Abstract
Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is strictly controlled by several interrelated regulatory mechanisms. Enzyme synthesis is controlled by epigenetic factors, transcription factors, and mRNA levels. Enzyme activity is regulated by end-product feedback inhibition. Phosphorylation of the enzyme is catalyzed by several protein kinases and dephosphorylation is mediated by two protein phosphatases that establish a sensitive process for regulating enzyme activity on a minute-to-minute basis. Interactions between tyrosine hydroxylase and other proteins introduce additional layers to the already tightly controlled production of catecholamines. Tyrosine hydroxylase degradation by the ubiquitin–proteasome coupled pathway represents yet another mechanism of regulation. Here, we revisit the myriad mechanisms that regulate tyrosine hydroxylase expression and activity and highlight their physiological importance in the control of catecholamine biosynthesis.
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Introduction
Tyrosine hydroxylase (TH) is an important component of the central and the peripheral nervous systems. It is essential for the production of three catecholamines (dopamine, norepinephrine, and epinephrine) whose functions include modulating autonomic reflexes, behavior, circulatory control, cognition, endocrine function, movement, pain, reward, and vigilance. The catecholamines function as neurotransmitters while norepinephrine and epinephrine, which are released from adrenal medullary chromaffin cells, function as hormones. If one performs a PubMed search using “tyrosine hydroxylase,” 19,972 citations (as of 4/4/2014) are retrieved. These citations encompass disparate subjects including enzyme properties, oxidative stress in neurodegenerative disorders, and the use of this enzyme as a marker of catecholaminergic cells. As would be expected from its functional importance, TH is under strict regulation at several distinct and overlapping levels. We have previously reviewed these mechanisms in detail (Kumer and Vrana 1996). Moreover, selected aspects 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 regulation of TH that includes approximately 200 selected articles since 1996 along with conclusions from several historically important papers.
Catecholamine biosynthesis and physiological relevance
Tyrosine hydroxylase (EC 1.14.16.2) catalyzes the first (Nagatsu et al. 1964) and rate-limiting step in catecholamine biosynthesis (Levitt et al. 1965) (Fig. 1). Tyrosine hydroxylase mediates the reaction of tetrahydrobiopterin (BH4), molecular oxygen, and tyrosine to form l-3,4-dihydroxyphenylalanine (l-DOPA) and pterin-4a-carbinolamine (4a-OH–BH3). Aromatic amino acid decarboxylase, which is the second enzyme in the catecholamine biosynthetic pathway, catalyzes the decarboxylation of DOPA to form dopamine, the first of the neurotransmitters. Dopamine β-hydroxylase catalyzes the conversion of DOPA to norepinephrine within vesicles in a reaction involving molecular oxygen and ascorbate; water and the ascorbate free radical are the other products. The ascorbate free radical is reconverted to ascorbate in a complex process involving cytosolic NADH, cytochrome b561, and intravesicular ascorbate free radical (Diliberto et al. 1991). S-adenosylmethionine is the methyl donor in the reaction converting norepinephrine to epinephrine. The other product, S-adenosylhomocysteine, is reconverted to S-adenosylmethionine in a multistep process.
Complete reaction pathway for the biosynthesis of the catecholamines. The rate-limiting step is the conversion of tyrosine to l-DOPA and is catalyzed by tyrosine hydroxylase. The regulatory events that affect TH are therefore thought to control the synthesis of catecholamines. l-DOPA is then converted to dopamine by AADC, which then is converted to norepinephrine through the action of DBH. In select cells, the norepinephrine is converted to epinephrine by PNMT
The physiological importance of TH is exemplified by its role in development. Disruption of the tyrosine hydroxylase gene in transgenic mice results in mid-gestational 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 nucleotide 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 neurodevelopmental disorder (Segawa disease) that can be managed, in part by administration of levodopa (the product of the TH reaction). (Ichinose et al. 1999; Segawa 2011; Segawa et al. 2003). In spite of the existence of high sequence homology among these enzymes, their modes of regulation, however, differ (Fitzpatrick 1999).
Mechanism of the TH reaction
The reaction mechanism for the hydroxylation of tyrosine has been studied extensively (Fitzpatrick 2003; Roberts and Fitzpatrick 2013). Fitzpatrick (1991) first determined the order of substrate binding to purified rat tyrosine hydroxylase by steady-state enzyme kinetics; the pterin substrate (BH4) binds first, followed by oxygen in rapid equilibrium, and then tyrosine. All three substrates must bind before any chemical reaction occurs. Additionally, a dead-end enzyme-tyrosine complex can form resulting in enzyme inhibition by high concentrations of tyrosine. Tyrosine hydroxylase is able to catalyze the hydroxylation of phenylalanine and tryptophan in addition to tyrosine. The specificity constant (V max/K m) of tyrosine hydroxylase for tyrosine is only 10-fold greater than that of phenylalanine (Daubner et al. 2000) and 30-fold greater than that of tryptophan (Daubner et al. 2002). In contrast, the specificity constant of phenylalanine hydroxylase for phenylalanine 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 His331, His336, and Glu376 in the rat enzyme (Goodwill et al. 1997). Ramsey et al. (1995) reported that each of these residues is required for iron binding and for catalytic activity. When the ferrous iron in TH undergoes oxidation to yield ferric iron, reduced BH4 converts the enzyme back to the active, or ferrous, state (Ramsey et al. 1995). During catalysis, a bridge is formed among pterin, iron, and oxygen, which results in reduction of molecular oxygen to a peroxy-pterin intermediate. This intermediate participates in the hydroxylation reaction as determined with the rat recombinant enzyme (Chow et al. 2009). Dopamine, norepinephrine, and epinephrine inhibit enzyme activity and it has been postulated that such feedback inhibition is physiologically important (Meyer-Klaucke et al. 1996; Nagatsu et al. 1964; Okuno and Fujisawa 1985), a hypothesis that is widely accepted today.
TH gene structure
The human TH gene contains 14 exons. Four different human TH isoforms are formed as a result of the alternative splicing of the hnRNA (Fig. 2). The rat and mouse TH gene contains 13 exons separated by 12 introns. In addition, there are two alternatively spliced isoforms in monkey that correspond to the most common human isoforms (hTH-1 and hTH-2; Ichinose et al. 1993). The TH promoter, which is located upstream of the gene, contains binding sites for several transcription factors (reviewed in Kumer and Vrana 1996; Lenartowski and Goc 2011). During development, TH expression is restricted to several discrete components of the central and peripheral nervous systems and to adrenal chromaffin cells (Schimmel et al. 1999). The balanced production of the different transcription factors mediates tissue-specific expression of the gene (Tinti et al. 1996).
Overview of the different modes of TH regulation. The human TH gene contains 14 exons that are alternatively spliced (to exclude exon 2 [short isoforms] or include exon 2 [long isoforms]). TH gene expression can be regulated by different transcription factors, as well as changes in RNA half-life. Numerous single nucleotide polymorphisms (SNPs) exist in the population that produces variant mRNAs and proteins. Once the transcript is translated, several post-translational modifications can occur to regulate catecholamine biosynthesis
TH protein structure
Native tyrosine hydroxylase exists as a tetramer with a molecular mass of ≈240 kDa (Kumer and Vrana 1996). In mouse and rat, each monomer is composed of 498 amino acids (Grima et al. 1985). The human enzyme consists of four isoforms that result from the alternative splicing of the primary RNA transcript although the primary form is cognate to the rodent enzymes. Each TH monomer consists of three components (Fig. 3). The first 165 residues constitute a regulatory segment, the next 280 residues make up the catalytic domain, and the last 40 residues make up a tetramerization domain (Abate et al. 1988; Fitzpatrick 2003; Liu and Vrana 1991; Lohse and Fitzpatrick 1993; Nakashima et al. 2009; Ota et al. 1995; Walker et al. 1994).
Molecular models prepared from the crystal structure of the rat isoform of tyrosine hydroxylase. The catalytic domain of the protein is about 50 % α-helix, 10 % β-sheet, and 40 % random coil (based on X-ray crystallography). Crystal structures of the regulatory domain, however, are not available. a Linear structure of the monomeric human tyrosine hydroxylase. The figure is prepared with DOG 2.0 software from NM_000360 (NCBI Nucleotide Database). b Molecular model of tetrameric (I–IV) rat tyrosine hydroxylase showing two of the four regulatory segments. The other two regulatory domains, which are in back of the structure, are obscured. c Same view as b depicting the tetramerization domains that are colored coded to match the catalytic domains; the regulatory domains are omitted for clarity. d A view directed into the active site of rat monomeric tyrosine hydroxylase. The active site contains iron and a bound substrate analog (7,8-tetrahydrobiopterin, or BH4). His331, His336, and Glu376, which bind iron, and the cofactors are shown as stick representations. b–d are adapted from Zhang et al. and Jaffe et al. and BH2 of d is superposed from PDB ID 2TOH. Ct carboxyterminus, Nt amino-terminus, TIZ tetramerization segment. The structures were prepared using the PyMOL Molecular Graphics System Version 1.5.0.4 Schrödinger, LLC
The X-ray crystal structure of the catalytic and tetramerization domains of rat (PDB ID 1TOH and 2TOH; Goodwill et al. 1997, 1998) and human (PDB ID 2XSN) TH have been determined. The overall structure of each monomer is a basket-like arrangement of helices and loops with a long carboxyl-terminal α-helix that forms the core of the tetramer (Fig. 3b).
The overall structure of each catalytic domain is a basket-like arrangement of helices and loops with a long C-terminal α-helix which forms the core of the tetramer (Fig. 3). Each monomer contains 16 α-helices and 12 β-strands including those of the regulatory segment (2 α-helices and 4 β-strands). The catalytic domain has a 30-Å wide and 17-Å deep active site within the basket-like arrangement. Site-directed mutagenesis studies have identified amino acid residues that are involved in crucial steps such as iron binding, feedback inhibition, tetramerization, and amino acid hydroxylation (Ellis et al. 2000; He et al. 1996; Quinsey et al. 1996; Ribeiro et al. 1993; Vrana et al. 1994; Yohrling et al. 2000). We should note, however, that crystallization studies have been unable to visualize the structure of the regulatory domain. This may indicate that this segment does not assume a stable conformation. Recently, a segment of the regulatory domain of rat TH has been determined via NMR studies (Zhang et al. 2014).
TH possesses a flexible loop (Fig. 3c) consisting of residues 177–191 that may form part of the tyrosine-binding site (Daubner et al. 2006). The conformation of the PAH homolog of this loop changes when an amino acid is bound, suggesting that this loop plays a role in amino acid binding. However, site-directed mutagenesis studies indicate that specific residues in the loop fail to play a dominant role in determining the amino acid substrate specificity of either TH or PAH. Sura et al. (2006) suggested that pterin binding results in a conformational change of this loop that supports formation of the amino acid binding site in TH. An iron atom is 10 Å below the enzyme surface within the active site cleft. The iron atom is coordinated by His331, His336, and Glu376. Each of these residues has been shown to be required for iron binding and for activity by site-directed mutagenesis (Ramsey et al. 1995). The crystal structure of TH with bound 7,8-dihydrobiopterin (PDB ID 2TOH) reveals no conformational changes when compared with the resting enzyme (PDB ID 1TOH) (Goodwill et al. 1997, 1998). Both of these structures show a five-coordinate ferric iron site with His 331 as the axial ligand and two water molecules joining the protein ligands in the equatorial positions with the iron atom in the plane of the equatorial ligands. Several other prokaryotic and eukaryotic oxygen-utilizing enzymes possess this 2-His-1-carboxylate facial triad (Que 2000). The facial triad binds iron in the active site and leaves the opposite face of the octahedron available to coordinate a variety of exogenous ligands. The biopterin binds close to iron; the 4a-carbon is 5.9 Å from it. 7,8-Dihydrobiopterin binds to several residues in the peptide backbone of TH through hydrogen bonding, while the only interaction with the side chain of an amino acid residue involves that of Glu322.
Zhang et al. (2014) compared the structure of the phosphorylated and unphosphorylated regulatory segment by NMR. These peptides exhibited the same chemical shifts except for Gly36, Arg37, Gln39, Ser40, Leu41, Ile42, and Glu43. The authors conclude that the core structure of the regulatory segment of TH remains the same as that in the unphosphorylated segment and a local structural change takes place around Ser40. At the present time there is no structural information on the catalytically relevant ferrous form of resting TH or of TH with tyrosine or tyrosine plus pterin bound. It would be of value to determine these structures and those of the unphosphorylated and Ser40-phosphorylated holoenzymes. Thus far structural studies aimed at defining that the structures of unphosphorylated and phosphorylated TH holoenzymes have been problematic.
The tetramerization domain of tyrosine hydroxylase consists of residues 457–498 and is formed by two β-strands and a 40 Å-long α-helix (Goodwill et al. 1997, 1998) (Fig. 3b). Based on the primary structure of the aromatic amino acid hydroxylases, Liu and Vrana hypothesized that these enzymes assemble into tetramers through the involvement of coiled-coil interactions of the carboxyl-terminal (Liu and Vrana 1991), which was then confirmed experimentally by deletion mutagenesis studies (Lohse and Fitzpatrick 1993; Vrana et al. 1994). Later crystallization data confirmed this notion and demonstrated that the 40-Å long α-helix forms a coiled-coil at the core of the tetramer (Goodwill et al. 1997) as depicted in Fig. 3. Deletion of the carboxyl-terminal 19 residues results in a protein that migrates as a dimer while the wild-type enzyme migrates as a tetramer (Yohrling et al. 2000). The tetramer consists of a dimer of dimers (I and II with III and IV). Subunits I and II interact via a salt bridge (Lys170–Glu282) and their tetramerization domains are in contact (Fig. 3c). The only contact between subunits I and III is by their tetramerization domains, and subunits I and IV have no direct contact. Zhang et al. found that residues 1–72 of the amino-terminal regulatory segment (residues 1–159) were unstructured. They performed more extensive studies on the regulatory segment consisting of residues 65–159. This segment forms a dimer in solution and contains an ACT domain (named after aspartate kinase, chorismate mutase and TyrA, or prephenate dehydrogenase), which consists of 2 α-helices and 4 β-strands. The secondary structure of the ACT domain consists of a β1-sheet, an α1-helix, the β2- and β3-sheets, the α2-helix and finally the β4-sheet (Fig. 3d). Zhang et al. combined their structural information with that of Jaffe et al. on phenylalanine hydroxylase and arrived at the model shown in Fig. 3b, c (Zhang et al. 2014; Jaffe et al. 2013). Two regulatory segments are in front of the complex (from subunits I and IV) and two are in the back of the complex (II and III) that cannot be seen.
X-ray studies identified hydrogen bonds and a salt bridge from Glu282 of one subunit to Lys170 in the adjacent subunit as a dimerization interface between the two subunits. Point mutants of residues that form the salt bridge result in a protein that migrates as a dimer, thus indicating that this salt bridge plays an essential role in the formation of a tetramer (Yohrling et al. 2000). Deletion of the carboxyl-terminal residues results in the formation of a protein that migrates as a dimer or monomer, while the wild-type enzyme migrates as a tetramer (Lohse and Fitzpatrick 1993; Vrana et al. 1994; Yohrling et al. 2000). Although it is unclear whether the monomers directly form a tetramer or they first form dimers that then come together to form a tetramer, the native enzyme exists as a tetramer. A point mutation (Leu435Ala) or deletion of multiple residues in the long carboxyl-terminal helix results in the formation of a protein dimer (Vrana et al. 1994; He et al. 1996).
Overview of TH regulation
Owing to the physiological importance of the catecholamine end products, TH is under stringent control at the transcriptional, translational, and post-translational levels (Kumer and Vrana 1996) (Fig. 2). An important element necessitating strict control of TH is the observation that catecholamines undergo reactions with molecular oxygen to generate toxic catechol-quinones (Hastings and Zigmond 1994). These reactions occur at neutral pH, but not under acidic conditions. Cells minimize the generation of these toxic metabolites by maintaining production of catecholamines at near-optimal levels, by providing on-demand regulation of synthesis, and by storing them in acidic synaptic vesicles. TH gene expression is strictly controlled by numerous promoter sequences that are located upstream of the 5′ transcription initiation site (Lenartowski and Goc 2011). These elements mediate quantitative tissue-specific expression and maintenance of TH (Kessler et al. 2003). Alterations in TH mRNA stability and the existence of different TH mRNA transcripts that are produced by alternative hnRNA splicing provide additional avenues for regulation (Kumer and Vrana 1996).
Four human TH mRNAs and proteins occur physiologically, and several others are observed under pathological conditions. Expression can also be regulated in a cell type-specific fashion by neuropeptides and by depolarization (Zigmond 1998). TH regulation at the protein level is mediated by phosphorylation, dephosphorylation, changes in enzyme stability, substrate inhibition, and feedback inhibition by catecholamines (Daubner et al. 2011; Daubner and Piper 1995; Nakashima et al. 2002). Phosphorylation occurs at four different serine residues (Campbell et al. 1986), which activate the enzyme and alter its stability (Dunkley et al. 2004; Fujisawa and Okuno 2005; Martinez et al. 1996; Zigmond et al. 1989). These regulatory mechanisms will each be discussed in the following sections.
Overview of transcriptional regulation of the TH gene
TH plays important physiological roles in both the central and peripheral nervous systems and is under dynamic developmental and environmental transcriptional control. In the CNS, TH is expressed in dopaminergic cell-containing areas including the substantia nigra and ventral tegmental area in the midbrain, the diencephalon, the olfactory bulb, and retinal amacrine cells. TH is also expressed in adrenergic and noradrenergic cells found in the hypothalamus, medulla, and locus coeruleus (LC). TH expression occurs peripherally in sympathetic ganglia and also in adrenal chromaffin cells (Kumer and Vrana 1996).
TH gene transcription, mRNA stability, and SNP variants participate in the regulation of TH expression both temporally and spatially. These modalities of TH regulation have been reviewed in detail (Kumer and Vrana 1996; Lenartowski and Goc 2011). The TH promoter contains numerous transcription factor-binding sites that permit mRNA levels to be regulated by Ca2+, glucocorticoids, neuronal activity, oxygen levels, and stress. Extracellular stimuli acting through several protein kinases mediate transcriptional regulation of TH (Hebert et al. 2005; Seta and Millhorn 2004; Suzuki et al. 2004). Stress increases TH expression by increasing both TH gene transcription and mRNA stability (Tank et al. 2008). Recent advances in these regulatory mechanisms will be addressed in turn.
Tissue-specific and developmental regulation of TH gene expression
There is developing knowledge of the DNA elements and regulatory proteins responsible for tissue-specific expression of TH. Transgenic mice containing the 9-kb 5′-upstream segment of the rat TH promoter fused to the β-galactosidase reporter gene, but not mice bearing 0.15 or 2.4 kb of 5′ flanking sequence, are able to express the reporter at levels equivalent to endogenous TH in central catecholaminergic cells (Min et al. 1994). The authors concluded that the crucial catecholaminergic neuron-specific DNA elements reside between −9 and −2.4 kb of the 5′ flanking region of the TH gene. Moreover, stimuli that increase or reduce TH levels produce parallel changes in β-galactosidase expression in various brain regions in transgenic mice bearing the 5′ 9-kb rat TH promoter region indicating that this promoter sequence mediates cell type-specific regulation of reporter gene expression (Min et al. 1996).
Dopaminergic TH mRNA expression
Nuclear receptor related-1 (Nurr1) is an orphan nuclear receptor that is required for the development and maintenance of mouse midbrain dopaminergic neurons (Zetterstrom et al. 1997). Nurr-1 deficient mice fail to generate these midbrain neurons, are hypoactive, and die shortly after birth. Moreover, Nurr-1 expression mediates dopaminergic-specific TH gene expression. Satoh and Kuroda (2002) reported that Nurr1 interaction with the TH promoter is mediated by PKA and PKC in NTera2 human embryonic stem cells over-expressing the receptor protein. In rats, Nurr1 can bind directly to the TH promoter at any of three sites that are located 1 kb upstream of the transcription start site, and it increases gene expression in progenitor cells during their differentiation into midbrain dopaminergic neurons (Sakurada et al. 1999; Kim et al. 2003). Schimmel et al. (1999) reported that about 4.5 kb of the rat TH promoter is required for TH expression during development and that this region contains consensus sequences for neural restrictive silencer element (NRSE), Nurr1, Pitx1/3 and Gli1/2. The rat promoter contains a Nurr1 response element (Iwawaki et al. 2000). Jacobsen et al. (2008) identified a point mutation in a Nurr1 ERK1/2 phosphorylation site (Ser125Cys) in a Parkinson’s disease patient. They demonstrated that this mutation attenuated Nurr1-induced transcriptional activation in human neuroblastoma cells (SK-N-AS). Following activation, they found that ERK1/2 proteins enhance transcriptional activation by wild-type Nurr1, but not the Ser125Cys mutant.
A Pitx3 site is found 50 bp upstream of the transcription initiation site in the rat TH promoter (Cazorla et al. 2000) (Fig. 4). The mouse TH promoter also contains a Pitx3 response element while lacking a consensus sequence for Nurr1 (Lebel et al. 2001). In mice, deletion of Pitx3 results in the loss of TH in midbrain dopaminergic neurons (Maxwell et al. 2005). Nurr1 and Pitx3 are both required for the differentiation of mouse and human embryonic stem cells into dopaminergic neurons (Martinat et al. 2006). However, there was no difference in the levels of TH expression in human and mouse embryonic stem cells when these transcription factors are expressed alone or in combination (Messmer et al. 2007). Jacobs et al. (2009) suggested that Nurr1 is bound by a repressor protein and upon interaction with Pitx3, Nurr1 is released, allowing it to act on the TH promoter. In addition to Pitx3, Brn4 can also work in concert with Nurr1 to mediate dopaminergic neuronal differentiation (Tan et al. 2011). Stott et al. (2013) reported that Foxa1 and Foxa2 transcription factors are required for the development and maintenance of the dopaminergic phenotype in mouse. Deletion of Foxa1 and Foxa2 significantly reduces the number of TH positive dopaminergic neurons, which they report is due to a reduction in the binding of Nurr1 to the promoter region of TH.
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)
Lazaroff et al. (1998) compared the expression of a transiently transfected chloramphenicol acetyltransferase (CAT) reporter gene under the transcriptional control of the TH 5′ flanking DNA in undifferentiated and differentiated mouse CNS CAD (Cath.a-differentiated) cells and found that TH expression varied as a function of their differentiated state. They reported that the cAMP response element (CRE) and the AP-1 site participate in TH expression in both states. In undifferentiated cells, however, the dyad/E-box element represses expression of the reporter gene to 25–33 % that of the differentiated cell. This corresponds to the decrease in TH protein that occurs in undifferentiated cells. Based upon immunoreactivity, Ghee et al. (1998) reported that CRE-associated transcription factors exist and regulate TH expression in rat midbrain dopaminergic cells, but that these cells lack Fos. In contrast, Fos expression in the olfactory bulb parallels TH expression while CRE-associated transcription factors remain constant. They conclude that Fos and CREB make differential contributions to TH gene activity in different cells.
Noradrenergic and adrenergic TH mRNA expression
Although many studies on the regulation of TH expression have focused on dopaminergic cells, owing to their importance in the pathogenesis of Parkinson’s disease, an extensive literature also exists on the regulation of TH in adrenergic and noradrenergic phenotypes, mainly focusing on the peripheral nervous system development (Apostolova and Dechant 2009; Ernsberger 2001).
The regulation of the rat promoter in vivo depends on the duration and nature of a stimulus. Sun et al. (2003) reported that chronic nicotine administration (14 days), acting through cholinergic receptors, results in a sustained increase in rat TH mRNA, protein, and activity in the adrenal medulla. A sustained transcriptional rate that lasted 1 week following chronic nicotine treatment correlated with a modest increase in adrenal TH gene AP-1 binding, but not in the levels of Fra-2 or other Fos or Jun proteins. A single nicotine injection elicited only a small and transient increase in TH mRNA, but not protein. Sun et al. (2004) found that chronic nicotine treatment induced TH mRNA and protein in rat LC neurons. These increases lasted for 3 days in LC cell bodies, but for at least 1 week in LC nerve terminals. In contrast to the adrenal medulla, these investigators found no sustained transcriptional response in the LC and suggested that post-transcriptional mechanisms may play a role in this long-term response.
The response to acute and chronic stimuli may be mediated through the differential activation of several transcription factors. For example, Sabban et al. (2004) reported that the AP-1 factor Fra-2 increases in the rat adrenal medulla (but not in LC) after 6 days of repeated immobilization stress. However, Fos increases in both the adrenal medulla and LC following acute immobilization stress. The changes of Fos and Fra-2 observed in this study, but not in response to chronic nicotine treatment (Sun et al. 2003), most likely reflect the complexity of the immobilization stress paradigm. In the same study by Sun et al., both acute and chronic immobilization stresses increase early growth response protein 1 (Egr1) in the medulla, but not in the LC. Furthermore, short- and long-term immobilization increases CREB phosphorylation in the LC. In studies from the same laboratory, Hebert et al. (2005) observed an increase in Fos levels and CREB phosphorylation following a single immobilization without changing the expression of Egr1, Fra-2, or phosphorylated activating transcription factor-2. Repeated immobilization induced Fos and Fra-2. These investigators reported that repeated immobilization stress increased phosphorylation of several mitogen-activated protein kinases (MAPK) including p38, c-Jun N-terminal kinases (JNK1/2/3) and extracellular signal-regulated kinases (ERK1/2). It is clear that transcription factor binding to the promoter region in TH is an important regulatory mechanism that determines catecholamine levels in different tissues. Not surprisingly, however, the specific elements are different depending on the type of cells and their functions.
TH promoter and regulatory elements
Regulatory elements in the TH promoter include the glucocorticoid response element (GRE; 450 bp upstream), activator protein-1 (AP-1; 200 bp upstream), cAMP response elements (CRE1/2; 40 and 90 bp upstream), a cis-acting dyad element (200 bp upstream), and an inhibitory heptamer sequence (HEPT; 160 bp upstream) along with other factors, that are depicted in Fig. 4, and initially discussed in Kumer and Vrana (1996). We will describe recent studies investigating the regulatory elements in the TH promoter and other regions on TH mRNA. The sections below will focus on some of the well-established stimuli and the promoter sequences through which they act. Interestingly, studies with the rat promoter display a cooperation between different regulatory sequences. However, it should be noted that there are differences in the types of promoter elements utilized by the rodent and human promoter, as will be discussed below.
Ca2+ and cAMP
Nankova et al. (1996) measured the induction of the bacterial CAT reporter fused to wild-type or mutant 5′ flanking sequences of the rat TH gene in rat PC12 cells in response to the Ca2+ ionophore, ionomycin. Point mutations in CRE abolished the ionomycin-induced reporter gene induction that was not overcome by an intact AP-1 site. These investigators found that ionomycin rapidly increased the phosphorylation of the CREB transcription factor. KN62, which is a CaMPK inhibitor, prevented the ionomycin induction of the reporter gene. These investigators concluded that Ca2+ activation of the rat promoter is mediated mainly through CaMPKII and CREB phosphorylation.
Nagamoto-Combs et al. (1997) studied constructs of the CAT reporter gene expressed in rat PC12 cells in response to 50-mM KCl-induced Ca2+ influx. They found that TH reporter gene induction by KCl is dependent upon both CRE and the AP-1 sites. Both sites are required for the KCl induction, but either site will support induction mediated by the A23187 calcium ionophore. In a CREB-deficient PC12 cell line, the response to cAMP is greatly inhibited, but the response to A23187 is robust. Thus, calcium-dependent increases in TH expression can also occur through a mechanism that is independent of CREB phosphorylation, suggesting that there are multiple pathways that regulate calcium-dependent TH expression. Both PKA and CaMPKII catalyze the phosphorylation of CREB and increase TH mRNA expression.
Approximately 40 bp upstream of the transcription start site on the rat TH promoter is a CRE/CaRE site that regulates TH expression in rat PC12 cells (Osaka and Sabban 1997) (Fig. 4). CRE is one of the most highly studied and important factors for the control of TH expression, and its significance has been established by mutagenesis of a rat TH promoter CAT reporter gene construct transfected in the human SK-N-BE(2)C neuroblastoma cell line (Tinti et al. 1997). A second rat CRE site about 95 bp upstream of the transcription start site can also be activated by phorbol esters in a rat CAT reporter construct expressed in rat PC12 cells (Best and Tank 1998). In the mouse, ATF-2 binds to the CRE site to regulate TH promoter activity (Suzuki et al. 2002). Increases in cAMP and activation of PKA can also result in the repression of the TH promoter through inducible cAMP early repressor (ICER) as demonstrated in transfected PC12 cells (Tinti et al. 1996). Hiremagalur et al. (1993) reported that nicotine treatment of PC12 cells for 1–2 days increased both TH and dopamine β-hydroxylase mRNA levels. Mutagenesis of the CRE site abolished the response to nicotine as determined in TH reporter constructs. Nicotine treatment elevated intracellular Ca2+ in PC12-derived cells lacking PKA, but it failed to induce TH mRNA levels. These results indicate that PKA is required for the nicotine-stimulated TH induction (Nankova et al. 1996).
In addition to their separate roles, AP-1 and CRE can work in concert with a partial dyad element (the partial dyad involves symmetry between the sequences GAATAC and GTATTC that is hypothesized to mediate positive transcriptional regulation) that regulates basal rat TH expression in seven TH-expressing cell lines: CATH.a and CATH.b (mouse CNS tumor), PATH.2 (mouse adrenal tumor), CAD (mouse CNS), PC12 (rat adrenal medulla tumor), SK-N-BE(2)C (human neuroblastoma), and B103 (rat CNS tumor) (Patankar et al. 1997). The partial dyad is required for TH expression in cell lines that rely on CRE or the AP-1 dyad element. The partial dyad is not required for the cAMP-mediated TH induction in PC8b cells (derived from PC12 cells) or in CATH.a cells, nor is it required for the KCl induction of TH in CATH.a cells.
Calcium and cAMP are important regulators of TH promoter activity. As discussed, they work through multiple regions on the promoter. In addition, they are also involved in several other cellular events, which make the regulation of TH gene expression more complex. Even though these stimuli have been characterized rather early on, there is still more to be known regarding their role in the maintenance of catecholamine production.
Phorbol esters
Yang et al. (1998) identified seven rat cis-regulatory elements by DNAse I footprint analysis from extracts prepared from SK-N-BE(2) and CATH.a cells within the first 0.5 kb upstream promoter region. They found that the element located from −124 to −107 bp proximal to the transcription start site interacts with Specificity protein 1 (Sp1) and contributes to the transcriptional activation of the TH gene in cooperation with CRE. Papanikolaou and Sabban reported that the Sp1 region and an Egr1 motif (Fig. 4) of the TH promoter bind rat adrenomedullary protein factors following immobilization stress (Papanikolaou and Sabban 1999, 2000). They also found that phorbol esters increase Egr1/Sp1 response element binding activity, and that this can work in concert with the AP-1 element.
Nakashima et al. (2003) found that phorbol esters induce both Egr1 and AP-1 factors in rat PC12 cells. They also reported that the insertion of 10 bp between the Spr/Egr1 and AP-1/E-box reduced the ability of EGR1 to upregulate luciferase reporter activity controlled by the proximal 272 nucleotides of the rat TH promoter in these cells. Although there is no competition between the Sp1/Egr1 site and the AP-1 site, the Sp1/Egr1 site can still be acted upon by AP-1 factors to modulate the rat TH promoter linked to a luciferase reporter gene. This observation is supported by the finding that binding to Egr1 increases upon the treatment of the tumorigenic mouse brain noradrenergic CATH.a cell line with phorbol esters (Stefano et al. 2006).
Piech-Dumas et al. (2001) found that both wild-type AP-1 and CRE sites are required for the complete activation of gene expression by phorbol esters in rat PC12 cells. Phorbol esters lead to the phosphorylation of CREB and to the activation of PKA. Phorbol esters also lead to the activation of ERK1/2 following activation of PKC (rev. in Roskoski 2012), and ERK1/2 activation may also participate in regulating TH expression. AP-1 activation occurs via stimulation of the upstream ERK1/2 pathway in neurons of the developing rat brain striatum (Guo et al. 1998). Cazorla et al. (2000) reported that CRE in mouse neuroblastoma Neuro2A cells does not interact with the previously mentioned Pitx3 site. It appears, therefore, that there may be several differences regarding the inter-species mechanisms for the regulation of TH gene expression as we note for the human gene below.
Hypoxia
In rat PC12 cells, hypoxia increases Fos expression, AP-1 promoter activity, and results in increases in TH mRNA levels (Mishra et al. 1998). Fos expression is required in depolarization and phorbol ester activation of this promoter (Sun and Tank 2003). However, this hypoxic response is not observed in a human neuroblastoma cell line. Hypoxia also increases CRE activity to increase TH mRNA levels (Beitner-Johnson and Millhorn 1998). A study investigating the effect of multiple stimuli on rat TH promoter activity demonstrated that basal expression is mediated through the partial dyad involving CRE and AP-1 elements while inducible expression occurs mainly via CRE and to a lesser extent through AP-1 and hypoxia response element 1 (HRE1) (Lewis-Tuffin et al. 2004). Moreover, Rani et al. (2009) found a novel 7-bp AP-1 like element about −5.7 kb upstream from the TH transcription start site that mediates the response to glucocorticoids in rat PC12 cells transfected with a rat 9-kb TH promoter-luciferase construct. Deletion of all but 100 bp surrounding the −5.7 kb upstream sequence from the 9 kb construct resulted in a promoter that fully maintained the response to dexamethasone, providing strong evidence that this sequence is responsible for glucocorticoid sensitivity.
von Hippel–Lindau protein (VHL) is a tumor suppressor protein, and its loss is frequently observed in vascular tumors of the CNS, renal cell carcinomas, and pheochromocytomas (Chou et al. 2013). Overexpression of VHL in rat pheochromocytoma PC12 cells decreases TH mRNA levels by reducing mRNA elongation (Kroll et al. 1999). Inhibition of the endogenous levels of VHL in PC12 cells with antisense RNA results in an increase in TH expression (Bauer et al. 2002). This increase may be mediated through the inhibition of a response that results in increased ubiquitination and degradation of hypoxia-inducible transcription factors (HIFs) that normally bind to the hypoxia-responsive element (HRE-1) (Fig. 4) and activate the TH promoter (Schnell et al. 2003).
Gender
Gender also plays a role in the differential regulation of TH expression. Sry, a gene found on the Y chromosome, encodes a protein that binds to the rat TH promoter (presumably to an AP-1 site) about 300 bp upstream of the transcription start site (Milsted et al. 2004). Sry is expressed in catecholaminergic regions in male, but not female, rats. Cotransfection of rat PC12 cells with an expression vector for Sry and a luciferase reporter construct containing 773 of the proximal nucleotides of the TH promoter led to the elevation of reporter activity. However, a reporter construct lacking the canonical Sry site also responded to Sry. Mutation of the AP-1 site in the TH promoter reduced induction suggesting that regulation occurs primarily at this motif.
Estrogen and progesterone participate in the regulation of TH expression. Estradiol increases promoter activity in rat PC12 cells transfected with estrogen receptors-α/β (ER-α/β) through an estrogen-response element (ERE) that overlaps with the CRE/CaRE site (Maharjan et al. 2005) (Fig. 4). The action of 17 β-estradiol can also be mediated at the plasma membrane as demonstrated by experiments performed with an impermeable estradiol derivative (Maharjan et al. 2010). An estradiol/ER-α complex can activate PKA/MEK signaling that results in the phosphorylation of CREB and the activation of CRE/CaRE, where MEK refers to Mitogen-activated/ERK Kinase (Roskoski 2012). Progesterone receptors also bind to the TH promoter 1.4 kb upstream of the transcription start site in experiments performed with mouse neuronal CAD cells (Jensik and Arbogast 2011). In addition, intracerebroventricular injection of progesterone receptor antisense RNA decreases progesterone receptor levels and increases TH levels in rat hypothalamus (Gonzalez-Flores et al. 2011). The latter authors suggest that their findings indicate a direct role for progesterone in the neuroendocrine regulation of dopaminergic neurons in the CNS through progesterone receptors.
Introns as regulators of TH expression
Kelly et al. (2006) replaced the first exon and first intron of one allele of the mouse TH gene with yellow fluorescent protein. The reporter gene failed to identify functional TH-expressing cells with complete accuracy in a mouse transgenic knock-in model. These investigators suggested that the first intron of the mouse TH gene functions as a cis-regulatory element. Romano et al. (2007) analyzed the transcriptional profile of the promoter, the first exon, and the first intron of the human TH gene in human neuroblastoma BE(2)-C-16 cells. The addition of a 1.2-kb fragment of the first intron enhanced transcriptional activity of the recombinant promoter.
Epigenetic regulation
The epigenetic states of the chromosome and histone acetylation are also important factors in determining TH transcription levels (Lenartowski et al. 2003). In addition to the investigation of the role of introns, Romano et al. (2007) also found, through chromatin immunoprecipitation, that extensive histone H3 and H4 acetylation occurs in nucleosomes isolated from the TH promoter region of BE(2)-C-16 cells. In human renal carcinoma 293FT cells that fail to express TH, histone acetylation in the TH promoter region was minimal. The effect of acetylation on the regulation of TH mRNA levels occurs through regulation of RNA stability, as TH mRNA stability was shown to decrease upon treatment of PC12 cells with sodium butyrate, a histone deacetylase inhibitor (Aranyi et al. 2007). Detailed discussion of these mechanisms and their physiological and developmental roles is provided elsewhere (Lenartowski and Goc 2011).
Human TH promoter
Although most transcriptional regulation studies have been performed in rat and mouse models, important differences exist in human and rodent TH promoters. Studies of the human TH promoter in human neuroblastoma cell cultures indicate that nucleotides 513 bp upstream of the transcription initiation site and 976 bp downstream from the 3′ end are required for TH expression (Gardaneh et al. 2000). However, transgenic mice that contain the 5′ 11 kb region of the human TH promoter express a reporter in TH positive cells in vivo (Kessler et al. 2003). Sequence analysis of this region led to the identification of five common consensus sites that are similar to those of rat and mouse sequences: GR, AP-1, HOXA4/HOXA5, TBF-1, and AP-3. The nearest (GR) is 2.3 kb and the farthest (AP-3) is 8.8 kb from the transcription start site.
Constructs prepared with minimal promoters that contain the five conserved consensus sites were able to drive TH expression in a commercially available human neuronal progenitor cell line, but they failed to do so in mouse primary striatal or substantia nigral cells in culture (Romano et al. 2005). This finding indicates that there are differences in the promoter-based regulation of TH gene expression in human and mouse cells and suggests that extra caution be used when extrapolating from rodent to human biology. Despite its important developmental role in rodents, studies from the same laboratory indicate that Nurr1 lacks a consensus binding site on the human TH promoter (Jin et al. 2006). This is in agreement with the finding of Satoh and Kuroda (2002) who reported that human neuroblastoma SK-N-SH cells fail to express Nurr1 mRNA.
A neuron-restrictive silencer element/repressor element 1 (NRSE/RE1) occurs about 2 kb upstream in the human TH promoter, binds neuron-restrictive silencer factor/repressor element transcription factor (NRSF/REST) and inhibits TH mRNA synthesis in human HB1.F3 neural stem cells (Kim et al. 2006). This repression is alleviated by either mutating or deleting NRSE/RE1. In SH-SY5Y human dopaminergic neuroblastoma cells that express TH, mutating or deleting NRSE/RE1 has no effect on TH expression, implying that this element regulates TH expression in a cell type-specific manner.
Despite the myriad studies available on the structure and regulation of the rodent promoter, few data are available regarding the control of human TH expression. This presents a strong need in the field for the understanding of the TH promoter and how it will affect TH gene regulation. Having insight into these mechanisms will allow us to be able to propose ways to reach optimal levels of catecholamines in both the central and peripheral nervous systems. In addition, such knowledge may also contribute to a further understanding of the pathological conditions that affect catecholaminergic tissues.
Regulation of TH mRNA stability
TH mRNA can be stabilized by interacting with selected proteins, leading to increased translation of TH and, hence, increased TH protein and activity (Roe et al. 2004). Expression of these mRNA-binding proteins and their combined interaction with TH mRNA is altered by cellular activity. Several TH splice variants have been identified, and their role in regulating and maintaining catecholamine levels are reviewed here (and will be discussed in detail in the following sections) and have been addressed elsewhere (Haavik et al. 2008; Kobayashi and Nagatsu 2005; Willemsen et al. 2010).
Several factors that control TH promoter activity regulate mRNA stability including hypoxia and stress (reviewed in Kumer and Vrana 1996). The stabilization of rat PC12 cell TH mRNA occurs through binding of hypoxia-inducible protein (HIP) to a 27-bp binding site (HIPBS) in the 3′-UTR (untranslated region) of TH mRNA (Czyzyk-Krzeska and Beresh 1996). The HIPBS element is conserved in bovine, human, mouse, and rat TH mRNA. This 27-bp stabilizing domain contains a pyrimidine-rich sequence that increases rat TH mRNA stability in PC12 cells during hypoxic conditions (Paulding and Czyzyk-Krzeska 1999). Sustained hypoxia increases TH mRNA in rat cerebral cortex, which is expected with increased mRNA stability (Gozal et al. 2005). The levels of TH protein measured immunochemically, however, remain unchanged. The reason for the discrepancy between TH mRNA and protein levels is unclear. However, tyrosine hydroxylase activity is increased in response to hypoxia owing to the phosphorylation of Ser40 as described later.
Nicotine treatment of bovine adrenal chromaffin cells increases TH mRNA stability and induces the binding of novel proteins to the 3′ UTR (Roe et al. 2004). Long-term stress also induces protein binding to the poly-pyrimidine rich region in the 3′-UTR (Tank et al. 2008). Chen et al. (2008) reported that cAMP leads to the induction of TH protein and activity, but not TH mRNA, in rat ventral midbrain slice cultures and in MN9D cell cultures, which are hybrids of mouse neuroblastoma and embryonic mouse mesencephalic neurons. cAMP increases the translational activation of TH mRNA that is mediated by sequences within the 3′-UTR. These authors suggest that cAMP-dependent increases in expression of stabilizing trans-activating factors occur in these rodent systems. Xu et al. (2009) reported that cAMP increases levels of the poly(C)-binding protein-2 (PCBP2) in MN9D cells. PCBP2 binds to the poly-pyrimidine rich region of the 3′-UTR and thereby stabilizes TH mRNA. It previously has been suggested that 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 the promoter activity can regulate mRNA stability.
Alternative RNA processing
TH exists in various isoforms in a few species, including humans, as a result of alternative splicing of hnRNA (Kumer and Vrana 1996). Such splicing changes the regulatory, but not catalytic domains of TH. Mogi et al. (1984) purified tyrosine hydroxylase from human adrenal medulla and resolved two fractions with different specific activities. This was the first suggestion of the existence of different TH isoforms. Two splice variants occur in non-human primates and Drosophila (Birman et al. 1994; Ichikawa et al. 1990; Ichinose et al. 1993; Lewis et al. 1994). Two splice variants have also been reported in rat (Laniece et al. 1996), but this finding has not been corroborated (Haycock 2002b; Ichikawa et al. 1990).
Human TH has four splice variants that are denoted as hTH-1, hTH-2, hTH-3, and hTH-4, and were named in the order that they were discovered. hTH-1 is composed of 13 exons, consists of 1,494 nucleotides and shows 89 % identity to its rat counterpart. Specifically, hTH-1 is the most abundant isoform and the closest homolog to the rat enzyme (the rat enzyme is 498 and the human ortholog is 497 amino acids long). hTH-2, hTH-3, and hTH-4 contain the addition of 4, 27, and 31 (4 + 27) amino acids, respectively (derived from exon 1, plus or minus exon 2) (Grima et al. 1987; Kaneda et al. 1987). Computer-assisted analysis of the secondary structure of the primary RNA transcript led to the prediction of four stable hairpin loops in introns 1 and 2 (Kobayashi et al. 1988). This analysis indicates that these secondary structures may account for the inclusion/exclusion of exon 2 that occurs during hTH-1 and hTH-2 generation. Other minor TH mRNAs were identified that lack exons 3, 4, 8, and 9 (Bodeau-Pean et al. 1999; Ohye et al. 2001; Parareda et al. 2003). Splicing patterns resulting in the major mRNAs and TH protein isoforms are shown in Fig. 5.
a Schematic representation of the differential regulation of TH through several protein kinases and phosphatases. b Alignment of the first 50 amino acids of different rat and human TH mRNAs. Human TH lacks one of the serines (position 8) that is present in the rat mRNA. In addition, while hTH2 contains the equivalent of serine 31, it is not phosphorylated by ERK1/2. Note that the most abundant isoform (hTH-1) is also most closely homologous to the rat TH protein. The sequences were obtained from NCBI Nucleotide Database (rTH: NM_012740.3, hTH-1: NM_000360.3, hTH-2: XM_005253099.1, hTH-3: NM_199293.2, hTH-4: NM_199292.2)
All of the human TH isoforms are found in adrenal chromaffin cells (Haycock 1991) and in brain catecholaminergic neurons with hTH-1 and hTH-2 accounting for about 90 % of human brain TH (Lewis et al. 1993). Moreover, all isoforms are expressed in human pheochromocytomas (Haycock 2002b) and neuroblastomas (Haycock 1993). Among the major four isoforms, hTH-1 and hTH-2 mRNA transcripts are detected in highest abundance (Ichinose et al. 1994). Human pheochromocytomas contain the highest relative abundance of hTH-3 and hTH-4 (Coker et al. 1990; Grima et al. 1987; Haycock 1991, 1993; Le Bourdelles et al. 1988; Lewis et al. 1993). Several additional isoforms occur in human neuroblastomas and in brain samples from patients with progressive supranuclear palsy (Bodeau-Pean et al. 1999; Dumas et al. 1996; Ohye et al. 2001; Parareda et al. 2003; Roma et al. 2007).
After identification of different TH mRNAs, investigators sought to characterize enzymes produced from the different splice variants. Eukaryotic expression systems such as COS cells and Xenopus oocytes have been utilized, along with expression of the enzyme in E. coli (Horellou et al. 1988; Kobayashi et al. 1988). In spite of varying specific values for enzyme activities that most likely reflect different assay conditions, all reports indicate that hTH-1 has the highest specific activity. Nasrin et al. (1994) reported a regulatory effect of BH4 on enzyme activity at high concentrations of tyrosine substrate. These investigators reported that hTH-2 and hTH-4 are more stable at elevated temperatures than hTH-1 and hTH-3. Bodeau-Pean et al. (1999) found an isoform that lacks exon 3 in a human neuroblastoma that has 30 % of the activity of hTH-1 but exhibits a tenfold increase in its K i for dopamine.
The physiological significance of the four human TH isoforms remains unclear. Although hTH-1 has the highest specific activity, it is not that much different from that of the other isoforms. Perhaps the major difference in the isoforms is the sequence variation in the regulatory ERK1/2 protein kinase Ser31 phosphorylation site (Kumer and Vrana 1996), which is described below.
Feedback inhibition of TH
Inhibition of TH by pathway end products is an important regulatory mechanism that acts as a sensor to maintain required levels of the catecholamines. Excessive catecholamines can be harmful, as these substances have been shown to form toxic quinones as noted earlier. For example, dopamine forms reactive quinone metabolites (Hastings and Zigmond 1994; reviewed in Stokes et al. 1999). Reactive quinones (1) form reactive oxygen species, (2) mediate the covalent modification of DNA and proteins, and (3) activate apoptotic pathways (Stokes et al. 1999). Direct effects of catechol-quinones on TH protein have been characterized in vitro (Kuhn et al. 1999; Xu et al. 1998a). The extent that such modifications occur in vivo is unclear.
Dopamine exerts direct inhibitory effect on TH at concentrations that are in the nanomolar range. This low K i of TH for DA allows for the strict control of intracellular catecholamine levels. Feedback inhibition of TH can be considered in two parts. The first is concentration dependent, reversible, and results from direct binding of DA to TH. DA is a competitive inhibitor of TH against BH4 and a non-competitive inhibitor against tyrosine (Fitzpatrick 1988). Binding of DA prevents BH4 from binding to the active site, thereby causing a dramatic increase in the K m value for this substrate (Ribeiro et al. 1992). The second mechanism of inhibition of TH by DA results from the formation of a tight enzyme–iron–catecholamine complex. DA interacts with the active site iron atom, but only when the iron is in its ferric form (Andersson et al. 1988; Okuno and Fujisawa 1985; Ramsey and Fitzpatrick 1998). The ferric form occurs as a result of oxidation by molecular oxygen in the cell or during enzyme isolation. DA binds tightly to ferric iron, which is located in the active site cleft. As described above, TH requires its iron atom to be in the ferrous form during the reaction. The pterin co-substrate reduces the ferric iron to enable formation of an active enzyme.
Alterations in feedback inhibition occur as a result of enzyme phosphorylation as catalyzed by various protein kinases as noted here and in the following section. PKA catalyzed phosphorylation of TH at Ser40 increases the K i value for DA and decreases the K m for BH4 (Daubner et al. 1992; Ramsey and Fitzpatrick 1998; Ribeiro et al. 1992). Note that an increase in K i and a decrease in K m increase TH activity. Inhibition of TH purified from rat PC12 cells and from bovine adrenal by catecholamine end products and relief of this inhibition following Ser40 phosphorylation as catalyzed by PKA have been shown in vitro (Andersson 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 ferrous to ferric iron during isolation with the subsequent formation of the DA–enzyme complex. All four isoforms of human TH are subject to feedback inhibition to the same extent when assayed in vitro (Almas et al. 1992).
Structural modeling (Maass et al. 2003) and kinetic studies (Ramsey and Fitzpatrick 2000) of rat TH have been used to decipher the nature of catecholamine binding to the enzyme. Dopamine interacts with iron and a negatively charged carboxyl group in the active site cleft (Haavik et al. 1990). Molecular modeling predicts two planar conformations for DA, whose binding is favored by neutral pH, while DOPA has only one conformation that is energetically favorable. Electrostatic repulsion is hypothesized to occur between the carboxyl group of DOPA and the side chain of nearby Asp425. This explains the twofold increase in K i values for DOPA when compared with DA. A short stretch of the regulatory domain, whose involvement in DA inhibition has been supported by the role of phosphorylation in reversing the inhibition, is hypothesized to interact with DA while closing on the catalytic domain. However, there is no structural evidence for this prediction due to the unavailability of a crystal structure for the complete enzyme. Nonetheless, mutagenesis studies have identified residues in the regulatory segment including Gly36, Arg37, and Arg38 as the critical regions involved in mediating catecholamine binding to recombinant rat (McCulloch and Fitzpatrick 1999; Nakashima et al. 1999, 2000; Ota et al. 1997).
Gordon et al. (2008) have suggested the existence of a second binding site for dopamine with much lower affinity. They reported that all four human isoforms possess this second site. They came to this conclusion by fitting their data to a two-site binding model. They followed a similar approach to reproduce their results in rat PC12 cells in situ and showed that this site also resides in the catalytic domain (Gordon et al. 2009b). Using site-directed mutagenesis of selected residues in the carboxyl-terminal catalytic domain of recombinant hTH-1, they concluded that the second binding site is located in a region close to the first, which was later confirmed by the same group (Briggs et al. 2011). They conclude that the two sites may be present on different monomers. However, it is also possible that there are distinct versions of the enzyme with dissimilar dissociation constants, rather than two different binding sites. These differing K D values may arise owing to distinctive enzyme conformations. These conformations might have different affinities, and as there is not a single enzyme conformation, dose–response curves appear shallow. Further investigation is warranted to differentiate between these possibilities.
Phosphorylation of TH serine residues
Phosphorylation and regulation of TH have been well established in vitro, in situ with cell cultures and brain slices, and in vivo using complex systems such as brain or retina in intact animals. In the present context, we will focus most of our attention on the mechanistic consequences of phosphorylation on enzyme activity, but note that there is a considerable recent literature on the physiological signals responsible for the dynamic regulation of phosphorylation state (reviewed in Daubner et al. 2011; Dickson and Briggs 2013; Dunkley et al. 2004; Nakashima et al. 2013).
Rat TH is phosphorylated at four different sites following cellular stimulation with cAMP, growth factors, phorbol esters, or depolarization with KCl or neurotransmitters (McTigue et al. 1985). These sites are comprised of serines 8, 19, 31 and 40 in the rat enzyme and serines 19, 31, and 40 in the human enzyme in vitro (Campbell et al. 1986; Haycock 1990; Haycock and Wakade 1992; Waymire et al. 1988), in situ (Haycock 1990), and in vivo (Haycock and Haycock 1991). A threonine occurs at position 8 in the human enzyme isoforms. Although threonine is a substrate for protein serine/threonine kinases, phosphorylation of Ser/Thr8 appears to play no role in the regulation of TH.
Regulation of enzyme activity by phosphorylation is one of the most extensively studied forms of metabolic control of cellular processes in general (Cohen 2002) and of TH in particular (Daubner et al. 2011; Dickson and Briggs 2013; Dunkley et al. 2004; Fujisawa and Okuno 2005; Kumer and Vrana 1996; Nakashima et al. 2009, 2013). Phosphorylation as catalyzed by protein kinases and dephosphorylation as catalyzed by protein phosphatases allow for a rapid and reversible regulation of TH and are critical for maintaining optimal catecholamine levels in cells (Fig. 5).
Early phosphorylation studies of TH focused on the rat enzyme because of its ready availability. Following the production of recombinant enzymes, data obtained initially from the rat enzyme were confirmed and extended with human TH isoforms. To avoid confusion, however, specific residues will be denoted by their positions in the rat protein (recalling that there are four human isoform proteins generated by alternative splicing). Four sites have been identified, in the rat enzyme, that are phosphorylated by various protein kinases. Sequences of the amino-terminal segments of rat TH and the human TH isoforms are shown in Fig. 5, and consensus motif sequences are highlighted. Note that the rat enzyme and four human isoforms contain two serine residues embedded in consensus phosphorylation sites (the rat Ser19 and Ser40 equivalents). The amino acid sequence 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 to ERK1/2 catalyzed phosphorylation as discussed later.
Phosphorylation of TH at different residues produces differing molecular effects. However, TH phosphorylation generally results in an increase in catecholamine production. Throughout this section, we will discuss individual serine residues and (1) the effect of their phosphorylation on enzyme activity, (2) the different kinases implicated in phosphorylation, and (3) dephosphorylation by phosphatases. We will describe the physiological importance of these events on regulation of brain catecholamine levels. It is beyond the scope of the present review to explore all of the various stimuli (stress, drugs, behavior, etc.) that trigger phosphorylation; however, selected exemplars will be provided.
There are three important regulatory phosphorylation sites in TH (Ser19/31/40). The phosphorylation of Ser8 in the rat enzyme has little, if any, regulatory effect and a threonine occurs in its place in the human enzyme isoforms. A large number of protein kinases are involved in phosphorylation–regulation of TH (summarized in Table 1).
Phosphorylation and regulation of rat and bovine TH
Serine-40
Initial reports of TH phosphorylation showed PKA to be a central component of enzyme regulation. Increases in enzyme activity were reported when rat brain or bovine adrenal TH was incubated in vitro with ATP and (1) PKA or (2) cAMP (Joh et al. 1978; Morgenroth et al. 1975; Yamauchi and Fujisawa 1979; Vrana et al. 1981; Vrana and Roskoski 1983). The phosphorylation-dependent increase in rat or bovine TH activity occurs as a result of lowering the K m for BH4 and an increase in the K i for dopamine both in vitro (Daubner et al. 1992; Lovenberg et al. 1975; Okuno and Fujisawa 1985; Ramsey and Fitzpatrick 1998; Vrana et al. 1981; Vrana and Roskoski 1983; Vulliet et al. 1980) and in vivo (Haavik et al. 1990). However, Ribeiro et al. (1992) suggested that the increased PKA-dependent activation occurs after the rat recombinant enzyme, which is isolated in a catecholamine-free state, is treated with and inhibited by DA. The hydroxyl group of recombinant rat TH serine residue 40 has been hypothesized to interact with DA, which is reversed upon phosphorylation (McCulloch et al. 2001). The PKA-mediated decrease in the K m for BH4 and the resulting increase in activity have also been demonstrated in bovine adrenal chromaffin cells in situ (Meligeni et al. 1982). Phosphorylation of TH, isolated from bovine striatum, by PKA stabilizes the interactions occurring between the protein backbone in the region surrounding the active site and the hydroxyl groups of BH4 (Bailey et al. 1989).
Interestingly, the K m of PKA for rat TH and for peptides corresponding to the TH Ser40 phosphorylation site has been shown to be about 100 μM, which is rather high (Roskoski and Ritchie 1991). Almas et al. (1992) reported that the K m of PKA for bovine TH was about 150 μM in vitro. These high values can be explained in part by an evolutionary mechanism of the cell adapting itself to the relatively high intracellular TH concentration (Roskoski and Ritchie 1991). When expressed and purified in vitro, hTH-1/2/4 isoforms are catecholamine free and required the addition of FeSO4 for detectable activity (Almas et al. 1992). These proteins were good substrates for PKA with K m values of only 5 μM. However, the addition of Fe2+ and DA increased the K m about threefold. This suggests that the presence of tightly bound iron and DA in the enzyme isolated from mammalian cells contributes to the higher K m observed for the rat pheochromocytoma and bovine adrenal enzymes.
Funakoshi et al. (1991) reported that TH phosphorylation at Ser40 by PKA results in an increase in activity, while phosphorylation of this site by CaMPKII or PKC fails to increase activity in vitro. The stoichiometry of rat Ser40 phosphorylation by PKA was 0.78 mol/mol of subunit, while that for PKC and CaMPKII were both 0.4 mol/mol of subunit. The authors suggest that phosphorylation of Ser40 in all four subunits is required for activation, and such extensive phosphorylation is not achieved by PKC or CaMPKII.
PKC phosphorylation has been shown to produce effects similar to those induced by PKA. This is, in part, because they both phosphorylate Ser40 in the rat enzyme (or its equivalent in the human enzyme isoforms). Albert et al. (1984) reported that PKC-mediated phosphorylation of partially purified rat TH decreases the K m for BH4 and increases the K i for DA, which leads to an increase in enzyme activity. They found that PKC and PKA catalyze the phosphorylation of the same serine residue. However, Cahill et al. (1989) reported that treatment of rat PC12 cells with phorbol ester leads to the phosphorylation of a different serine residue than that mediated by cAMP as determined by 32Pi labeling followed by trypsin digestion. The explanation for this difference in in vitro versus in situ labeling is unclear. Rat TH is also phosphorylated and activated by PKG via cGMP in a manner similar to PKA (via cAMP) in situ (Roskoski and Roskoski 1987).
Harada et al. (1996) found that elimination of rat TH Ser40 by site-directed mutagenesis, which they expressed in non-neuronal mouse AtT20 cells, is still activated by phosphorylation of other residues. The effect of ERK 1/2-mediated phosphorylation was confirmed by the observed increase in catecholamine synthesis in bovine adrenal chromaffin cells in situ following treatment with acetylcholine, an ERK1/2 (p42/p44) MAP kinase activator (Luke and Hexum 2008; Thomas et al. 1997; Yu et al. 2011). TH is phosphorylated by ERK1/2 at Ser40 to a lesser extent, as will be discussed further in the following sections (Haycock 2002a). Royo and Colette Daubner (2006) reported that the phosphorylation of recombinant rat tyrosine hydroxylase at Ser40 by purified bovine heart PKA was diminished in the presence of dopamine. Ser40 phosphorylation also may occur through the action of mitogen and stress-activated kinase (MSK1) (Toska et al. 2002a).
As it has been established to be a pivotal mechanism for the activation of TH, the effects of phosphorylation at Ser40 on the enzyme structure have been an active area of more recent investigation. It has been suggested, through conformation studies, that Ser40 phosphorylation of the rat enzyme will result in the promotion of an open conformation in vitro (Bevilaqua et al. 2001; Wang et al. 2011). Moreover, while it is beyond the scope of this review, considerable other work has been reported on what physiological insults and influences mediate Ser40 phosphorylation and TH activation. Such effectors include depolarization, hormones, receptor stimulation, and pathological conditions (reviewed in Daubner et al. 2011; Dickson and Briggs 2013; Dunkley et al. 2004; Nakashima et al. 2013).
Serine-31
The ERK1/2 serine kinases have also been shown to be important components in the regulation of catecholamine biosynthesis (Haycock et al. 1992; Luke and Hexum 2008; Yu et al. 2011). ERK 1 and 2 are proline-directed protein serine/threonine kinases that are members of the mitogen-activated protein kinase (MAPK) family that characteristically function downstream of growth factor receptors (Roskoski 2012). ERK 1 and 2 occur together in most cells where they act in concert. Halloran and Vulliet (1994) reported that depolarization of bovine adrenal chromaffin cells in culture leads to the phosphorylation of TH Ser31. They found that the depolarization-activated kinase shares biochemical properties with ERK1/2 proline-directed protein kinases. This phosphorylation leads to a twofold increase in TH enzyme activity. In addition, unlike the phosphorylation at Ser40, phosphorylation of this site by recombinant ERK2 is unaffected by dopamine (Royo and Colette Daubner 2006).
Cyclin-dependent kinase 5 (Cdk5) can phosphorylate TH in vitro and in vivo and this phosphorylation occurs at Ser31 (Kansy et al. 2004). In transgenic animals, Cdk5 expression correlates with the preservation of TH protein levels (Moy and Tsai 2004). However, the mechanism through which Cdk5 may regulate TH expression warrants further investigation.
Serine-19
CaMPKII catalyzes the phosphorylation of TH at Ser19 in the presence of calcium; however, phosphorylation of TH by CaMPKII fails to activate the enzyme in the same manner as PKA. An additional protein, identified as a member of the 14-3-3 chaperone protein family, is required to activate human (Itagaki et al. 1999), rat (Funakoshi et al. 1991; Ichimura et al. 1987) and bovine TH (Yamauchi and Fujisawa 1981; Yamauchi et al. 1981) phosphorylated by CaMPKII in vitro. The γ-isoform of 14-3-3 protein is abundant in brain, suggesting a role for this chaperone protein isoform in the regulation of TH (Isobe et al. 1991). Nonetheless, CaMPKII seems to be an important regulator of catecholamine synthesis, as suggested by studies in which cellular inhibition of calcium channels leads to a decrease in DA production in rat PC12h cells, (a subclone of PC12 cells that undergoes differentiation following treatment with epidermal growth factor) (Sumi et al. 1991). However, it is unlikely that this is solely Ser19 mediated, as mutagenesis studies have eliminated a direct role for this residue in controlling catecholamine amounts in other PC12 cell lines (Haycock et al. 1998). TH can be phosphorylated at this site by other kinases as well, as seen following inhibition of CaMPKII in situ (Goncalves et al. 1997). MAPK, for instance, has been shown to phosphorylate TH at Ser19 (Bobrovskaya et al. 2004).
Despite the known effects of the phosphorylation at this site (enzyme activation through allowing the binding of activator proteins), phosphorylation itself is not thought to alter the enzyme’s conformation (Bevilaqua et al. 2001). However, the binding of 14-3-3 protein to the Ser19 phosphorylated TH has been shown to produce a more extended and relaxed conformation (Skjevik et al. 2014). Ser19 phosphorylation also increases the rate of phosphorylation at Ser40, a process described as hierarchical phosphorylation. Such hierarchical phosphorylation has been observed upon phosphorylation of Ser19 in bovine adrenal chromaffin cells (Bobrovskaya et al. 2004).
Phosphorylation of recombinant human TH
Given that the alternative splicing of human TH occurs within the regulatory domain, it is likely that the resulting isoforms are differentially phosphorylated (recalling that hTH-1 is the closest homolog to rat TH; see Fig. 5b—all residues are referred to as the number of the rat TH/human TH-1 homologous residue). Upon expression in E. coli, hTH-1, hTH-2, and hTH-3 are phosphorylated by PKA at Ser40, and they are phosphorylated at Ser19 and Ser40 by CaMPKII (Almas et al. 1992; Alterio et al. 1998). The extent of dopamine binding is reduced upon Ser40 phosphorylation in hTH-1 (Sura et al. 2004). MAPKAP Kinase 1 and MAPKAP Kinase 2 phosphorylate serine residues 19 and 40, respectively, in all four isoforms. Among the four isoforms, hTH-3 and hTH-4 phosphorylation by recombinant mouse ERK2 at Ser31 occurs at a much higher rate (Sutherland et al. 1993). Activation by phosphorylation of isoforms 3 and 4 (twofold increase in activity) was also higher than that of isoform 1, whose activity was increased by 40 %. hTH-1 is phosphorylated at Ser31 by ERK2 in vitro, whereas hTH-2 is not. Differential phosphorylation by ERK1/2 was investigated in neuronal cells (Gordon et al. 2009a). Human neuroblastoma SH-SY5Y dopaminergic cells were transfected with hTH-1 or hTH-2. hTH-1 displayed variable levels of phosphorylation upon stimulation of the cells with epidermal growth factor (EGF), the receptor of which is the protein-tyrosine kinase that is upstream of ERK1/2. hTH-2 was not phosphorylated at a residue that corresponds to Ser-31 in hTH-1.
Lehmann et al. (2006) reported that the hierarchical activation of Ser40 upon phosphorylation of Ser19 as catalyzed by CaMPKII was higher in hTH-2 than hTH-1. Furthermore, ERK1/2-catalyzed phosphorylation of Ser31 in hTH-1 increased the phosphorylation rate of Ser40 by ninefold. This effect was not observed in hTH-3 and hTH-4. When ERK1/2 was inhibited with UO126, the decrease in the phosphorylation of hTH-1 at Ser31 resulted in a 50 % decrease in the phosphorylation at Ser40. The authors concluded that hierarchical phosphorylation provides a mechanism whereby the two major human TH isoforms (1 and 2) can be differentially regulated with only hTH-1 responding to the ERK1/2 pathway, whereas hTH-2 is more sensitive to calcium-mediated events.
Possible mechanisms for the regulation of TH activity via phosphorylation
Up to this point, we have discussed the mechanisms for TH phosphorylation separately. However, it should be considered that all these mechanism may act on TH in an overlapping manner in cells and tissues. This section will discuss the evidence for the existence for these multiple mechanisms, and provide a brief discussion of the physiological importance and relevance of this type of molecular regulation of TH activity. Incubation of purified rat TH with both PKA and CaMPKII results in additive incorporation of phosphate under in vitro assay conditions suggesting that these kinases act on distinct residues (Vulliet et al. 1984). PKC, on the other hand, phosphorylates the same site as PKA in vitro (Vulliet et al. 1985). Griffith and Schulman (1988) stimulated rat PC12 cells with A23187 (a calcium ionophore), carbachol, or high KCl, each of which leads to the phosphorylation and activation of TH. They showed that the concentration of cAMP is not elevated by any of these treatments. They also found that cells deficient in PKC exhibit TH phosphorylation and increase in activity following stimulation. They found that the sites of TH phosphorylation catalyzed by CaMPKII most closely mimic those observed in vivo and conclude that this enzyme mediates TH phosphorylation induced by hormonal and electrical stimuli that elevate intracellular Ca2+. Tachikawa et al. (1987) found that the incubation of rat PC12 cells with ionomycin (a calcium ionophore) leads to an increase in phosphorylation of three TH peptides derived from TH following tryptic digestion. In contrast, incubation with phorbol ester (an activator of PKC) or forskolin (an adenylyl cyclase and hence PKA activator) leads to increased phosphorylation of only one TH peptide. Surprisingly in this study, the resulting single tryptic peptides were different and the explanation for this finding is unclear. PKG phosphorylation also occurs at the same site as that of PKA as demonstrated in PC12 cells (Roskoski et al. 1987) and bovine adrenal chromaffin cells (Rodriguez-Pascual et al. 1999). Both Ser19 and Ser40 are phosphorylated by MAPKAP2 in situ as well (Toska et al. 2002b). In addition, new kinases have been implicated in the phosphorylation of TH at multiple residues such as the AMP-activated protein kinase (AMPK) (Fukuda et al. 2007). These results might be due to the activation of downstream protein kinases, and experiments with purified AMPK and TH should be performed to determine whether the kinase acts directly on TH, and if so, which residues are phosphorylated.
Differential phosphorylation of TH occurs in a cell location-specific manner. That is, differing phosphorylation patterns occur depending on where in the cell the enzyme is located. Phosphorylation of different residues causes distinct effects on catecholamine biosynthesis and provides cell and cell location-specific regulatory mechanisms in the grand scheme of catecholamine neurotransmission. Xu et al. (1998b) examined the state of phosphorylation of TH in different rat brain regions and different cellular locations using immunohistochemical studies performed with phospho-specific antibodies against TH. They reported that the noradrenergic locus coeruleus (LC) and the A5 cell bodies had high levels of TH phosphorylated at Ser40 and Ser19, but that the nerve terminals lacked TH that was phosphorylated at either residue. Moreover, different subpopulations of dopamine neurons in the ventral tegmental area express different amounts of Ser40-phosphorylated TH, while Ser19 phosphorylation was more uniform. Lindgren et al. (2002) reported that ERK1/2-mediated Ser31 phosphorylation increased in rat striatal slices upon depolarization, but that PKA-mediated Ser40 phosphorylation was not as great. The authors suggested that there might be a threshold for the effects obtained from Ser40 phosphorylation-dependent activation. When the phosphorylation stoichiometries are low, there is little or no activation until the threshold is reached. In light of the suggestion that Ser40-mediated activation requires dopamine binding to TH, Ser40 phosphorylation may mainly be involved in the relief of feedback inhibition. Using phosphorylation site-specific antibodies and an antibody against total TH, Salvatore et al. (2000) measured the phosphorylation stoichiometry of TH at different amino acid residues in rat nigrostriatal and mesolimbic dopaminergic cell bodies (substantia nigra and ventral tegmental area) and nerve terminals (corpus striatum and nucleus accumbens). The extent of phosphorylation of Ser19 and Ser31 differs between the cell bodies and terminal fields. Ser19 phosphorylation is greater in the cell bodies (1.5-fold higher) and Ser31 phosphorylation is greater in the nerve terminals (two- to fourfold). Ser40 phosphorylation is similar in these two compartments and has the lowest value with only 3 % of the TH Ser40 sites bearing a phosphate group (Table 2). Ser40 phosphorylation is 1/3rd to 1/10th the level of Ser19 or Ser31 phosphorylation in any region. Haloperidol treatment of rats for 30–40 min caused an approximate 230 % increase in total phosphorylation (Ser19/31/40) in nerve terminals, while there was a smaller increase in their phosphorylation stoichiometry (around a 130 % increase) in the cell bodies. The authors suggest that the mechanism of haloperidol action on TH in vivo in the nigrostriatal system involves both pre- and post-synaptic DA receptors. Region-specific phosphorylation seems to be mostly neuronal, as this type of regulation is not observed in rat retina (Witkovsky et al. 2004). Salvatore et al. (2001) measured the phosphorylation stoichiometry in response to depolarization by 58 mM KCl in rat PC12 cells and in a PC12 cell line (A126-B1) that lacks PKA. They found that Ser19 and Ser31 phosphorylation increased three- to fourfold in both cell lines. Ser40 phosphorylation increased about 40 % following depolarization of PC12 cells, but there was little, if any, increase in the A126-B1 cell line. The depolarization-dependent increases in phosphorylation at all sites were dependent upon external Ca2+. MEK is upstream of ERK1/2, and inhibition of the former by PD98059 decreased basal Ser31 phosphorylation and completely prevented the KCl stimulation in both cell lines. The MEK inhibitor decreased basal Ser40 phosphorylation in PC12 cells, but not in A126-B1 cells, suggesting that there is cross-talk between ERK and PKA pathways in PC12 cells. They used a CaMPKII inhibitor in an effort to identify this protein kinase as being responsible for Ser19 phosphorylation. This proved impractical because its inactive congener had the same inhibitory effects. They reported that PKA is responsible for the increase in Ser40 phosphorylation in PC12 cells and they concluded that Ser31 phosphorylation is necessary and sufficient for depolarization-dependent increases in catecholamine synthesis in PC12 and A126-B1 cells. They concluded that Ser40 phosphorylation plays little or no role in this process. However, they suggested that PKA-mediated phosphorylation of Ser40 could play a role in regulating catecholamine biosynthesis under other conditions. In fact, these investigators concluded that the increases in catecholamine biosynthesis in rat brain following haloperidol treatment were due to activation of PKA (Salvatore et al. 2000).
In light of these data, we propose a model for regulation of dopamine synthesis in neurons (similar mechanisms may be partially active in noradrenergic and adrenergic neurons) (summarized in Fig. 6). Depolarization of the presynaptic terminal would activate ERK1/2 and increase DA production. Increases in calcium concentration at the nerve terminal to induce synaptic vesicle fusion to the presynaptic cell membrane will also simultaneously activate CaMPKII, causing an increase in DA production. Ser40 phosphorylation (established through studies performed with PKA) will mainly regulate DA-dependent changes in TH activity, in addition to responding to the cell’s requirement for rapid increases in the DA production by increasing enzyme activity (lower K m for BH4 and higher K i for DA). When the DA in the synaptic cleft is taken back up by the reuptake transporter, TH will be inhibited. Also, depending on the extent of stimulation of DA autoreceptors, PKA activation will be regulated. Hence when there is less DA present, TH is a better substrate for PKA, which increases TH phosphorylation and decreases feedback inhibition.
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+
The main effect exerted by phosphorylation of TH, regardless of the phosphorylation site and the kinase(s) involved, seems to be activation of the enzyme. Another effect of TH phosphorylation, however, has negative effects on the thermal stability of TH protein. Phosphorylation of rat TH is accompanied by a 50 % decrease in half-life of the protein (Gahn and Roskoski 1995; Lazar et al. 1981). Surprisingly, Royo et al. (2005) found that phosphorylation of recombinant rat TH residues other than Ser40 increases in protein stability. The role of phosphorylation as a regulator of thermal stability will be discussed later.
Dephosphorylation of TH
Phosphorylation-dependent TH activation is reversed by the removal of phosphate residues by phosphatases. In the case of bovine adrenal and corpus striatum, PP2A and PP2C (phosphoprotein phosphatases 2A and 2C) are the key enzymes that catalyze the dephosphorylation of bovine TH (Haavik et al. 1989). BH4 increases the rate of dephosphorylation of the rat striatal TH as catalyzed by PP2A (Ribeiro and Kaufman 1994). These experiments suggest that BH4 may play a dual role (activating and deactivating) in the regulation of TH activity. That PP2A is abundant in rat corpus striatum, while PP2C content is only 10 % that of PP2A, supports the role of the former in TH regulation (Berresheim and Kuhn 1994). Inhibition of PP2A by okadaic acid and microcystin leads to increased phosphorylation levels of bovine adrenal TH in digitonin-permeabilized chromaffin cells, especially at Ser19 and Ser40 (Goncalves et al. 1997). The rates of dephosphorylation of different phosphoserines by PP2A in bovine adrenal chromaffin cells have been shown to be highest for Ser40, followed by Ser19, Ser31, and Ser8, in descending order (Leal et al. 2002). PP2A from rat corpus striatum is the main TH phosphatase from brain as determined by measuring the dephosphorylation of recombinant rat TH, while PP2C is the chief TH protein phosphatase that occurs in adrenal chromaffin cells (Bevilaqua et al. 2003).
In vitro, α-synuclein, a protein that is known to accompany the pathology in Parkinson’s disease, increases the activity of PP2A, thereby decreasing TH activity through reduced Ser40 phosphorylation (Peng et al. 2005). However, a study that investigated the effects of α-synuclein on PP2A did not provide a similar observation in vivo (Drolet et al. 2006). Interestingly, α-synuclein overexpression and the presence of pathological mutations known to accompany the disease-related phenotype of this protein have been shown to be associated with increased TH phosphorylation, suggesting the loss of a necessary function for PP2A during neuronal loss (Alerte et al. 2008; Lou et al. 2010).
Recent evidence has suggested that the regulation of TH through dephosphorylation is more complicated than has been thought previously. First, there is a specific isoform of the PP2A regulatory subunit (PP2A/B′β) that is found predominantly in the brain and localizes to specific cellular compartments (nerve terminal; Saraf et al. 2007). Second, there appears to be specific glutamate residue on PP2A that interacts with the TH regulatory domain (Saraf et al. 2010). In addition, PP2A activity itself can be regulated through phosphorylation by a PKC isoform (Zhang et al. 2007) and the action of PKC on PP2A is more pronounced on a heterotrimeric regulatory subunit (Ahn et al. 2011). Finally, in vivo PP2A activity may be regulated via fluctuations in the levels of progesterone (increased PP2A activity accompanying an acute rise in progesterone levels) (Liu and Arbogast 2008) and hypoxia-dependent increases in reactive oxygen species (downregulation of PP2A) (Raghuraman et al. 2009).
TH-binding partners
The first identified binding partner of TH was a member of the 14-3-3 family of scaffolding proteins (Ichimura et al. 1987). These proteins bind to bovine adrenal medullary TH phosphorylated at Ser19 by CaMPKII, and this binding induces activation (Yamauchi and Fujisawa 1981; Yamauchi et al. 1981). Binding requires stimuli such as depolarization or an increase in calcium to activate CaMPKII. Itagaki et al. (1999) studied the interaction between recombinant hTH-1 and 14-3-3η. Phosphorylation of TH by purified rat brain CaMPKII resulted in Ser19 phosphorylation and binding of 14-3-3 with a K d of 3 nM. Phosphorylation by recombinant PKA leads to phosphorylation of Ser40 but not to 14-3-3 binding. The Ser40Ala TH mutant was a poor substrate for CaMPKII and failed to bind 14-3-3. The mutant possessed basal TH activity. Analysis of a PC12 cell line transfected with myc-tagged 14-3-3 showed that it formed a complex with endogenous TH after KCl-induced depolarization.
Kleppe et al. (2001) reported that binding of yeast and sheep brain 14-3-3 proteins to bovine and human TH isoforms occurs following phosphorylation of Ser40, but binding of bovine brain 14-3-3-ζ is not increased. Toska et al. (2002a), from the same laboratory, found that binding of 14-3-3 proteins to recombinant human TH decreases the rates for Ser19 and Ser40 dephosphorylation by 82 and 36 %, respectively. Obsilova et al. (2008) found that the interaction between 14-3-3 and Ser19- and Ser40-phosphorylated hTH-1 decreases the exposure of regulatory domain amino acids with the solvent as determined by time-resolved tryptophan fluorescence measurements. These investigators found no changes in hTH-1 secondary structure following 14-3-3 binding as determined by circular dichroism.
Many studies have been conducted to identify the specific 14-3-3 subtype(s) interacting with TH. The 14-3-3 γ isoform has been suggested as a binding partner owing to its predominant expression in bovine brain (Isobe et al. 1991). Halskau et al. (2009) found that recombinant human 14-3-3γ forms a triplet complex with bovine adrenal chromaffin membrane lipids and Ser19-phosphorylated recombinant hTH-1, suggesting a role for cellular localization and hTH-1 activation by this protein. These investigators reported that the amino-terminal regulatory segment of TH binds to membranes. This finding validates and perhaps explains the initial observations of membrane-associated and soluble forms of TH described in bovine brain homogenates (Kuczenski 1973, 1983). Interaction of TH with lipid bilayer fatty acids is thought to stabilize secondary structural elements, especially α-helices, hence protecting the enzyme against thermal denaturation. However, the membrane-bound form also displays lower catalytic activity (Thorolfsson et al. 2002).
Co-precipitation and knock down experiments on 14-3-3 and TH from rat midbrain and cultured dopaminergic cells indicated a role for the ζ isoform, and this isoform co-localized with TH on mitochondria in MN9D cells (Wang et al. 2009). There seems to be the requirement for a specific subcellular localization for TH interaction with different 14-3-3 isoforms. Whether there are different effects occurring following different isoforms has yet to be reported. Sachs and Vaillancourt (2004) reported that phosphorylation of recombinant rat TH catalyzed by casein kinase 2 or its downstream cyclin-dependent kinase inhibits interaction with 14-3-3 and thus diminishes TH activity. These investigators did not determine which residues of TH were phosphorylated by these enzymes. These experiments represent an unusual situation where TH phosphorylation is associated with a decrease in catalytic activity. The rat 14-3-3 η subtype may decrease hTH-1 stability upon interacting with its regulatory domain (Nakashima et al. 2007).
Other binding partners identified for TH have been investigated owing to their proposed roles in neurodegeneration. These include α-synuclein (α-Syn), which is a major protein in Lewy bodies, the intraneuronal protein aggregates characteristic of Parkinson’s disease (Spillantini et al. 1998). α-Syn, which has homology with the 14-3-3 chaperone, binds rat TH and decreases its activity (Leong et al. 2009; Perez et al. 2002). α-Syn co-immunoprecipitates with TH in rat brain and in mouse MN9D dopaminergic cells (Perez et al. 2002). α-Syn overexpression not only reduces TH activity, but it also causes a decrease in mouse MN9D cellular dopamine content. α-Syn is overexpressed upon increased dopamine oxidation; hence producing an increase in the amount of reactive oxygen species in MN9D cells that, in turn, promotes reduction of TH activity (Leong et al. 2009; Perez et al. 2002).
Lou et al. (2010) found that wild-type human α-syn expression in mouse MN9D cells reduces endogenous TH Ser19 phosphorylation. They also reported that either wild-type or mutant human Ser129Ala α-syn enhances PP2A activity in these cells with the Ser129Ala mutant producing greater activation. Phosphorylation of α-syn at Ser129 decreases its PP2A stimulatory activity. They also found that overexpression of wild-type human α-syn driven by the TH promoter in transgenic mice stimulates PP2A and reduces TH phosphorylation in dopaminergic cells. An increase in TH dephosphorylation is hypothesized to be the main mechanism through which α-syn depletes cellular dopamine content (Perez et al. 2002). Regulation of α-synuclein, therefore, provides an additional control point in catecholamine homeostasis.
TH activity in Drosophila is regulated by binding to GTP cyclohydrolase I (the rate-limiting enzyme for BH4 synthesis), and the interaction of these two enzymes promotes the activity of both partners (Bowling et al. 2008). The authors suggest that this interaction may provide a mechanism by which TH can obtain cofactor upon need. Vesicular monoamine transporter (VMAT) proteins interact with TH and aromatic amino acid decarboxylase in rat brain as demonstrated by co-immunoprecipitation, and complex formation involving these components provides a mechanism that links DA synthesis with synaptic vesicle filling (Cartier et al. 2010). This linkage may maintain low cytosolic dopamine levels and decreases the generation of reactive neurotoxic catechol-quinones. Identification of these binding partners provides additional insight into the functional regulation of catecholamine biosynthesis.
TH enzyme stability and proteasomal degradation
A number of regulatory factors or physiological conditions alter the inherent stability of the TH protein. Rat TH stability is decreased by enzyme phosphorylation or treatment with ascorbate or BH4 (Lazar et al. 1981; Vrana et al. 1981; Wilgus and Roskoski 1988). Binding of tyrosine or dopamine has a protective effect against enzyme inactivation (Roskoski et al. 1990). Binding of anionic heparin or RNA stabilizes the protein (Gahn and Roskoski 1995). The decrease in enzyme stability is ascribed to changes in the tertiary structure of the protein (Gahn and Roskoski 1995; Roskoski et al. 1993). Phosphorylation itself induces these aforementioned structural changes. Among the different phosphorylation states of TH, phospho-Ser 40 shows the least stability, while phospho-Ser19, phospho-Ser31, and phospho-Ser8 are even more stable than the wild-type enzyme (Royo et al. 2005).
Another mode of regulation for human (Doskeland and Flatmark 2002) and rat TH (Nakashima et al. 2011) involves the ubiquitination of TH followed by proteasomal enzyme degradation. Proteasomal degradation of TH seems to be triggered by enzyme phosphorylation. Serine phosphorylation at residues 19 and 40 is considered to be the main regulator of ubiquitination for rat TH as demonstrated in PC12D cells (a subline derived from PC12 cells), while Ser31 phosphorylation has no effect (Nakashima et al. 2011; Kawahata et al. 2009). One study reports the existence of a PEST (proline, glutamate, serine and threonine) motif in the human TH amino-terminal regulatory domain, which might be one of the underlying causes for the instability of the cellular enzyme observed in AtT20 cells (Nakashima et al. 2005). PEST sequences target proteins for proteasomal degradation.
Several candidate stimuli may lead to TH degradation via proteasomes including angiotensin in rat hypothalamus (Lopez Verrilli et al. 2009) and ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor inflammatory cytokines in mouse and rat sympathetic neurons and M17 human neuroblastoma cells in culture (Shi and Habecker 2012). Shi and Habecker (2012) reported that cytokine activation of gp130 increases the ubiquitination of TH. Moreover, they reported that the proteasome inhibitors MG-132 and lactacystin prevented the loss of TH in CNTF-treated sympathetic neuronal cells. In fact, binding of these inflammatory cytokines to the gp130 receptor results in TH degradation via activating ERK 1/2. This intriguing finding suggests an additional role for ERK1/2 in TH regulation in addition to increasing enzyme activity.
Future imperatives
TH serves as the central control mechanism in catecholamine biosynthesis. Despite the extensive study of the enzyme and its regulation, several areas remain to be tackled. Clearly, more work needs to be conducted to define the dynamic protein interactome involving TH and affecting its regulation. Considerable uncertainty exists around which specific 14-3-3 proteins are engaged in different cell types and what other proteins may contribute to TH regulation, stability, and turnover. Moreover, the recent reports from the Habecker laboratory (Shi and Habecker 2012) may provide important initial insights into the role of neuroinflammation in the regulation of catecholamine production in health and disease. One important future consideration will be the role of aberrant regulatory mechanisms that contribute to pathophysiological conditions. TH dysfunction has been implicated in several diseases including alcohol and drug addiction, bipolar disorder, hypertension, movement disorders (Parkinson’s disease), and schizophrenia (Bademci et al. 2012; Zhu et al. 2012; Sumi-Ichinose et al. 2010; Bellivier 2005). Note that this need not mean that TH causes the disorder; rather, aberrant regulation of the enzyme may exacerbate the disorder. Clearly, these situations will be very difficult to address in living organisms (especially human subjects). However, one can easily imagine a neurodegenerative disease in which TH regulation fails to compensate for reduced catecholamine function, thus exacerbating the symptoms or inducing more rapid progression.
In a similar vein, the completion of the human genome project and ongoing sequencing efforts (e.g., the 1000 Genome Project) are opening up new vistas for exploration. The NCBI dbSNP database of single nucleotide polymorphisms indicates that there are more than 50 genetic variants in human TH coding region that alter amino acids (Fig. 7). As we have noted elsewhere (Tekin and Vrana 2013), these reported SNP variants need to be carefully examined for validation, but they represent a natural laboratory for enzyme structure–function relationships. There is surprisingly little information on the functional consequences of these variants. While there are clearly a few genetic variants that are associated with frank disease (e.g., R202H and L205P in DOPA-responsive dystonia; Haavik et al. 2008), the vast majority of the SNPs have no associated pathology. Indeed, given that TH deletion is associated with embryonic lethality, we anticipate that there may be severe consequences for a few genetic variants. Moreover, genetically mediated changes in protein structure may influence the regulation of this pivotal enzyme and so modulate catecholamine levels. For this reason, future work in personalized medicine will need to incorporate knowledge of the regulatory effects of TH SNPs (as well as other enzymes) on catecholamine levels in health and disease.
Reported SNPs and their position on TH mRNA. The red dots represent single nucleotide polymorphisms that have been reported in the NCBI SNP database as of 2013. Note that the genetic variants are spread throughout the protein, although perhaps a little less densely in the center of the catalytic domain where they would be more likely to severely impact (or abolish) enzyme activity
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This work was supported by grants from the National Institutes of Health (GM38931) and the Penn State Institute for Personalized Medicine (04-017-52 HY 8A1HO; under a grant from the Pennsylvania Department of Health using Tobacco CURE Funds). The PA Department of Health specifically disclaims responsibility for any analyses, interpretations or conclusions.
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I. Tekin and R. Roskoski Jr. have contributed equally to preparation of this manuscript.
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Tekin, I., Roskoski, R., Carkaci-Salli, N. et al. Complex molecular regulation of tyrosine hydroxylase. J Neural Transm 121, 1451–1481 (2014). https://doi.org/10.1007/s00702-014-1238-7
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DOI: https://doi.org/10.1007/s00702-014-1238-7