanisms in biosynthesis Feedback inhibition Phosphorylation detail (Kumer and Vrana 1996). Moreover, selected aspects Promoter Transcription factor of this regulation have also been discussed by others (Dunkley et al. 2004; Haavik et al. 2008; Nakashima et al. 2009; Daubner et al. 2011; Lenartowski and Goc 2011). Here, however, we present an update of the intricate reg- I. Tekin and R. Roskoski Jr. have contributed equally to preparation ulation of TH that includes approximately 200 selected of this manuscript. articles since 1996 along with conclusions from several historically important papers. I. Tekin N. Carkaci-Salli K. E. Vrana (&) Department of Pharmacology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033-0850, USA Catecholamine biosynthesis and physiological relevance e-mail: [email protected] Tyrosine hydroxylase (EC 1.14.16.2) catalyzes the first R. Roskoski Jr. Blue Ridge Institute for Medical Research, Horse Shoe, NC, (Nagatsu et al. 1964) and rate-limiting step in catechol- USA amine biosynthesis (Levitt et al. 1965) (Fig. 1). Tyrosine 123 Author's personal copy

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reconverted to ascorbate in a complex process involving

cytosolic NADH, cytochrome b561, and intravesicular ascorbate free radical (Diliberto et al. 1991). S-adenosyl- methionine is the methyl donor in the reaction converting norepinephrine to epinephrine. The other product, S-adeno- sylhomocysteine, is reconverted to S-adenosylmethionine in a multistep process. The physiological importance of TH is exemplified by its role in development. Disruption of the tyrosine hydroxylase gene in transgenic mice results in mid-ges- tational lethality (Kobayashi et al. 1995; Zhou et al. 1995). Of the minority of embryos that survive, all exhibit stunted development and die within the first 5 weeks of life. In addition, more than 53 single nucle- otide polymorphisms (SNPs) in the coding region of human TH have been described (Haavik et al. 2008; Kobayashi and Nagatsu 2005; Rao et al. 2007; http:// www.ncbi.nlm.nih.gov/SNP/). Several of these SNPs are strongly associated with movement disorders and other conditions that arise from a deficiency in dopamine production, as will be discussed later. There are also numerous polymorphisms observed in the noncoding (intronic) regions of the TH gene. These polymorphisms will not be considered in this review primarily because of a lack of functional data. Tyrosine hydroxylase, phenylalanine hydroxylase, and tryptophan hydroxylase form a family of related enzymes called aromatic amino acid hydroxylases (Grenett et al. 1987). With few exceptions, the mechanism of action of these enzymes and the tertiary structure of the catalytic domains are nearly identical and they use the common co-

substrate tetrahydrobiopterin (BH4). Interestingly, genetic defects in BH4 biosynthesis manifest as a neurodevelop- mental disorder (Segawa disease) that can be managed, in Fig. 1 Complete reaction pathway for the biosynthesis of the part by administration of levodopa (the product of the TH catecholamines. The rate-limiting step is the conversion of tyrosine reaction). (Ichinose et al. 1999; Segawa 2011; Segawa to L-DOPA and is catalyzed by tyrosine hydroxylase. The regulatory events that affect TH are therefore thought to control the synthesis of et al. 2003). In spite of the existence of high sequence catecholamines. L-DOPA is then converted to dopamine by AADC, homology among these enzymes, their modes of regula- which then is converted to norepinephrine through the action of DBH. tion, however, differ (Fitzpatrick 1999). In select cells, the norepinephrine is converted to epinephrine by PNMT

Mechanism of the TH reaction hydroxylase mediates the reaction of tetrahydrobiopterin

(BH4), molecular oxygen, and tyrosine to form L-3,4-dihy- The reaction mechanism for the hydroxylation of tyrosine droxyphenylalanine (L-DOPA) and pterin-4a-carbinolamine has been studied extensively (Fitzpatrick 2003; Roberts (4a-OH–BH3). Aromatic amino acid decarboxylase, which and Fitzpatrick 2013). Fitzpatrick (1991) first determined is the second enzyme in the catecholamine biosynthetic the order of substrate binding to purified rat tyrosine pathway, catalyzes the decarboxylation of DOPA to form hydroxylase by steady-state enzyme kinetics; the pterin dopamine, the first of the neurotransmitters. Dopamine b- substrate (BH4) binds first, followed by oxygen in rapid hydroxylase catalyzes the conversion of DOPA to norepi- equilibrium, and then tyrosine. All three substrates must nephrine within vesicles in a reaction involving molecular bind before any chemical reaction occurs. Additionally, a oxygen and ascorbate; water and the ascorbate free radical dead-end enzyme-tyrosine complex can form resulting in are the other products. The ascorbate free radical is enzyme inhibition by high concentrations of tyrosine. 123 Author's personal copy

Regulation of tyrosine hydroxylase activity 1453

Tyrosine hydroxylase is able to catalyze the hydroxylation of phenylalanine and tryptophan in addition to tyrosine.

The specificity constant (Vmax/Km) of tyrosine hydroxylase for tyrosine is only 10-fold greater than that of phenylal- anine (Daubner et al. 2000) and 30-fold greater than that of tryptophan (Daubner et al. 2002). In contrast, the speci- ficity constant of phenylalanine hydroxylase for phenylal- anine is 105-fold greater than that for tyrosine (Daubner et al. 2000). These data indicate that TH can be somewhat promiscuous in its amino acid selection. Tyrosine hydroxylase contains a mononuclear non-heme iron atom. Ferrous iron, but not ferric iron, promotes enzyme activity (Dix et al. 1987; Fitzpatrick 1989; Haavik et al. 1988). The iron content of each enzyme subunit is between 0.4 and 1 atom/subunit of protein (Almas et al. 1992; Haavik et al. 1991). The iron atom is coordinated by Fig. 2 Overview of the different modes of TH regulation. The human His331, His336, and Glu376 in the rat enzyme (Goodwill TH gene contains 14 exons that are alternatively spliced (to exclude et al. 1997). Ramsey et al. (1995) reported that each of exon 2 [short isoforms] or include exon 2 [long isoforms]). TH gene expression can be regulated by different transcription factors, as well these residues is required for iron binding and for catalytic as changes in RNA half-life. Numerous single nucleotide polymor- activity. When the ferrous iron in TH undergoes oxidation phisms (SNPs) exist in the population that produces variant mRNAs to yield ferric iron, reduced BH4 converts the enzyme back and proteins. Once the transcript is translated, several post-transla- to the active, or ferrous, state (Ramsey et al. 1995). During tional modifications can occur to regulate catecholamine biosynthesis catalysis, a bridge is formed among pterin, iron, and oxy- gen, which results in reduction of molecular oxygen to a TH protein structure peroxy-pterin intermediate. This intermediate participates in the hydroxylation reaction as determined with the rat Native tyrosine hydroxylase exists as a tetramer with a recombinant enzyme (Chow et al. 2009). Dopamine, nor- molecular mass of &240 kDa (Kumer and Vrana 1996). In epinephrine, and epinephrine inhibit enzyme activity and it mouse and rat, each monomer is composed of 498 amino has been postulated that such feedback inhibition is phys- acids (Grima et al. 1985). The human enzyme consists of iologically important (Meyer-Klaucke et al. 1996; Nagatsu four isoforms that result from the alternative splicing of the et al. 1964; Okuno and Fujisawa 1985), a hypothesis that is primary RNA transcript although the primary form is widely accepted today. cognate to the rodent enzymes. Each TH monomer consists of three components (Fig. 3). The first 165 residues con- stitute a regulatory segment, the next 280 residues make up TH gene structure the catalytic domain, and the last 40 residues make up a tetramerization domain (Abate et al. 1988; Fitzpatrick The human TH gene contains 14 exons. Four different 2003; Liu and Vrana 1991; Lohse and Fitzpatrick 1993; human TH isoforms are formed as a result of the alternative Nakashima et al. 2009; Ota et al. 1995; Walker et al. 1994). splicing of the hnRNA (Fig. 2). The rat and mouse TH The X-ray crystal structure of the catalytic and tetra- gene contains 13 exons separated by 12 introns. In addi- merization domains of rat (PDB ID 1TOH and 2TOH; tion, there are two alternatively spliced isoforms in monkey Goodwill et al. 1997, 1998) and human (PDB ID 2XSN) that correspond to the most common human isoforms TH have been determined. The overall structure of each (hTH-1 and hTH-2; Ichinose et al. 1993). The TH pro- monomer is a basket-like arrangement of helices and loops moter, which is located upstream of the gene, contains with a long carboxyl-terminal a-helix that forms the core of binding sites for several transcription factors (reviewed in the tetramer (Fig. 3b). Kumer and Vrana 1996; Lenartowski and Goc 2011). The overall structure of each catalytic domain is a During development, TH expression is restricted to several basket-like arrangement of helices and loops with a long discrete components of the central and peripheral nervous C-terminal a-helix which forms the core of the tetramer systems and to adrenal chromaffin cells (Schimmel et al. (Fig. 3). Each monomer contains 16 a-helices and 12 b- 1999). The balanced production of the different transcrip- strands including those of the regulatory segment (2 a- tion factors mediates tissue-specific expression of the gene helices and 4 b-strands). The catalytic domain has a 30-A˚ (Tinti et al. 1996). wide and 17-A˚ deep active site within the basket-like

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Fig. 3 Molecular models prepared from the crystal structure of the colored coded to match the catalytic domains; the regulatory domains rat isoform of tyrosine hydroxylase. The catalytic domain of the are omitted for clarity. d A view directed into the active site of rat protein is about 50 % a-helix, 10 % b-sheet, and 40 % random coil monomeric tyrosine hydroxylase. The active site contains iron and a (based on X-ray crystallography). Crystal structures of the regulatory bound substrate analog (7,8-tetrahydrobiopterin, or BH4). His331, domain, however, are not available. a Linear structure of the His336, and Glu376, which bind iron, and the cofactors are shown as monomeric human tyrosine hydroxylase. The figure is prepared with stick representations. b–d are adapted from Zhang et al. and Jaffe DOG 2.0 software from NM_000360 (NCBI Nucleotide Database). et al. and BH2 of d is superposed from PDB ID 2TOH. Ct b Molecular model of tetrameric (I–IV) rat tyrosine hydroxylase carboxyterminus, Nt amino-terminus, TIZ tetramerization segment. showing two of the four regulatory segments. The other two The structures were prepared using the PyMOL Molecular Graphics regulatory domains, which are in back of the structure, are obscured. System Version 1.5.0.4 Schro¨dinger, LLC c Same view as b depicting the tetramerization domains that are arrangement. Site-directed mutagenesis studies have iden- PAH homolog of this loop changes when an amino acid is tified amino acid residues that are involved in crucial steps bound, suggesting that this loop plays a role in amino acid such as iron binding, feedback inhibition, tetramerization, binding. However, site-directed mutagenesis studies indi- and amino acid hydroxylation (Ellis et al. 2000; He et al. cate that specific residues in the loop fail to play a domi- 1996; Quinsey et al. 1996; Ribeiro et al. 1993; Vrana et al. nant role in determining the amino acid substrate 1994; Yohrling et al. 2000). We should note, however, that specificity of either TH or PAH. Sura et al. (2006) sug- crystallization studies have been unable to visualize the gested that pterin binding results in a conformational structure of the regulatory domain. This may indicate that change of this loop that supports formation of the amino this segment does not assume a stable conformation. acid binding site in TH. An iron atom is 10 A˚ below the Recently, a segment of the regulatory domain of rat TH has enzyme surface within the active site cleft. The iron atom is been determined via NMR studies (Zhang et al. 2014). coordinated by His331, His336, and Glu376. Each of these TH possesses a flexible loop (Fig. 3c) consisting of residues has been shown to be required for iron binding and residues 177–191 that may form part of the tyrosine- for activity by site-directed mutagenesis (Ramsey et al. binding site (Daubner et al. 2006). The conformation of the 1995). The crystal structure of TH with bound 7,8-

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Regulation of tyrosine hydroxylase activity 1455 dihydrobiopterin (PDB ID 2TOH) reveals no conforma- their tetramerization domains, and subunits I and IV have tional changes when compared with the resting enzyme no direct contact. Zhang et al. found that residues 1–72 of (PDB ID 1TOH) (Goodwill et al. 1997, 1998). Both of the amino-terminal regulatory segment (residues 1–159) these structures show a five-coordinate ferric iron site with were unstructured. They performed more extensive studies His 331 as the axial ligand and two water molecules joining on the regulatory segment consisting of residues 65–159. the protein ligands in the equatorial positions with the iron This segment forms a dimer in solution and contains an atom in the plane of the equatorial ligands. Several other ACT domain (named after aspartate kinase, chorismate prokaryotic and eukaryotic oxygen-utilizing enzymes pos- mutase and TyrA, or prephenate dehydrogenase), which sess this 2-His-1-carboxylate facial triad (Que 2000). The consists of 2 a-helices and 4 b-strands. The secondary facial triad binds iron in the active site and leaves the structure of the ACT domain consists of a b1-sheet, an a1- opposite face of the octahedron available to coordinate a helix, the b2- and b3-sheets, the a2-helix and finally the variety of exogenous ligands. The biopterin binds close to b4-sheet (Fig. 3d). Zhang et al. combined their structural iron; the 4a-carbon is 5.9 A˚ from it. 7,8-Dihydrobiopterin information with that of Jaffe et al. on phenylalanine binds to several residues in the peptide backbone of TH hydroxylase and arrived at the model shown in Fig. 3b, c through hydrogen bonding, while the only interaction with (Zhang et al. 2014; Jaffe et al. 2013). Two regulatory the side chain of an amino acid residue involves that of segments are in front of the complex (from subunits I and Glu322. IV) and two are in the back of the complex (II and III) that Zhang et al. (2014) compared the structure of the cannot be seen. phosphorylated and unphosphorylated regulatory segment X-ray studies identified hydrogen bonds and a salt by NMR. These peptides exhibited the same chemical bridge from Glu282 of one subunit to Lys170 in the shifts except for Gly36, Arg37, Gln39, Ser40, Leu41, Ile42, adjacent subunit as a dimerization interface between the and Glu43. The authors conclude that the core structure of two subunits. Point mutants of residues that form the salt the regulatory segment of TH remains the same as that in bridge result in a protein that migrates as a dimer, thus the unphosphorylated segment and a local structural indicating that this salt bridge plays an essential role in the change takes place around Ser40. At the present time there formation of a tetramer (Yohrling et al. 2000). Deletion of is no structural information on the catalytically relevant the carboxyl-terminal residues results in the formation of a ferrous form of resting TH or of TH with tyrosine or protein that migrates as a dimer or monomer, while the tyrosine plus pterin bound. It would be of value to deter- wild-type enzyme migrates as a tetramer (Lohse and Fitz- mine these structures and those of the unphosphorylated patrick 1993; Vrana et al. 1994; Yohrling et al. 2000). and Ser40-phosphorylated holoenzymes. Thus far struc- Although it is unclear whether the monomers directly form tural studies aimed at defining that the structures of a tetramer or they first form dimers that then come together unphosphorylated and phosphorylated TH holoenzymes to form a tetramer, the native enzyme exists as a tetramer. have been problematic. A point mutation (Leu435Ala) or deletion of multiple The tetramerization domain of tyrosine hydroxylase residues in the long carboxyl-terminal helix results in the consists of residues 457–498 and is formed by two b- formation of a protein dimer (Vrana et al. 1994; He et al. strands and a 40 A˚ -long a-helix (Goodwill et al. 1997, 1996). 1998) (Fig. 3b). Based on the primary structure of the aromatic amino acid hydroxylases, Liu and Vrana hypothesized that these enzymes assemble into tetramers Overview of TH regulation through the involvement of coiled-coil interactions of the carboxyl-terminal (Liu and Vrana 1991), which was then Owing to the physiological importance of the catechol- confirmed experimentally by deletion mutagenesis studies amine end products, TH is under stringent control at the (Lohse and Fitzpatrick 1993; Vrana et al. 1994). Later transcriptional, translational, and post-translational levels crystallization data confirmed this notion and demonstrated (Kumer and Vrana 1996) (Fig. 2). An important element that the 40-A˚ long a-helix forms a coiled-coil at the core of necessitating strict control of TH is the observation that the tetramer (Goodwill et al. 1997) as depicted in Fig. 3. catecholamines undergo reactions with molecular oxygen Deletion of the carboxyl-terminal 19 residues results in a to generate toxic catechol-quinones (Hastings and Zig- protein that migrates as a dimer while the wild-type mond 1994). These reactions occur at neutral pH, but not enzyme migrates as a tetramer (Yohrling et al. 2000). The under acidic conditions. Cells minimize the generation of tetramer consists of a dimer of dimers (I and II with III and these toxic metabolites by maintaining production of cat- IV). Subunits I and II interact via a salt bridge (Lys170– echolamines at near-optimal levels, by providing on- Glu282) and their tetramerization domains are in contact demand regulation of synthesis, and by storing them in (Fig. 3c). The only contact between subunits I and III is by acidic synaptic vesicles. TH gene expression is strictly 123 Author's personal copy

1456 I. Tekin et al. controlled by numerous promoter sequences that are loca- Tissue-specific and developmental regulation of TH ted upstream of the 50 transcription initiation site (Lenar- gene expression towski and Goc 2011). These elements mediate quantitative tissue-specific expression and maintenance of There is developing knowledge of the DNA elements and TH (Kessler et al. 2003). Alterations in TH mRNA stability regulatory proteins responsible for tissue-specific expres- and the existence of different TH mRNA transcripts that sion of TH. Transgenic mice containing the 9-kb 50- are produced by alternative hnRNA splicing provide upstream segment of the rat TH promoter fused to the b- additional avenues for regulation (Kumer and Vrana 1996). galactosidase reporter gene, but not mice bearing 0.15 or Four human TH mRNAs and proteins occur physio- 2.4 kb of 50 flanking sequence, are able to express the logically, and several others are observed under patholog- reporter at levels equivalent to endogenous TH in central ical conditions. Expression can also be regulated in a cell catecholaminergic cells (Min et al. 1994). The authors type-specific fashion by neuropeptides and by depolariza- concluded that the crucial catecholaminergic neuron-spe- tion (Zigmond 1998). TH regulation at the protein level is cific DNA elements reside between -9 and -2.4 kb of the mediated by phosphorylation, dephosphorylation, changes 50 flanking region of the TH gene. Moreover, stimuli that in enzyme stability, substrate inhibition, and feedback increase or reduce TH levels produce parallel changes in b- inhibition by catecholamines (Daubner et al. 2011; Daub- galactosidase expression in various brain regions in trans- ner and Piper 1995; Nakashima et al. 2002). Phosphory- genic mice bearing the 50 9-kb rat TH promoter region lation occurs at four different serine residues (Campbell indicating that this promoter sequence mediates cell type- et al. 1986), which activate the enzyme and alter its sta- specific regulation of reporter gene expression (Min et al. bility (Dunkley et al. 2004; Fujisawa and Okuno 2005; 1996). Martinez et al. 1996; Zigmond et al. 1989). These regula- tory mechanisms will each be discussed in the following Dopaminergic TH mRNA expression sections. Nuclear receptor related-1 (Nurr1) is an orphan nuclear receptor that is required for the development and mainte- Overview of transcriptional regulation of the TH gene nance of mouse midbrain dopaminergic neurons (Zetter- strom et al. 1997). Nurr-1 deficient mice fail to generate TH plays important physiological roles in both the central these midbrain neurons, are hypoactive, and die shortly and peripheral nervous systems and is under dynamic after birth. Moreover, Nurr-1 expression mediates dopa- developmental and environmental transcriptional control. minergic-specific TH gene expression. Satoh and Kuroda In the CNS, TH is expressed in dopaminergic cell-con- (2002) reported that Nurr1 interaction with the TH pro- taining areas including the substantia nigra and ventral moter is mediated by PKA and PKC in NTera2 human tegmental area in the midbrain, the diencephalon, the embryonic stem cells over-expressing the receptor protein. olfactory bulb, and retinal amacrine cells. TH is also In rats, Nurr1 can bind directly to the TH promoter at any expressed in adrenergic and noradrenergic cells found in of three sites that are located 1 kb upstream of the tran- the hypothalamus, medulla, and locus coeruleus (LC). scription start site, and it increases gene expression in TH expression occurs peripherally in sympathetic ganglia progenitor cells during their differentiation into midbrain andalsoinadrenalchromaffincells(KumerandVrana dopaminergic neurons (Sakurada et al. 1999; Kim et al. 1996). 2003). Schimmel et al. (1999) reported that about 4.5 kb of TH gene transcription, mRNA stability, and SNP vari- the rat TH promoter is required for TH expression during ants participate in the regulation of TH expression both development and that this region contains consensus temporally and spatially. These modalities of TH regula- sequences for neural restrictive silencer element (NRSE), tion have been reviewed in detail (Kumer and Vrana 1996; Nurr1, Pitx1/3 and Gli1/2. The rat promoter contains a Lenartowski and Goc 2011). The TH promoter contains Nurr1 response element (Iwawaki et al. 2000). Jacobsen numerous transcription factor-binding sites that permit et al. (2008) identified a point mutation in a Nurr1 ERK1/2 mRNA levels to be regulated by Ca2?, glucocorticoids, phosphorylation site (Ser125Cys) in a Parkinson’s disease neuronal activity, oxygen levels, and stress. Extracellular patient. They demonstrated that this mutation attenuated stimuli acting through several protein kinases mediate Nurr1-induced transcriptional activation in human neuro- transcriptional regulation of TH (Hebert et al. 2005; Seta blastoma cells (SK-N-AS). Following activation, they and Millhorn 2004; Suzuki et al. 2004). Stress increases found that ERK1/2 proteins enhance transcriptional acti- TH expression by increasing both TH gene transcription vation by wild-type Nurr1, but not the Ser125Cys mutant. and mRNA stability (Tank et al. 2008). Recent advances in A Pitx3 site is found 50 bp upstream of the transcription these regulatory mechanisms will be addressed in turn. initiation site in the rat TH promoter (Cazorla et al. 2000) 123 Author's personal copy

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Fig. 4 Schematic representation and overview of the regulation of the rat TH promoter. Some of the well- known stimuli and their corresponding sites on the promoter are depicted with the black arrows (GRE glucocorticoid response element, HRE hypoxia response element, E-box enhancer box, AP-1 activator protein-1, Egr/ Sp1 early growth response protein 1/steroidogenic factor 1, CRE1/2 cAMP response element 1/2, ERE estrogen- response element, CaRE calcium response element, pDSE partial dyad symmetry element)

(Fig. 4). The mouse TH promoter also contains a Pitx3 box element represses expression of the reporter gene to response element while lacking a consensus sequence for 25–33 % that of the differentiated cell. This corresponds to Nurr1 (Lebel et al. 2001). In mice, deletion of Pitx3 results the decrease in TH protein that occurs in undifferentiated in the loss of TH in midbrain dopaminergic neurons cells. Based upon immunoreactivity, Ghee et al. (1998) (Maxwell et al. 2005). Nurr1 and Pitx3 are both required reported that CRE-associated transcription factors exist and for the differentiation of mouse and human embryonic stem regulate TH expression in rat midbrain dopaminergic cells, cells into dopaminergic neurons (Martinat et al. 2006). but that these cells lack Fos. In contrast, Fos expression in However, there was no difference in the levels of TH the olfactory bulb parallels TH expression while CRE- expression in human and mouse embryonic stem cells associated transcription factors remain constant. They when these transcription factors are expressed alone or in conclude that Fos and CREB make differential contribu- combination (Messmer et al. 2007). Jacobs et al. (2009) tions to TH gene activity in different cells. suggested that Nurr1 is bound by a repressor protein and upon interaction with Pitx3, Nurr1 is released, allowing it Noradrenergic and adrenergic TH mRNA expression to act on the TH promoter. In addition to Pitx3, Brn4 can also work in concert with Nurr1 to mediate dopaminergic Although many studies on the regulation of TH expression neuronal differentiation (Tan et al. 2011). Stott et al. (2013) have focused on dopaminergic cells, owing to their reported that Foxa1 and Foxa2 transcription factors are importance in the pathogenesis of Parkinson’s disease, an required for the development and maintenance of the extensive literature also exists on the regulation of TH in dopaminergic phenotype in mouse. Deletion of Foxa1 and adrenergic and noradrenergic phenotypes, mainly focusing Foxa2 significantly reduces the number of TH positive on the peripheral nervous system development (Apostolova dopaminergic neurons, which they report is due to a and Dechant 2009; Ernsberger 2001). reduction in the binding of Nurr1 to the promoter region of The regulation of the rat promoter in vivo depends on TH. the duration and nature of a stimulus. Sun et al. (2003) Lazaroff et al. (1998) compared the expression of a reported that chronic nicotine administration (14 days), transiently transfected chloramphenicol acetyltransferase acting through cholinergic receptors, results in a sustained (CAT) reporter gene under the transcriptional control of the increase in rat TH mRNA, protein, and activity in the TH 50 flanking DNA in undifferentiated and differentiated adrenal medulla. A sustained transcriptional rate that lasted mouse CNS CAD (Cath.a-differentiated) cells and found 1 week following chronic nicotine treatment correlated that TH expression varied as a function of their differen- with a modest increase in adrenal TH gene AP-1 binding, tiated state. They reported that the cAMP response element but not in the levels of Fra-2 or other Fos or Jun proteins. A (CRE) and the AP-1 site participate in TH expression in single nicotine injection elicited only a small and transient both states. In undifferentiated cells, however, the dyad/E- increase in TH mRNA, but not protein. Sun et al. (2004)

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1458 I. Tekin et al. found that chronic nicotine treatment induced TH mRNA Interestingly, studies with the rat promoter display a and protein in rat LC neurons. These increases lasted for cooperation between different regulatory sequences. 3 days in LC cell bodies, but for at least 1 week in LC However, it should be noted that there are differences in nerve terminals. In contrast to the adrenal medulla, these the types of promoter elements utilized by the rodent and investigators found no sustained transcriptional response in human promoter, as will be discussed below. the LC and suggested that post-transcriptional mechanisms may play a role in this long-term response. Ca2? and cAMP The response to acute and chronic stimuli may be mediated through the differential activation of several Nankova et al. (1996) measured the induction of the bac- transcription factors. For example, Sabban et al. (2004) terial CAT reporter fused to wild-type or mutant 50 flanking reported that the AP-1 factor Fra-2 increases in the rat sequences of the rat TH gene in rat PC12 cells in response adrenal medulla (but not in LC) after 6 days of repeated to the Ca2? ionophore, ionomycin. Point mutations in CRE immobilization stress. However, Fos increases in both the abolished the ionomycin-induced reporter gene induction adrenal medulla and LC following acute immobilization that was not overcome by an intact AP-1 site. These stress. The changes of Fos and Fra-2 observed in this study, investigators found that ionomycin rapidly increased the but not in response to chronic nicotine treatment (Sun et al. phosphorylation of the CREB transcription factor. KN62, 2003), most likely reflect the complexity of the immobili- which is a CaMPK inhibitor, prevented the ionomycin zation stress paradigm. In the same study by Sun et al., induction of the reporter gene. These investigators con- both acute and chronic immobilization stresses increase cluded that Ca2? activation of the rat promoter is mediated early growth response protein 1 (Egr1) in the medulla, but mainly through CaMPKII and CREB phosphorylation. not in the LC. Furthermore, short- and long-term immo- Nagamoto-Combs et al. (1997) studied constructs of the bilization increases CREB phosphorylation in the LC. In CAT reporter gene expressed in rat PC12 cells in response studies from the same laboratory, Hebert et al. (2005) to 50-mM KCl-induced Ca2? influx. They found that TH observed an increase in Fos levels and CREB phosphory- reporter gene induction by KCl is dependent upon both lation following a single immobilization without changing CRE and the AP-1 sites. Both sites are required for the KCl the expression of Egr1, Fra-2, or phosphorylated activating induction, but either site will support induction mediated transcription factor-2. Repeated immobilization induced by the A23187 calcium ionophore. In a CREB-deficient Fos and Fra-2. These investigators reported that repeated PC12 cell line, the response to cAMP is greatly inhibited, immobilization stress increased phosphorylation of several but the response to A23187 is robust. Thus, calcium- mitogen-activated protein kinases (MAPK) including p38, dependent increases in TH expression can also occur c-Jun N-terminal kinases (JNK1/2/3) and extracellular through a mechanism that is independent of CREB phos- signal-regulated kinases (ERK1/2). It is clear that tran- phorylation, suggesting that there are multiple pathways scription factor binding to the promoter region in TH is an that regulate calcium-dependent TH expression. Both PKA important regulatory mechanism that determines cate- and CaMPKII catalyze the phosphorylation of CREB and cholamine levels in different tissues. Not surprisingly, increase TH mRNA expression. however, the specific elements are different depending on Approximately 40 bp upstream of the transcription start the type of cells and their functions. site on the rat TH promoter is a CRE/CaRE site that reg- ulates TH expression in rat PC12 cells (Osaka and Sabban 1997) (Fig. 4). CRE is one of the most highly studied and TH promoter and regulatory elements important factors for the control of TH expression, and its significance has been established by mutagenesis of a rat Regulatory elements in the TH promoter include the glu- TH promoter CAT reporter gene construct transfected in cocorticoid response element (GRE; 450 bp upstream), the human SK-N-BE(2)C neuroblastoma cell line (Tinti activator protein-1 (AP-1; 200 bp upstream), cAMP et al. 1997). A second rat CRE site about 95 bp upstream of response elements (CRE1/2; 40 and 90 bp upstream), a cis- the transcription start site can also be activated by phorbol acting dyad element (200 bp upstream), and an inhibitory esters in a rat CAT reporter construct expressed in rat PC12 heptamer sequence (HEPT; 160 bp upstream) along with cells (Best and Tank 1998). In the mouse, ATF-2 binds to other factors, that are depicted in Fig. 4, and initially dis- the CRE site to regulate TH promoter activity (Suzuki et al. cussed in Kumer and Vrana (1996). We will describe 2002). Increases in cAMP and activation of PKA can also recent studies investigating the regulatory elements in the result in the repression of the TH promoter through TH promoter and other regions on TH mRNA. The sections inducible cAMP early repressor (ICER) as demonstrated in below will focus on some of the well-established stimuli transfected PC12 cells (Tinti et al. 1996). Hiremagalur and the promoter sequences through which they act. et al. (1993) reported that nicotine treatment of PC12 cells 123 Author's personal copy

Regulation of tyrosine hydroxylase activity 1459 for 1–2 days increased both TH and dopamine b-hydrox- luciferase reporter activity controlled by the proximal 272 ylase mRNA levels. Mutagenesis of the CRE site abolished nucleotides of the rat TH promoter in these cells. Although the response to nicotine as determined in TH reporter there is no competition between the Sp1/Egr1 site and the constructs. Nicotine treatment elevated intracellular Ca2? AP-1 site, the Sp1/Egr1 site can still be acted upon by AP-1 in PC12-derived cells lacking PKA, but it failed to induce factors to modulate the rat TH promoter linked to a lucif- TH mRNA levels. These results indicate that PKA is erase reporter gene. This observation is supported by the required for the nicotine-stimulated TH induction (Nank- finding that binding to Egr1 increases upon the treatment of ova et al. 1996). the tumorigenic mouse brain noradrenergic CATH.a cell In addition to their separate roles, AP-1 and CRE can line with phorbol esters (Stefano et al. 2006). work in concert with a partial dyad element (the partial Piech-Dumas et al. (2001) found that both wild-type AP- dyad involves symmetry between the sequences GAATAC 1 and CRE sites are required for the complete activation of and GTATTC that is hypothesized to mediate positive gene expression by phorbol esters in rat PC12 cells. transcriptional regulation) that regulates basal rat TH Phorbol esters lead to the phosphorylation of CREB and to expression in seven TH-expressing cell lines: CATH.a and the activation of PKA. Phorbol esters also lead to the CATH.b (mouse CNS tumor), PATH.2 (mouse adrenal activation of ERK1/2 following activation of PKC (rev. in tumor), CAD (mouse CNS), PC12 (rat adrenal medulla Roskoski 2012), and ERK1/2 activation may also partici- tumor), SK-N-BE(2)C (human neuroblastoma), and B103 pate in regulating TH expression. AP-1 activation occurs (rat CNS tumor) (Patankar et al. 1997). The partial dyad is via stimulation of the upstream ERK1/2 pathway in neu- required for TH expression in cell lines that rely on CRE or rons of the developing rat brain striatum (Guo et al. 1998). the AP-1 dyad element. The partial dyad is not required for Cazorla et al. (2000) reported that CRE in mouse neuro- the cAMP-mediated TH induction in PC8b cells (derived blastoma Neuro2A cells does not interact with the previ- from PC12 cells) or in CATH.a cells, nor is it required for ously mentioned Pitx3 site. It appears, therefore, that there the KCl induction of TH in CATH.a cells. may be several differences regarding the inter-species Calcium and cAMP are important regulators of TH mechanisms for the regulation of TH gene expression as we promoter activity. As discussed, they work through multi- note for the human gene below. ple regions on the promoter. In addition, they are also involved in several other cellular events, which make the Hypoxia regulation of TH gene expression more complex. Even though these stimuli have been characterized rather early In rat PC12 cells, hypoxia increases Fos expression, AP-1 on, there is still more to be known regarding their role in promoter activity, and results in increases in TH mRNA the maintenance of catecholamine production. levels (Mishra et al. 1998). Fos expression is required in depolarization and phorbol ester activation of this promoter Phorbol esters (Sun and Tank 2003). However, this hypoxic response is not observed in a human neuroblastoma cell line. Hypoxia Yang et al. (1998) identified seven rat cis-regulatory ele- also increases CRE activity to increase TH mRNA levels ments by DNAse I footprint analysis from extracts pre- (Beitner-Johnson and Millhorn 1998). A study investigat- pared from SK-N-BE(2) and CATH.a cells within the first ing the effect of multiple stimuli on rat TH promoter 0.5 kb upstream promoter region. They found that the activity demonstrated that basal expression is mediated element located from -124 to -107 bp proximal to the through the partial dyad involving CRE and AP-1 elements transcription start site interacts with Specificity protein 1 while inducible expression occurs mainly via CRE and to a (Sp1) and contributes to the transcriptional activation of the lesser extent through AP-1 and hypoxia response element 1 TH gene in cooperation with CRE. Papanikolaou and (HRE1) (Lewis-Tuffin et al. 2004). Moreover, Rani et al. Sabban reported that the Sp1 region and an Egr1 motif (2009) found a novel 7-bp AP-1 like element about (Fig. 4) of the TH promoter bind rat adrenomedullary -5.7 kb upstream from the TH transcription start site that protein factors following immobilization stress (Papa- mediates the response to glucocorticoids in rat PC12 cells nikolaou and Sabban 1999, 2000). They also found that transfected with a rat 9-kb TH promoter-luciferase con- phorbol esters increase Egr1/Sp1 response element binding struct. Deletion of all but 100 bp surrounding the -5.7 kb activity, and that this can work in concert with the AP-1 upstream sequence from the 9 kb construct resulted in a element. promoter that fully maintained the response to dexameth- Nakashima et al. (2003) found that phorbol esters induce asone, providing strong evidence that this sequence is both Egr1 and AP-1 factors in rat PC12 cells. They also responsible for glucocorticoid sensitivity. reported that the insertion of 10 bp between the Spr/Egr1 von Hippel–Lindau protein (VHL) is a tumor suppressor and AP-1/E-box reduced the ability of EGR1 to upregulate protein, and its loss is frequently observed in vascular 123 Author's personal copy

1460 I. Tekin et al. tumors of the CNS, renal cell carcinomas, and pheochro- Introns as regulators of TH expression mocytomas (Chou et al. 2013). Overexpression of VHL in rat pheochromocytoma PC12 cells decreases TH mRNA Kelly et al. (2006) replaced the first exon and first intron of levels by reducing mRNA elongation (Kroll et al. 1999). one allele of the mouse TH gene with yellow fluorescent Inhibition of the endogenous levels of VHL in PC12 cells protein. The reporter gene failed to identify functional TH- with antisense RNA results in an increase in TH expression expressing cells with complete accuracy in a mouse (Bauer et al. 2002). This increase may be mediated through transgenic knock-in model. These investigators suggested the inhibition of a response that results in increased ubiq- that the first intron of the mouse TH gene functions as a cis- uitination and degradation of hypoxia-inducible transcrip- regulatory element. Romano et al. (2007) analyzed the tion factors (HIFs) that normally bind to the hypoxia- transcriptional profile of the promoter, the first exon, and responsive element (HRE-1) (Fig. 4) and activate the TH the first intron of the human TH gene in human neuro- promoter (Schnell et al. 2003). blastoma BE(2)-C-16 cells. The addition of a 1.2-kb frag- ment of the first intron enhanced transcriptional activity of Gender the recombinant promoter.

Gender also plays a role in the differential regulation of TH Epigenetic regulation expression. Sry, a gene found on the Y chromosome, encodes a protein that binds to the rat TH promoter (pre- The epigenetic states of the chromosome and histone sumably to an AP-1 site) about 300 bp upstream of the acetylation are also important factors in determining TH transcription start site (Milsted et al. 2004). Sry is transcription levels (Lenartowski et al. 2003). In addition to expressed in catecholaminergic regions in male, but not the investigation of the role of introns, Romano et al. female, rats. Cotransfection of rat PC12 cells with an (2007) also found, through chromatin immunoprecipita- expression vector for Sry and a luciferase reporter con- tion, that extensive histone H3 and H4 acetylation occurs in struct containing 773 of the proximal nucleotides of the TH nucleosomes isolated from the TH promoter region of promoter led to the elevation of reporter activity. However, BE(2)-C-16 cells. In human renal carcinoma 293FT cells a reporter construct lacking the canonical Sry site also that fail to express TH, histone acetylation in the TH pro- responded to Sry. Mutation of the AP-1 site in the TH moter region was minimal. The effect of acetylation on the promoter reduced induction suggesting that regulation regulation of TH mRNA levels occurs through regulation occurs primarily at this motif. of RNA stability, as TH mRNA stability was shown to Estrogen and progesterone participate in the regulation decrease upon treatment of PC12 cells with sodium buty- of TH expression. Estradiol increases promoter activity in rate, a histone deacetylase inhibitor (Aranyi et al. 2007). rat PC12 cells transfected with estrogen receptors-a/b Detailed discussion of these mechanisms and their physi- (ER-a/b) through an estrogen-response element (ERE) that ological and developmental roles is provided elsewhere overlaps with the CRE/CaRE site (Maharjan et al. 2005) (Lenartowski and Goc 2011). (Fig. 4). The action of 17 b-estradiol can also be mediated at the plasma membrane as demonstrated by experiments Human TH promoter performed with an impermeable estradiol derivative (Ma- harjan et al. 2010). An estradiol/ER-a complex can acti- Although most transcriptional regulation studies have been vate PKA/MEK signaling that results in the performed in rat and mouse models, important differences phosphorylation of CREB and the activation of CRE/ exist in human and rodent TH promoters. Studies of the CaRE, where MEK refers to Mitogen-activated/ERK human TH promoter in human neuroblastoma cell cultures Kinase (Roskoski 2012). Progesterone receptors also bind indicate that nucleotides 513 bp upstream of the tran- to the TH promoter 1.4 kb upstream of the transcription scription initiation site and 976 bp downstream from the 30 start site in experiments performed with mouse neuronal end are required for TH expression (Gardaneh et al. 2000). CAD cells (Jensik and Arbogast 2011). In addition, However, transgenic mice that contain the 50 11 kb region intracerebroventricular injection of progesterone receptor of the human TH promoter express a reporter in TH posi- antisense RNA decreases progesterone receptor levels and tive cells in vivo (Kessler et al. 2003). Sequence analysis of increases TH levels in rat hypothalamus (Gonzalez-Flores this region led to the identification of five common con- et al. 2011). The latter authors suggest that their findings sensus sites that are similar to those of rat and mouse indicate a direct role for progesterone in the neuroendo- sequences: GR, AP-1, HOXA4/HOXA5, TBF-1, and AP-3. crine regulation of dopaminergic neurons in the CNS The nearest (GR) is 2.3 kb and the farthest (AP-3) is 8.8 kb through progesterone receptors. from the transcription start site.

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Regulation of tyrosine hydroxylase activity 1461

Constructs prepared with minimal promoters that con- Several factors that control TH promoter activity regu- tain the five conserved consensus sites were able to drive late mRNA stability including hypoxia and stress TH expression in a commercially available human neuro- (reviewed in Kumer and Vrana 1996). The stabilization of nal progenitor cell line, but they failed to do so in mouse rat PC12 cell TH mRNA occurs through binding of primary striatal or substantia nigral cells in culture hypoxia-inducible protein (HIP) to a 27-bp binding site (Romano et al. 2005). This finding indicates that there are (HIPBS) in the 30-UTR (untranslated region) of TH mRNA differences in the promoter-based regulation of TH gene (Czyzyk-Krzeska and Beresh 1996). The HIPBS element is expression in human and mouse cells and suggests that conserved in bovine, human, mouse, and rat TH mRNA. extra caution be used when extrapolating from rodent to This 27-bp stabilizing domain contains a pyrimidine-rich human biology. Despite its important developmental role in sequence that increases rat TH mRNA stability in PC12 rodents, studies from the same laboratory indicate that cells during hypoxic conditions (Paulding and Czyzyk- Nurr1 lacks a consensus binding site on the human TH Krzeska 1999). Sustained hypoxia increases TH mRNA in promoter (Jin et al. 2006). This is in agreement with the rat cerebral cortex, which is expected with increased finding of Satoh and Kuroda (2002) who reported that mRNA stability (Gozal et al. 2005). The levels of TH human neuroblastoma SK-N-SH cells fail to express Nurr1 protein measured immunochemically, however, remain mRNA. unchanged. The reason for the discrepancy between TH A neuron-restrictive silencer element/repressor element mRNA and protein levels is unclear. However, tyrosine 1 (NRSE/RE1) occurs about 2 kb upstream in the human hydroxylase activity is increased in response to hypoxia TH promoter, binds neuron-restrictive silencer factor/ owing to the phosphorylation of Ser40 as described later. repressor element transcription factor (NRSF/REST) and Nicotine treatment of bovine adrenal chromaffin cells inhibits TH mRNA synthesis in human HB1.F3 neural increases TH mRNA stability and induces the binding of stem cells (Kim et al. 2006). This repression is alleviated novel proteins to the 30 UTR (Roe et al. 2004). Long-term by either mutating or deleting NRSE/RE1. In SH-SY5Y stress also induces protein binding to the poly-pyrimidine human dopaminergic neuroblastoma cells that express TH, rich region in the 30-UTR (Tank et al. 2008). Chen et al. mutating or deleting NRSE/RE1 has no effect on TH (2008) reported that cAMP leads to the induction of TH expression, implying that this element regulates TH protein and activity, but not TH mRNA, in rat ventral expression in a cell type-specific manner. midbrain slice cultures and in MN9D cell cultures, which Despite the myriad studies available on the structure and are hybrids of mouse neuroblastoma and embryonic mouse regulation of the rodent promoter, few data are available mesencephalic neurons. cAMP increases the translational regarding the control of human TH expression. This pre- activation of TH mRNA that is mediated by sequences sents a strong need in the field for the understanding of the within the 30-UTR. These authors suggest that cAMP- TH promoter and how it will affect TH gene regulation. dependent increases in expression of stabilizing trans- Having insight into these mechanisms will allow us to be activating factors occur in these rodent systems. Xu et al. able to propose ways to reach optimal levels of catechol- (2009) reported that cAMP increases levels of the poly(C)- amines in both the central and peripheral nervous systems. binding protein-2 (PCBP2) in MN9D cells. PCBP2 binds to In addition, such knowledge may also contribute to a fur- the poly-pyrimidine rich region of the 30-UTR and thereby ther understanding of the pathological conditions that stabilizes TH mRNA. It previously has been suggested that affect catecholaminergic tissues. TH mRNA stability may be important for the maintenance of mRNA levels (Kumer and Vrana 1996). In addition, it is interesting that the same environmental factors that affect Regulation of TH mRNA stability the promoter activity can regulate mRNA stability.

TH mRNA can be stabilized by interacting with selected proteins, leading to increased translation of TH and, hence, Alternative RNA processing increased TH protein and activity (Roe et al. 2004). Expression of these mRNA-binding proteins and their TH exists in various isoforms in a few species, including combined interaction with TH mRNA is altered by cellular humans, as a result of alternative splicing of hnRNA activity. Several TH splice variants have been identified, (Kumer and Vrana 1996). Such splicing changes the reg- and their role in regulating and maintaining catecholamine ulatory, but not catalytic domains of TH. Mogi et al. (1984) levels are reviewed here (and will be discussed in detail in purified tyrosine hydroxylase from human adrenal medulla the following sections) and have been addressed elsewhere and resolved two fractions with different specific activities. (Haavik et al. 2008; Kobayashi and Nagatsu 2005; Wil- This was the first suggestion of the existence of different lemsen et al. 2010). TH isoforms. Two splice variants occur in non-human 123 Author's personal copy

1462 I. Tekin et al.

Fig. 5 a Schematic representation of the differential regulation of TH it is not phosphorylated by ERK1/2. Note that the most abundant through several protein kinases and phosphatases. b Alignment of the isoform (hTH-1) is also most closely homologous to the rat TH first 50 amino acids of different rat and human TH mRNAs. Human protein. The sequences were obtained from NCBI Nucleotide TH lacks one of the serines (position 8) that is present in the rat Database (rTH: NM_012740.3, hTH-1: NM_000360.3, hTH-2: mRNA. In addition, while hTH2 contains the equivalent of serine 31, XM_005253099.1, hTH-3: NM_199293.2, hTH-4: NM_199292.2) primates and Drosophila (Birman et al. 1994; Ichikawa patterns resulting in the major mRNAs and TH protein et al. 1990; Ichinose et al. 1993; Lewis et al. 1994). Two isoforms are shown in Fig. 5. splice variants have also been reported in rat (Laniece et al. All of the human TH isoforms are found in adrenal 1996), but this finding has not been corroborated (Haycock chromaffin cells (Haycock 1991) and in brain catechol- 2002b; Ichikawa et al. 1990). aminergic neurons with hTH-1 and hTH-2 accounting for Human TH has four splice variants that are denoted as about 90 % of human brain TH (Lewis et al. 1993). hTH-1, hTH-2, hTH-3, and hTH-4, and were named in the Moreover, all isoforms are expressed in human pheochro- order that they were discovered. hTH-1 is composed of 13 mocytomas (Haycock 2002b) and neuroblastomas (Hay- exons, consists of 1,494 nucleotides and shows 89 % cock 1993). Among the major four isoforms, hTH-1 and identity to its rat counterpart. Specifically, hTH-1 is the hTH-2 mRNA transcripts are detected in highest abun- most abundant isoform and the closest homolog to the rat dance (Ichinose et al. 1994). Human pheochromocytomas enzyme (the rat enzyme is 498 and the human ortholog is contain the highest relative abundance of hTH-3 and hTH- 497 amino acids long). hTH-2, hTH-3, and hTH-4 contain 4 (Coker et al. 1990; Grima et al. 1987; Haycock 1991, the addition of 4, 27, and 31 (4 ? 27) amino acids, 1993; Le Bourdelles et al. 1988; Lewis et al. 1993). Several respectively (derived from exon 1, plus or minus exon 2) additional isoforms occur in human neuroblastomas and in (Grima et al. 1987; Kaneda et al. 1987). Computer-assisted brain samples from patients with progressive supranuclear analysis of the secondary structure of the primary RNA palsy (Bodeau-Pean et al. 1999; Dumas et al. 1996; Ohye transcript led to the prediction of four stable hairpin loops et al. 2001; Parareda et al. 2003; Roma et al. 2007). in introns 1 and 2 (Kobayashi et al. 1988). This analysis After identification of different TH mRNAs, investiga- indicates that these secondary structures may account for tors sought to characterize enzymes produced from the the inclusion/exclusion of exon 2 that occurs during hTH-1 different splice variants. Eukaryotic expression systems and hTH-2 generation. Other minor TH mRNAs were such as COS cells and Xenopus oocytes have been utilized, identified that lack exons 3, 4, 8, and 9 (Bodeau-Pean et al. along with expression of the enzyme in E. coli (Horellou 1999; Ohye et al. 2001; Parareda et al. 2003). Splicing et al. 1988; Kobayashi et al. 1988). In spite of varying

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Regulation of tyrosine hydroxylase activity 1463 specific values for enzyme activities that most likely reflect tightly to ferric iron, which is located in the active site different assay conditions, all reports indicate that hTH-1 cleft. As described above, TH requires its iron atom to be in has the highest specific activity. Nasrin et al. (1994) the ferrous form during the reaction. The pterin co-sub- reported a regulatory effect of BH4 on enzyme activity at strate reduces the ferric iron to enable formation of an high concentrations of tyrosine substrate. These investi- active enzyme. gators reported that hTH-2 and hTH-4 are more stable at Alterations in feedback inhibition occur as a result of elevated temperatures than hTH-1 and hTH-3. Bodeau- enzyme phosphorylation as catalyzed by various protein Pean et al. (1999) found an isoform that lacks exon 3 in a kinases as noted here and in the following section. PKA human neuroblastoma that has 30 % of the activity of hTH- catalyzed phosphorylation of TH at Ser40 increases the Ki 1 but exhibits a tenfold increase in its Ki for dopamine. value for DA and decreases the Km for BH4 (Daubner et al. The physiological significance of the four human TH 1992; Ramsey and Fitzpatrick 1998; Ribeiro et al. 1992). isoforms remains unclear. Although hTH-1 has the highest Note that an increase in Ki and a decrease in Km increase specific activity, it is not that much different from that of TH activity. Inhibition of TH purified from rat PC12 cells the other isoforms. Perhaps the major difference in the and from bovine adrenal by catecholamine end products isoforms is the sequence variation in the regulatory ERK1/ and relief of this inhibition following Ser40 phosphoryla- 2 protein kinase Ser31 phosphorylation site (Kumer and tion as catalyzed by PKA have been shown in vitro (An- Vrana 1996), which is described below. dersson et al. 1992). Inhibition of TH also occurs in vivo, as the enzyme isolated from brain is found in a complex with DA. However, this may result from oxidation of fer- Feedback inhibition of TH rous to ferric iron during isolation with the subsequent formation of the DA–enzyme complex. All four isoforms Inhibition of TH by pathway end products is an important of human TH are subject to feedback inhibition to the same regulatory mechanism that acts as a sensor to maintain extent when assayed in vitro (Almas et al. 1992). required levels of the catecholamines. Excessive cate- Structural modeling (Maass et al. 2003) and kinetic cholamines can be harmful, as these substances have been studies (Ramsey and Fitzpatrick 2000) of rat TH have been shown to form toxic quinones as noted earlier. For exam- used to decipher the nature of catecholamine binding to the ple, dopamine forms reactive quinone metabolites (Has- enzyme. Dopamine interacts with iron and a negatively tings and Zigmond 1994; reviewed in Stokes et al. 1999). charged carboxyl group in the active site cleft (Haavik Reactive quinones (1) form reactive oxygen species, (2) et al. 1990). Molecular modeling predicts two planar con- mediate the covalent modification of DNA and proteins, formations for DA, whose binding is favored by neutral and (3) activate apoptotic pathways (Stokes et al. 1999). pH, while DOPA has only one conformation that is ener- Direct effects of catechol-quinones on TH protein have getically favorable. Electrostatic repulsion is hypothesized been characterized in vitro (Kuhn et al. 1999; Xu et al. to occur between the carboxyl group of DOPA and the side 1998a). The extent that such modifications occur in vivo is chain of nearby Asp425. This explains the twofold increase unclear. in Ki values for DOPA when compared with DA. A short Dopamine exerts direct inhibitory effect on TH at con- stretch of the regulatory domain, whose involvement in DA centrations that are in the nanomolar range. This low Ki of inhibition has been supported by the role of phosphoryla- TH for DA allows for the strict control of intracellular tion in reversing the inhibition, is hypothesized to interact catecholamine levels. Feedback inhibition of TH can be with DA while closing on the catalytic domain. However, considered in two parts. The first is concentration depen- there is no structural evidence for this prediction due to the dent, reversible, and results from direct binding of DA to unavailability of a crystal structure for the complete

TH. DA is a competitive inhibitor of TH against BH4 and a enzyme. Nonetheless, mutagenesis studies have identified non-competitive inhibitor against tyrosine (Fitzpatrick residues in the regulatory segment including Gly36, Arg37,

1988). Binding of DA prevents BH4 from binding to the and Arg38 as the critical regions involved in mediating active site, thereby causing a dramatic increase in the Km catecholamine binding to recombinant rat (McCulloch and value for this substrate (Ribeiro et al. 1992). The second Fitzpatrick 1999; Nakashima et al. 1999, 2000; Ota et al. mechanism of inhibition of TH by DA results from the 1997). formation of a tight enzyme–iron–catecholamine complex. Gordon et al. (2008) have suggested the existence of a DA interacts with the active site iron atom, but only when second binding site for dopamine with much lower affinity. the iron is in its ferric form (Andersson et al. 1988; Okuno They reported that all four human isoforms possess this and Fujisawa 1985; Ramsey and Fitzpatrick 1998). The second site. They came to this conclusion by fitting their ferric form occurs as a result of oxidation by molecular data to a two-site binding model. They followed a similar oxygen in the cell or during enzyme isolation. DA binds approach to reproduce their results in rat PC12 cells in situ 123 Author's personal copy

1464 I. Tekin et al. and showed that this site also resides in the catalytic Early phosphorylation studies of TH focused on the rat domain (Gordon et al. 2009b). Using site-directed muta- enzyme because of its ready availability. Following the genesis of selected residues in the carboxyl-terminal cata- production of recombinant enzymes, data obtained initially lytic domain of recombinant hTH-1, they concluded that from the rat enzyme were confirmed and extended with the second binding site is located in a region close to the human TH isoforms. To avoid confusion, however, specific first, which was later confirmed by the same group (Briggs residues will be denoted by their positions in the rat protein et al. 2011). They conclude that the two sites may be (recalling that there are four human isoform proteins gen- present on different monomers. However, it is also possible erated by alternative splicing). Four sites have been iden- that there are distinct versions of the enzyme with dis- tified, in the rat enzyme, that are phosphorylated by various similar dissociation constants, rather than two different protein kinases. Sequences of the amino-terminal segments binding sites. These differing KD values may arise owing to of rat TH and the human TH isoforms are shown in Fig. 5, distinctive enzyme conformations. These conformations and consensus motif sequences are highlighted. Note that might have different affinities, and as there is not a single the rat enzyme and four human isoforms contain two serine enzyme conformation, dose–response curves appear shal- residues embedded in consensus phosphorylation sites (the low. Further investigation is warranted to differentiate rat Ser19 and Ser40 equivalents). The amino acid sequence between these possibilities. preceding the Ser31 site in human isoforms 2, 3, and 4 differs from that of isoform 1 as a result of alternative splicing (Fig. 5). The significance of this finding is related Phosphorylation of TH serine residues to ERK1/2 catalyzed phosphorylation as discussed later. Phosphorylation of TH at different residues produces Phosphorylation and regulation of TH have been well differing molecular effects. However, TH phosphorylation established in vitro, in situ with cell cultures and brain generally results in an increase in catecholamine produc- slices, and in vivo using complex systems such as brain or tion. Throughout this section, we will discuss individual retina in intact animals. In the present context, we will serine residues and (1) the effect of their phosphorylation focus most of our attention on the mechanistic conse- on enzyme activity, (2) the different kinases implicated in quences of phosphorylation on enzyme activity, but note phosphorylation, and (3) dephosphorylation by phospha- that there is a considerable recent literature on the physi- tases. We will describe the physiological importance of ological signals responsible for the dynamic regulation of these events on regulation of brain catecholamine levels. It phosphorylation state (reviewed in Daubner et al. 2011; is beyond the scope of the present review to explore all of Dickson and Briggs 2013; Dunkley et al. 2004; Nakashima the various stimuli (stress, drugs, behavior, etc.) that trigger et al. 2013). phosphorylation; however, selected exemplars will be Rat TH is phosphorylated at four different sites following provided. cellular stimulation with cAMP, growth factors, phorbol There are three important regulatory phosphorylation esters, or depolarization with KCl or neurotransmitters sites in TH (Ser19/31/40). The phosphorylation of Ser8 in (McTigue et al. 1985). These sites are comprised of serines the rat enzyme has little, if any, regulatory effect and a 8, 19, 31 and 40 in the rat enzyme and serines 19, 31, and 40 threonine occurs in its place in the human enzyme iso- in the human enzyme in vitro (Campbell et al. 1986; Hay- forms. A large number of protein kinases are involved in cock 1990; Haycock and Wakade 1992; Waymire et al. phosphorylation–regulation of TH (summarized in 1988), in situ (Haycock 1990), and in vivo (Haycock and Table 1). Haycock 1991). A threonine occurs at position 8 in the human enzyme isoforms. Although threonine is a substrate Phosphorylation and regulation of rat and bovine TH for protein serine/threonine kinases, phosphorylation of Ser/ Thr8 appears to play no role in the regulation of TH. Serine-40 Regulation of enzyme activity by phosphorylation is one of the most extensively studied forms of metabolic control Initial reports of TH phosphorylation showed PKA to be a of cellular processes in general (Cohen 2002) and of TH in central component of enzyme regulation. Increases in particular (Daubner et al. 2011; Dickson and Briggs 2013; enzyme activity were reported when rat brain or bovine Dunkley et al. 2004; Fujisawa and Okuno 2005; Kumer and adrenal TH was incubated in vitro with ATP and (1) PKA Vrana 1996; Nakashima et al. 2009, 2013). Phosphoryla- or (2) cAMP (Joh et al. 1978; Morgenroth et al. 1975; tion as catalyzed by protein kinases and dephosphorylation Yamauchi and Fujisawa 1979; Vrana et al. 1981; Vrana as catalyzed by protein phosphatases allow for a rapid and and Roskoski 1983). The phosphorylation-dependent reversible regulation of TH and are critical for maintaining increase in rat or bovine TH activity occurs as a result of optimal catecholamine levels in cells (Fig. 5). lowering the Km for BH4 and an increase in the Ki for 123 Author's personal copy

Regulation of tyrosine hydroxylase activity 1465

Table 1 Tyrosine hydroxylase phosphorylation sites and protein in vitro. These high values can be explained in part by an kinases evolutionary mechanism of the cell adapting itself to the Site Protein kinase References relatively high intracellular TH concentration (Roskoski and Ritchie 1991). When expressed and purified in vitro, Ser40 PKA Edelman et al. (1978), Joh et al. hTH-1/2/4 isoforms are catecholamine free and required (1978), Campbell et al. (1986) the addition of FeSO for detectable activity (Almas et al. PKG Roskoski et al. (1987) 4 1992). These proteins were good substrates for PKA with PKC Albert et al. (1984) 2? Km values of only 5 lM. However, the addition of Fe CaMPKII Vulliet et al. (1984) and DA increased the Km about threefold. This suggests MSK1 Toska et al. (2002a) that the presence of tightly bound iron and DA in the MAPKAPK1 Sutherland et al. (1993) enzyme isolated from mammalian cells contributes to the MAPKAPK2 Sutherland et al. (1993) higher Km observed for the rat pheochromocytoma and MAPKAPK5/PRAK Toska et al. (2002a) bovine adrenal enzymes. Ser19 CaMPKII Schworer and Soderling (1983), Funakoshi et al. (1991) reported that TH phosphoryla- Campbell et al. (1986) tion at Ser40 by PKA results in an increase in activity, MAPKAPK2 Sutherland et al. (1993) while phosphorylation of this site by CaMPKII or PKC MAPKAPK5/PRAK Toska et al. (2002a, b) fails to increase activity in vitro. The stoichiometry of rat Cdk11 Sachs and Vaillancourt (2004) Ser40 phosphorylation by PKA was 0.78 mol/mol of sub- Ser31 ERK1/2 Haycock et al. (1992) unit, while that for PKC and CaMPKII were both 0.4 mol/ Cdk5 Kansy et al. (2004) mol of subunit. The authors suggest that phosphorylation of The three serine residues are numbered as per the rat enzyme and Ser40 in all four subunits is required for activation, and human TH-1 (see Fig. 5) such extensive phosphorylation is not achieved by PKC or CaMPKII calcium–calmodulin-dependent protein kinase II, Cdk CaMPKII. cyclin-dependent kinase, ERK1/2 extracellular signal-regulated kinase 1 and 2, or microtubule-associated protein kinases 1 and 2, PKC phosphorylation has been shown to produce effects MAPKAPK microtubule-associated protein kinase-activated protein similar to those induced by PKA. This is, in part, because kinase, MSK1 mitogen and stress-activated kinase 1, PKA protein they both phosphorylate Ser40 in the rat enzyme (or its kinase A, or cyclic AMP-dependent protein kinase, PKC protein equivalent in the human enzyme isoforms). Albert et al. kinase C, where C refers to calcium ion, PKG protein kinase G, or cyclic GMP-dependent protein kinase, PRAK p38-regulated/activated (1984) reported that PKC-mediated phosphorylation of protein kinase partially purified rat TH decreases the Km for BH4 and increases the Ki for DA, which leads to an increase in dopamine both in vitro (Daubner et al. 1992; Lovenberg enzyme activity. They found that PKC and PKA catalyze et al. 1975; Okuno and Fujisawa 1985; Ramsey and Fitz- the phosphorylation of the same serine residue. However, patrick 1998; Vrana et al. 1981; Vrana and Roskoski 1983; Cahill et al. (1989) reported that treatment of rat PC12 cells Vulliet et al. 1980) and in vivo (Haavik et al. 1990). with phorbol ester leads to the phosphorylation of a dif- However, Ribeiro et al. (1992) suggested that the increased ferent serine residue than that mediated by cAMP as 32 PKA-dependent activation occurs after the rat recombinant determined by Pi labeling followed by trypsin digestion. enzyme, which is isolated in a catecholamine-free state, is The explanation for this difference in in vitro versus in situ treated with and inhibited by DA. The hydroxyl group of labeling is unclear. Rat TH is also phosphorylated and recombinant rat TH serine residue 40 has been hypothe- activated by PKG via cGMP in a manner similar to PKA sized to interact with DA, which is reversed upon phos- (via cAMP) in situ (Roskoski and Roskoski 1987). phorylation (McCulloch et al. 2001). The PKA-mediated Harada et al. (1996) found that elimination of rat TH decrease in the Km for BH4 and the resulting increase in Ser40 by site-directed mutagenesis, which they expressed activity have also been demonstrated in bovine adrenal in non-neuronal mouse AtT20 cells, is still activated by chromaffin cells in situ (Meligeni et al. 1982). Phosphor- phosphorylation of other residues. The effect of ERK 1/2- ylation of TH, isolated from bovine striatum, by PKA mediated phosphorylation was confirmed by the observed stabilizes the interactions occurring between the protein increase in catecholamine synthesis in bovine adrenal backbone in the region surrounding the active site and the chromaffin cells in situ following treatment with acetyl- hydroxyl groups of BH4 (Bailey et al. 1989). choline, an ERK1/2 (p42/p44) MAP kinase activator (Luke Interestingly, the Km of PKA for rat TH and for peptides and Hexum 2008; Thomas et al. 1997; Yu et al. 2011). TH corresponding to the TH Ser40 phosphorylation site has is phosphorylated by ERK1/2 at Ser40 to a lesser extent, as been shown to be about 100 lM, which is rather high will be discussed further in the following sections (Hay- (Roskoski and Ritchie 1991). Almas et al. (1992) reported cock 2002a). Royo and Colette Daubner (2006) reported that the Km of PKA for bovine TH was about 150 lM that the phosphorylation of recombinant rat tyrosine 123 Author's personal copy

1466 I. Tekin et al. hydroxylase at Ser40 by purified bovine heart PKA was manner as PKA. An additional protein, identified as a diminished in the presence of dopamine. Ser40 phosphor- member of the 14-3-3 chaperone protein family, is required ylation also may occur through the action of mitogen and to activate human (Itagaki et al. 1999), rat (Funakoshi et al. stress-activated kinase (MSK1) (Toska et al. 2002a). 1991; Ichimura et al. 1987) and bovine TH (Yamauchi and As it has been established to be a pivotal mechanism for Fujisawa 1981; Yamauchi et al. 1981) phosphorylated by the activation of TH, the effects of phosphorylation at CaMPKII in vitro. The c-isoform of 14-3-3 protein is Ser40 on the enzyme structure have been an active area of abundant in brain, suggesting a role for this chaperone more recent investigation. It has been suggested, through protein isoform in the regulation of TH (Isobe et al. 1991). conformation studies, that Ser40 phosphorylation of the rat Nonetheless, CaMPKII seems to be an important regulator enzyme will result in the promotion of an open confor- of catecholamine synthesis, as suggested by studies in mation in vitro (Bevilaqua et al. 2001; Wang et al. 2011). which cellular inhibition of calcium channels leads to a Moreover, while it is beyond the scope of this review, decrease in DA production in rat PC12h cells, (a subclone considerable other work has been reported on what phys- of PC12 cells that undergoes differentiation following iological insults and influences mediate Ser40 phosphory- treatment with epidermal growth factor) (Sumi et al. 1991). lation and TH activation. Such effectors include However, it is unlikely that this is solely Ser19 mediated, depolarization, hormones, receptor stimulation, and path- as mutagenesis studies have eliminated a direct role for this ological conditions (reviewed in Daubner et al. 2011; residue in controlling catecholamine amounts in other Dickson and Briggs 2013; Dunkley et al. 2004; Nakashima PC12 cell lines (Haycock et al. 1998). TH can be phos- et al. 2013). phorylated at this site by other kinases as well, as seen following inhibition of CaMPKII in situ (Goncalves et al. Serine-31 1997). MAPK, for instance, has been shown to phosphor- ylate TH at Ser19 (Bobrovskaya et al. 2004). The ERK1/2 serine kinases have also been shown to be Despite the known effects of the phosphorylation at this important components in the regulation of catecholamine site (enzyme activation through allowing the binding of biosynthesis (Haycock et al. 1992; Luke and Hexum 2008; activator proteins), phosphorylation itself is not thought to Yu et al. 2011). ERK 1 and 2 are proline-directed protein alter the enzyme’s conformation (Bevilaqua et al. 2001). serine/threonine kinases that are members of the mitogen- However, the binding of 14-3-3 protein to the Ser19 activated protein kinase (MAPK) family that characteris- phosphorylated TH has been shown to produce a more tically function downstream of growth factor receptors extended and relaxed conformation (Skjevik et al. 2014). (Roskoski 2012). ERK 1 and 2 occur together in most cells Ser19 phosphorylation also increases the rate of phos- where they act in concert. Halloran and Vulliet (1994) phorylation at Ser40, a process described as hierarchical reported that depolarization of bovine adrenal chromaffin phosphorylation. Such hierarchical phosphorylation has cells in culture leads to the phosphorylation of TH Ser31. been observed upon phosphorylation of Ser19 in bovine They found that the depolarization-activated kinase shares adrenal chromaffin cells (Bobrovskaya et al. 2004). biochemical properties with ERK1/2 proline-directed pro- tein kinases. This phosphorylation leads to a twofold Phosphorylation of recombinant human TH increase in TH enzyme activity. In addition, unlike the phosphorylation at Ser40, phosphorylation of this site by Given that the alternative splicing of human TH occurs recombinant ERK2 is unaffected by dopamine (Royo and within the regulatory domain, it is likely that the resulting Colette Daubner 2006). isoforms are differentially phosphorylated (recalling that Cyclin-dependent kinase 5 (Cdk5) can phosphorylate hTH-1 is the closest homolog to rat TH; see Fig. 5b—all TH in vitro and in vivo and this phosphorylation occurs at residues are referred to as the number of the rat TH/human Ser31 (Kansy et al. 2004). In transgenic animals, Cdk5 TH-1 homologous residue). Upon expression in E. coli, expression correlates with the preservation of TH protein hTH-1, hTH-2, and hTH-3 are phosphorylated by PKA at levels (Moy and Tsai 2004). However, the mechanism Ser40, and they are phosphorylated at Ser19 and Ser40 by through which Cdk5 may regulate TH expression warrants CaMPKII (Almas et al. 1992; Alterio et al. 1998). The further investigation. extent of dopamine binding is reduced upon Ser40 phos- phorylation in hTH-1 (Sura et al. 2004). MAPKAP Kinase Serine-19 1 and MAPKAP Kinase 2 phosphorylate serine residues 19 and 40, respectively, in all four isoforms. Among the four CaMPKII catalyzes the phosphorylation of TH at Ser19 in isoforms, hTH-3 and hTH-4 phosphorylation by recombi- the presence of calcium; however, phosphorylation of TH nant mouse ERK2 at Ser31 occurs at a much higher rate by CaMPKII fails to activate the enzyme in the same (Sutherland et al. 1993). Activation by phosphorylation of 123 Author's personal copy

Regulation of tyrosine hydroxylase activity 1467 isoforms 3 and 4 (twofold increase in activity) was also hormonal and electrical stimuli that elevate intracellular higher than that of isoform 1, whose activity was increased Ca2?. Tachikawa et al. (1987) found that the incubation of by 40 %. hTH-1 is phosphorylated at Ser31 by ERK2 rat PC12 cells with ionomycin (a calcium ionophore) leads in vitro, whereas hTH-2 is not. Differential phosphoryla- to an increase in phosphorylation of three TH peptides tion by ERK1/2 was investigated in neuronal cells (Gordon derived from TH following tryptic digestion. In contrast, et al. 2009a). Human neuroblastoma SH-SY5Y dopami- incubation with phorbol ester (an activator of PKC) or nergic cells were transfected with hTH-1 or hTH-2. hTH-1 forskolin (an adenylyl cyclase and hence PKA activator) displayed variable levels of phosphorylation upon stimu- leads to increased phosphorylation of only one TH peptide. lation of the cells with epidermal growth factor (EGF), the Surprisingly in this study, the resulting single tryptic pep- receptor of which is the protein-tyrosine kinase that is tides were different and the explanation for this finding is upstream of ERK1/2. hTH-2 was not phosphorylated at a unclear. PKG phosphorylation also occurs at the same site residue that corresponds to Ser-31 in hTH-1. as that of PKA as demonstrated in PC12 cells (Roskoski Lehmann et al. (2006) reported that the hierarchical et al. 1987) and bovine adrenal chromaffin cells (Rodri- activation of Ser40 upon phosphorylation of Ser19 as cat- guez-Pascual et al. 1999). Both Ser19 and Ser40 are alyzed by CaMPKII was higher in hTH-2 than hTH-1. phosphorylated by MAPKAP2 in situ as well (Toska et al. Furthermore, ERK1/2-catalyzed phosphorylation of Ser31 2002b). In addition, new kinases have been implicated in in hTH-1 increased the phosphorylation rate of Ser40 by the phosphorylation of TH at multiple residues such as the ninefold. This effect was not observed in hTH-3 and hTH- AMP-activated protein kinase (AMPK) (Fukuda et al. 4. When ERK1/2 was inhibited with UO126, the decrease 2007). These results might be due to the activation of in the phosphorylation of hTH-1 at Ser31 resulted in a downstream protein kinases, and experiments with purified 50 % decrease in the phosphorylation at Ser40. The authors AMPK and TH should be performed to determine whether concluded that hierarchical phosphorylation provides a the kinase acts directly on TH, and if so, which residues are mechanism whereby the two major human TH isoforms (1 phosphorylated. and 2) can be differentially regulated with only hTH-1 Differential phosphorylation of TH occurs in a cell responding to the ERK1/2 pathway, whereas hTH-2 is location-specific manner. That is, differing phosphorylation more sensitive to calcium-mediated events. patterns occur depending on where in the cell the enzyme is located. Phosphorylation of different residues causes dis- Possible mechanisms for the regulation of TH activity tinct effects on catecholamine biosynthesis and provides via phosphorylation cell and cell location-specific regulatory mechanisms in the grand scheme of catecholamine neurotransmission. Xu Up to this point, we have discussed the mechanisms for TH et al. (1998b) examined the state of phosphorylation of TH phosphorylation separately. However, it should be con- in different rat brain regions and different cellular locations sidered that all these mechanism may act on TH in an using immunohistochemical studies performed with phos- overlapping manner in cells and tissues. This section will pho-specific antibodies against TH. They reported that the discuss the evidence for the existence for these multiple noradrenergic locus coeruleus (LC) and the A5 cell bodies mechanisms, and provide a brief discussion of the physi- had high levels of TH phosphorylated at Ser40 and Ser19, ological importance and relevance of this type of molecular but that the nerve terminals lacked TH that was phos- regulation of TH activity. Incubation of purified rat TH phorylated at either residue. Moreover, different subpopu- with both PKA and CaMPKII results in additive incorpo- lations of dopamine neurons in the ventral tegmental area ration of phosphate under in vitro assay conditions sug- express different amounts of Ser40-phosphorylated TH, gesting that these kinases act on distinct residues (Vulliet while Ser19 phosphorylation was more uniform. Lindgren et al. 1984). PKC, on the other hand, phosphorylates the et al. (2002) reported that ERK1/2-mediated Ser31 phos- same site as PKA in vitro (Vulliet et al. 1985). Griffith and phorylation increased in rat striatal slices upon depolar- Schulman (1988) stimulated rat PC12 cells with A23187 (a ization, but that PKA-mediated Ser40 phosphorylation was calcium ionophore), carbachol, or high KCl, each of which not as great. The authors suggested that there might be a leads to the phosphorylation and activation of TH. They threshold for the effects obtained from Ser40 phosphor- showed that the concentration of cAMP is not elevated by ylation-dependent activation. When the phosphorylation any of these treatments. They also found that cells deficient stoichiometries are low, there is little or no activation until in PKC exhibit TH phosphorylation and increase in activity the threshold is reached. In light of the suggestion that following stimulation. They found that the sites of TH Ser40-mediated activation requires dopamine binding to phosphorylation catalyzed by CaMPKII most closely TH, Ser40 phosphorylation may mainly be involved in the mimic those observed in vivo and conclude that this relief of feedback inhibition. Using phosphorylation site- enzyme mediates TH phosphorylation induced by specific antibodies and an antibody against total TH, 123 Author's personal copy

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Salvatore et al. (2000) measured the phosphorylation PKA is responsible for the increase in Ser40 phosphory- stoichiometry of TH at different amino acid residues in rat lation in PC12 cells and they concluded that Ser31 phos- nigrostriatal and mesolimbic dopaminergic cell bodies phorylation is necessary and sufficient for depolarization- (substantia nigra and ventral tegmental area) and nerve dependent increases in catecholamine synthesis in PC12 terminals (corpus striatum and nucleus accumbens). The and A126-B1 cells. They concluded that Ser40 phosphor- extent of phosphorylation of Ser19 and Ser31 differs ylation plays little or no role in this process. However, they between the cell bodies and terminal fields. Ser19 phos- suggested that PKA-mediated phosphorylation of Ser40 phorylation is greater in the cell bodies (1.5-fold higher) could play a role in regulating catecholamine biosynthesis and Ser31 phosphorylation is greater in the nerve terminals under other conditions. In fact, these investigators con- (two- to fourfold). Ser40 phosphorylation is similar in these cluded that the increases in catecholamine biosynthesis in two compartments and has the lowest value with only 3 % rat brain following haloperidol treatment were due to of the TH Ser40 sites bearing a phosphate group (Table 2). activation of PKA (Salvatore et al. 2000). Ser40 phosphorylation is 1/3rd to 1/10th the level of Ser19 In light of these data, we propose a model for regulation or Ser31 phosphorylation in any region. Haloperidol of dopamine synthesis in neurons (similar mechanisms may treatment of rats for 30–40 min caused an approximate be partially active in noradrenergic and adrenergic neurons) 230 % increase in total phosphorylation (Ser19/31/40) in (summarized in Fig. 6). Depolarization of the presynaptic nerve terminals, while there was a smaller increase in their terminal would activate ERK1/2 and increase DA produc- phosphorylation stoichiometry (around a 130 % increase) tion. Increases in calcium concentration at the nerve ter- in the cell bodies. The authors suggest that the mechanism minal to induce synaptic vesicle fusion to the presynaptic of haloperidol action on TH in vivo in the nigrostriatal cell membrane will also simultaneously activate CaMPKII, system involves both pre- and post-synaptic DA receptors. causing an increase in DA production. Ser40 phosphoryla- Region-specific phosphorylation seems to be mostly neu- tion (established through studies performed with PKA) will ronal, as this type of regulation is not observed in rat retina mainly regulate DA-dependent changes in TH activity, in (Witkovsky et al. 2004). Salvatore et al. (2001) measured addition to responding to the cell’s requirement for rapid the phosphorylation stoichiometry in response to depolar- increases in the DA production by increasing enzyme ization by 58 mM KCl in rat PC12 cells and in a PC12 cell activity (lower Km for BH4 and higher Ki for DA). When the line (A126-B1) that lacks PKA. They found that Ser19 and DA in the synaptic cleft is taken back up by the reuptake Ser31 phosphorylation increased three- to fourfold in both transporter, TH will be inhibited. Also, depending on the cell lines. Ser40 phosphorylation increased about 40 % extent of stimulation of DA autoreceptors, PKA activation following depolarization of PC12 cells, but there was little, will be regulated. Hence when there is less DA present, TH if any, increase in the A126-B1 cell line. The depolariza- is a better substrate for PKA, which increases TH phos- tion-dependent increases in phosphorylation at all sites phorylation and decreases feedback inhibition. were dependent upon external Ca2?. MEK is upstream of The main effect exerted by phosphorylation of TH, ERK1/2, and inhibition of the former by PD98059 regardless of the phosphorylation site and the kinase(s) decreased basal Ser31 phosphorylation and completely involved, seems to be activation of the enzyme. Another prevented the KCl stimulation in both cell lines. The MEK effect of TH phosphorylation, however, has negative effects inhibitor decreased basal Ser40 phosphorylation in PC12 on the thermal stability of TH protein. Phosphorylation of rat cells, but not in A126-B1 cells, suggesting that there is TH is accompanied by a 50 % decrease in half-life of the cross-talk between ERK and PKA pathways in PC12 cells. protein (Gahn and Roskoski 1995; Lazar et al. 1981). Sur- They used a CaMPKII inhibitor in an effort to identify this prisingly, Royo et al. (2005) found that phosphorylation of protein kinase as being responsible for Ser19 phosphory- recombinant rat TH residues other than Ser40 increases in lation. This proved impractical because its inactive con- protein stability. The role of phosphorylation as a regulator gener had the same inhibitory effects. They reported that of thermal stability will be discussed later.

Table 2 Phosphorylation stoichiometry of tyrosine hydroxylase SN Striatum VTA NA Hypothalamus PC12 cells A126-B1

Ser19 0.25 0.10 0.25 0.15 0.26 0.05 0.05 Ser31 0.08 0.30 0.10 0.20 0.13 0.09 0.08 Ser40 0.03 0.03 0.04 0.04 0.05 0.035 0.01 Number of moles of phosphate/mole of tyrosine hydroxylase subunit; data from Salvatore et al. (2000, 2001) SN substantia nigra, VTA ventral tegmental area, NA nucleus accumbens

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Regulation of tyrosine hydroxylase activity 1469

Fig. 6 Schematic depiction of the summary of the proposed physiological effect of TH phosphorylation. a The relative amount of TH phosphorylated at different sites is depicted in accordance with their subcellular locations. b The role of Ser40 phosphorylation and its regulation in maintaining dopamine in the nerve terminal are shown for stimuli originating from autoreceptors, reuptake transporters, cAMP, and Ca2?

Dephosphorylation of TH the activity of PP2A, thereby decreasing TH activity through reduced Ser40 phosphorylation (Peng et al. 2005). Phosphorylation-dependent TH activation is reversed by However, a study that investigated the effects of a-synuc- the removal of phosphate residues by phosphatases. In the lein on PP2A did not provide a similar observation in vivo case of bovine adrenal and corpus striatum, PP2A and (Drolet et al. 2006). Interestingly, a-synuclein overex- PP2C (phosphoprotein phosphatases 2A and 2C) are the pression and the presence of pathological mutations known key enzymes that catalyze the dephosphorylation of bovine to accompany the disease-related phenotype of this protein TH (Haavik et al. 1989). BH4 increases the rate of have been shown to be associated with increased TH dephosphorylation of the rat striatal TH as catalyzed by phosphorylation, suggesting the loss of a necessary func- PP2A (Ribeiro and Kaufman 1994). These experiments tion for PP2A during neuronal loss (Alerte et al. 2008; Lou suggest that BH4 may play a dual role (activating and et al. 2010). deactivating) in the regulation of TH activity. That PP2A is Recent evidence has suggested that the regulation of TH abundant in rat corpus striatum, while PP2C content is only through dephosphorylation is more complicated than has 10 % that of PP2A, supports the role of the former in TH been thought previously. First, there is a specific isoform of regulation (Berresheim and Kuhn 1994). Inhibition of the PP2A regulatory subunit (PP2A/B0b) that is found PP2A by okadaic acid and microcystin leads to increased predominantly in the brain and localizes to specific cellular phosphorylation levels of bovine adrenal TH in digitonin- compartments (nerve terminal; Saraf et al. 2007). Second, permeabilized chromaffin cells, especially at Ser19 and there appears to be specific glutamate residue on PP2A that Ser40 (Goncalves et al. 1997). The rates of dephospho- interacts with the TH regulatory domain (Saraf et al. 2010). rylation of different phosphoserines by PP2A in bovine In addition, PP2A activity itself can be regulated through adrenal chromaffin cells have been shown to be highest for phosphorylation by a PKC isoform (Zhang et al. 2007) and Ser40, followed by Ser19, Ser31, and Ser8, in descending the action of PKC on PP2A is more pronounced on a order (Leal et al. 2002). PP2A from rat corpus striatum is heterotrimeric regulatory subunit (Ahn et al. 2011). Finally, the main TH phosphatase from brain as determined by in vivo PP2A activity may be regulated via fluctuations in measuring the dephosphorylation of recombinant rat TH, the levels of progesterone (increased PP2A activity while PP2C is the chief TH protein phosphatase that occurs accompanying an acute rise in progesterone levels) (Liu in adrenal chromaffin cells (Bevilaqua et al. 2003). and Arbogast 2008) and hypoxia-dependent increases in In vitro, a-synuclein, a protein that is known to reactive oxygen species (downregulation of PP2A) (Rag- accompany the pathology in Parkinson’s disease, increases huraman et al. 2009).

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TH-binding partners However, the membrane-bound form also displays lower catalytic activity (Thorolfsson et al. 2002). The first identified binding partner of TH was a member of Co-precipitation and knock down experiments on 14-3-3 the 14-3-3 family of scaffolding proteins (Ichimura et al. and TH from rat midbrain and cultured dopaminergic cells 1987). These proteins bind to bovine adrenal medullary TH indicated a role for the f isoform, and this isoform co- phosphorylated at Ser19 by CaMPKII, and this binding localized with TH on mitochondria in MN9D cells (Wang induces activation (Yamauchi and Fujisawa 1981; Yama- et al. 2009). There seems to be the requirement for a spe- uchi et al. 1981). Binding requires stimuli such as depo- cific subcellular localization for TH interaction with dif- larization or an increase in calcium to activate CaMPKII. ferent 14-3-3 isoforms. Whether there are different effects Itagaki et al. (1999) studied the interaction between occurring following different isoforms has yet to be recombinant hTH-1 and 14-3-3g. Phosphorylation of TH reported. Sachs and Vaillancourt (2004) reported that by purified rat brain CaMPKII resulted in Ser19 phos- phosphorylation of recombinant rat TH catalyzed by casein phorylation and binding of 14-3-3 with a Kd of 3 nM. kinase 2 or its downstream cyclin-dependent kinase Phosphorylation by recombinant PKA leads to phosphor- inhibits interaction with 14-3-3 and thus diminishes TH ylation of Ser40 but not to 14-3-3 binding. The Ser40Ala activity. These investigators did not determine which res- TH mutant was a poor substrate for CaMPKII and failed to idues of TH were phosphorylated by these enzymes. These bind 14-3-3. The mutant possessed basal TH activity. experiments represent an unusual situation where TH Analysis of a PC12 cell line transfected with myc-tagged phosphorylation is associated with a decrease in catalytic 14-3-3 showed that it formed a complex with endogenous activity. The rat 14-3-3 g subtype may decrease hTH-1 TH after KCl-induced depolarization. stability upon interacting with its regulatory domain Kleppe et al. (2001) reported that binding of yeast and (Nakashima et al. 2007). sheep brain 14-3-3 proteins to bovine and human TH iso- Other binding partners identified for TH have been forms occurs following phosphorylation of Ser40, but investigated owing to their proposed roles in neurodegen- binding of bovine brain 14-3-3-f is not increased. Toska eration. These include a-synuclein (a-Syn), which is a et al. (2002a), from the same laboratory, found that binding major protein in Lewy bodies, the intraneuronal protein of 14-3-3 proteins to recombinant human TH decreases the aggregates characteristic of Parkinson’s disease (Spillantini rates for Ser19 and Ser40 dephosphorylation by 82 and et al. 1998). a-Syn, which has homology with the 14-3-3 36 %, respectively. Obsilova et al. (2008) found that the chaperone, binds rat TH and decreases its activity (Leong interaction between 14-3-3 and Ser19- and Ser40-phos- et al. 2009; Perez et al. 2002). a-Syn co-immunoprecipi- phorylated hTH-1 decreases the exposure of regulatory tates with TH in rat brain and in mouse MN9D dopami- domain amino acids with the solvent as determined by nergic cells (Perez et al. 2002). a-Syn overexpression not time-resolved tryptophan fluorescence measurements. only reduces TH activity, but it also causes a decrease in These investigators found no changes in hTH-1 secondary mouse MN9D cellular dopamine content. a-Syn is over- structure following 14-3-3 binding as determined by cir- expressed upon increased dopamine oxidation; hence pro- cular dichroism. ducing an increase in the amount of reactive oxygen Many studies have been conducted to identify the spe- species in MN9D cells that, in turn, promotes reduction of cific 14-3-3 subtype(s) interacting with TH. The 14-3-3 c TH activity (Leong et al. 2009; Perez et al. 2002). isoform has been suggested as a binding partner owing to Lou et al. (2010) found that wild-type human a-syn its predominant expression in bovine brain (Isobe et al. expression in mouse MN9D cells reduces endogenous TH 1991). Halskau et al. (2009) found that recombinant human Ser19 phosphorylation. They also reported that either wild- 14-3-3c forms a triplet complex with bovine adrenal type or mutant human Ser129Ala a-syn enhances PP2A chromaffin membrane lipids and Ser19-phosphorylated activity in these cells with the Ser129Ala mutant producing recombinant hTH-1, suggesting a role for cellular locali- greater activation. Phosphorylation of a-syn at Ser129 zation and hTH-1 activation by this protein. These inves- decreases its PP2A stimulatory activity. They also found tigators reported that the amino-terminal regulatory that overexpression of wild-type human a-syn driven by segment of TH binds to membranes. This finding validates the TH promoter in transgenic mice stimulates PP2A and and perhaps explains the initial observations of membrane- reduces TH phosphorylation in dopaminergic cells. An associated and soluble forms of TH described in bovine increase in TH dephosphorylation is hypothesized to be the brain homogenates (Kuczenski 1973, 1983). Interaction of main mechanism through which a-syn depletes cellular TH with lipid bilayer fatty acids is thought to stabilize dopamine content (Perez et al. 2002). Regulation of a- secondary structural elements, especially a-helices, hence synuclein, therefore, provides an additional control point in protecting the enzyme against thermal denaturation. catecholamine homeostasis.

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Regulation of tyrosine hydroxylase activity 1471

TH activity in Drosophila is regulated by binding to Several candidate stimuli may lead to TH degradation

GTP cyclohydrolase I (the rate-limiting enzyme for BH4 via proteasomes including angiotensin in rat hypothalamus synthesis), and the interaction of these two enzymes (Lopez Verrilli et al. 2009) and ciliary neurotrophic factor promotes the activity of both partners (Bowling et al. (CNTF) and leukemia inhibitory factor inflammatory 2008). The authors suggest that this interaction may cytokines in mouse and rat sympathetic neurons and M17 provide a mechanism by which TH can obtain cofactor human neuroblastoma cells in culture (Shi and Habecker upon need. Vesicular monoamine transporter (VMAT) 2012). Shi and Habecker (2012) reported that cytokine proteins interact with TH and aromatic amino acid activation of gp130 increases the ubiquitination of TH. decarboxylase in rat brain as demonstrated by co-immu- Moreover, they reported that the proteasome inhibitors noprecipitation, and complex formation involving these MG-132 and lactacystin prevented the loss of TH in components provides a mechanism that links DA syn- CNTF-treated sympathetic neuronal cells. In fact, binding thesis with synaptic vesicle filling (Cartier et al. 2010). of these inflammatory cytokines to the gp130 receptor This linkage may maintain low cytosolic dopamine levels results in TH degradation via activating ERK 1/2. This and decreases the generation of reactive neurotoxic cate- intriguing finding suggests an additional role for ERK1/2 in chol-quinones. Identification of these binding partners TH regulation in addition to increasing enzyme activity. provides additional insight into the functional regulation of catecholamine biosynthesis. Future imperatives

TH enzyme stability and proteasomal degradation TH serves as the central control mechanism in catechol- amine biosynthesis. Despite the extensive study of the A number of regulatory factors or physiological conditions enzyme and its regulation, several areas remain to be alter the inherent stability of the TH protein. Rat TH sta- tackled. Clearly, more work needs to be conducted to bility is decreased by enzyme phosphorylation or treatment define the dynamic protein interactome involving TH and with ascorbate or BH4 (Lazar et al. 1981; Vrana et al. 1981; affecting its regulation. Considerable uncertainty exists Wilgus and Roskoski 1988). Binding of tyrosine or dopa- around which specific 14-3-3 proteins are engaged in dif- mine has a protective effect against enzyme inactivation ferent cell types and what other proteins may contribute to (Roskoski et al. 1990). Binding of anionic heparin or RNA TH regulation, stability, and turnover. Moreover, the recent stabilizes the protein (Gahn and Roskoski 1995). The reports from the Habecker laboratory (Shi and Habecker decrease in enzyme stability is ascribed to changes in the 2012) may provide important initial insights into the role of tertiary structure of the protein (Gahn and Roskoski 1995; neuroinflammation in the regulation of catecholamine Roskoski et al. 1993). Phosphorylation itself induces these production in health and disease. One important future aforementioned structural changes. Among the different consideration will be the role of aberrant regulatory phosphorylation states of TH, phospho-Ser 40 shows the mechanisms that contribute to pathophysiological condi- least stability, while phospho-Ser19, phospho-Ser31, and tions. TH dysfunction has been implicated in several dis- phospho-Ser8 are even more stable than the wild-type eases including alcohol and drug addiction, bipolar enzyme (Royo et al. 2005). disorder, hypertension, movement disorders (Parkinson’s Another mode of regulation for human (Doskeland and disease), and schizophrenia (Bademci et al. 2012; Zhu et al. Flatmark 2002) and rat TH (Nakashima et al. 2011) 2012; Sumi-Ichinose et al. 2010; Bellivier 2005). Note that involves the ubiquitination of TH followed by proteasomal this need not mean that TH causes the disorder; rather, enzyme degradation. Proteasomal degradation of TH seems aberrant regulation of the enzyme may exacerbate the to be triggered by enzyme phosphorylation. Serine phos- disorder. Clearly, these situations will be very difficult to phorylation at residues 19 and 40 is considered to be the address in living organisms (especially human subjects). main regulator of ubiquitination for rat TH as demonstrated However, one can easily imagine a neurodegenerative in PC12D cells (a subline derived from PC12 cells), while disease in which TH regulation fails to compensate for Ser31 phosphorylation has no effect (Nakashima et al. reduced catecholamine function, thus exacerbating the 2011; Kawahata et al. 2009). One study reports the exis- symptoms or inducing more rapid progression. tence of a PEST (proline, glutamate, serine and threonine) In a similar vein, the completion of the human genome motif in the human TH amino-terminal regulatory domain, project and ongoing sequencing efforts (e.g., the 1000 which might be one of the underlying causes for the Genome Project) are opening up new vistas for exploration. instability of the cellular enzyme observed in AtT20 cells The NCBI dbSNP database of single nucleotide polymor- (Nakashima et al. 2005). PEST sequences target proteins phisms indicates that there are more than 50 genetic vari- for proteasomal degradation. ants in human TH coding region that alter amino acids 123 Author's personal copy

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Fig. 7 Reported SNPs and their position on TH mRNA. The red dots spread throughout the protein, although perhaps a little less densely in represent single nucleotide polymorphisms that have been reported in the center of the catalytic domain where they would be more likely to the NCBI SNP database as of 2013. Note that the genetic variants are severely impact (or abolish) enzyme activity

(Fig. 7). As we have noted elsewhere (Tekin and Vrana isozymes by catecholamine binding and phosphorylation. Struc- 2013), these reported SNP variants need to be carefully ture/activity studies and mechanistic implications. Eur J Bio- chem 209(1):249–255 examined for validation, but they represent a natural lab- Alterio J, Ravassard P, Haavik J, Le Caer JP, Biguet NF, Waksman G, oratory for enzyme structure–function relationships. There Mallet J (1998) Human tyrosine hydroxylase isoforms. Inhibition is surprisingly little information on the functional conse- by excess tetrahydropterin and unusual behavior of isoform 3 quences of these variants. While there are clearly a few after camp-dependent protein kinase phosphorylation. J Biol Chem 273(17):10196–10201 genetic variants that are associated with frank disease (e.g., Andersson KK, Cox DD, Que L Jr, Flatmark T, Haavik J (1988) R202H and L205P in DOPA-responsive dystonia; Haavik Resonance Raman studies on the blue-green-colored bovine et al. 2008), the vast majority of the SNPs have no asso- adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evi- ciated pathology. Indeed, given that TH deletion is asso- dence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron. J Biol Chem 263(35):18621–18626 ciated with embryonic lethality, we anticipate that there Andersson KK, Vassort C, Brennan BA, Que L Jr, Haavik J, Flatmark may be severe consequences for a few genetic variants. T, Gros F, Thibault J (1992) Purification and characterization of Moreover, genetically mediated changes in protein struc- the blue-green rat phaeochromocytoma (PC12) tyrosine hydrox- ture may influence the regulation of this pivotal enzyme ylase with a dopamine-Fe(III) complex. Reversal of the endog- enous feedback inhibition by phosphorylation of serine-40. and so modulate catecholamine levels. For this reason, Biochem J 284(Pt 3):687–695 future work in personalized medicine will need to incor- Apostolova G, Dechant G (2009) Development of neurotransmitter porate knowledge of the regulatory effects of TH SNPs (as phenotypes in sympathetic neurons. Auton Neurosci well as other enzymes) on catecholamine levels in health 151(1):30–38. doi:10.1016/j.autneu.2009.08.012 Aranyi T, Sarkis C, Berrard S, Sardin K, Siron V, Khalfallah O, and disease. Mallet J (2007) Sodium butyrate modifies the stabilizing complexes of tyrosine hydroxylase mRNA. Biochem Biophys Acknowledgments This work was supported by grants from the Res Commun 359(1):15–19. doi:10.1016/j.bbrc.2007.05.025 National Institutes of Health (GM38931) and the Penn State Institute Bademci G, Vance JM, Wang L (2012) Tyrosine hydroxylase gene: for Personalized Medicine (04-017-52 HY 8A1HO; under a grant another piece of the genetic puzzle of Parkinson’s disease. CNS from the Pennsylvania Department of Health using Tobacco CURE Neurol Disord Drug Targets 11(4):469–481 Funds). The PA Department of Health specifically disclaims Bailey SW, Dillard SB, Thomas KB, Ayling JE (1989) Changes in the responsibility for any analyses, interpretations or conclusions. cofactor binding domain of bovine striatal tyrosine hydroxylase at physiological pH upon cAMP-dependent phosphorylation mapped with tetrahydrobiopterin analogues. 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