complex molecular regulation of tyrosine hydroxylase

TRANSLATIONAL NEUROSCIENCES - REVIEW ARTICLE

Complex molecular regulation of tyrosine hydroxylase

Izel Tekin • Robert Roskoski Jr. • Nurgul Carkaci-Salli •

Kent E. Vrana

Received: 3 April 2014 / Accepted: 4 May 2014

� Springer-Verlag Wien 2014

Abstract Tyrosine hydroxylase, the rate-limiting enzyme

in catecholamine biosynthesis, is strictly controlled by

several interrelated regulatory mechanisms. Enzyme syn-

thesis 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 addi-

tional 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.

Keywords Alternative splicing � Catecholamine

biosynthesis � Feedback inhibition � Phosphorylation �Promoter � Transcription factor

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 func-

tions include modulating autonomic reflexes, behavior,

circulatory control, cognition, endocrine function, move-

ment, 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 reg-

ulation 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 catechol-

amine biosynthesis (Levitt et al. 1965) (Fig. 1). Tyrosine

I. Tekin and R. Roskoski Jr. have contributed equally to preparation

of this manuscript.

I. Tekin � N. Carkaci-Salli � K. E. Vrana (&)

Department of Pharmacology, Pennsylvania State University

College of Medicine, 500 University Drive, Hershey,

PA 17033-0850, USA

e-mail: [email protected]

R. Roskoski Jr.

Blue Ridge Institute for Medical Research, Horse Shoe, NC,

USA

123

J Neural Transm

DOI 10.1007/s00702-014-1238-7

hydroxylase mediates the reaction of tetrahydrobiopterin

(BH4), molecular oxygen, and tyrosine to form L-3,4-dihy-

droxyphenylalanine (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 b-

hydroxylase catalyzes the conversion of DOPA to norepi-

nephrine 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-adenosyl-

methionine is the methyl donor in the reaction converting

norepinephrine to epinephrine. The other product, S-adeno-

sylhomocysteine, is reconverted to S-adenosylmethionine in

a multistep process.

The physiological importance of TH is exemplified by

its role in development. Disruption of the tyrosine

hydroxylase gene in transgenic mice results in mid-ges-

tational lethality (Kobayashi et al. 1995; Zhou et al.

1995). Of the minority of embryos that survive, all

exhibit stunted development and die within the first

5 weeks of life. In addition, more than 53 single nucle-

otide polymorphisms (SNPs) in the coding region of

human TH have been described (Haavik et al. 2008;

Kobayashi and Nagatsu 2005; Rao et al. 2007; http://

www.ncbi.nlm.nih.gov/SNP/). Several of these SNPs are

strongly associated with movement disorders and other

conditions that arise from a deficiency in dopamine

production, as will be discussed later. There are also

numerous polymorphisms observed in the noncoding

(intronic) regions of the TH gene. These polymorphisms

will not be considered in this review primarily because of

a lack of functional data.

Tyrosine hydroxylase, phenylalanine hydroxylase, and

tryptophan hydroxylase form a family of related enzymes

called aromatic amino acid hydroxylases (Grenett et al.

1987). With few exceptions, the mechanism of action of

these enzymes and the tertiary structure of the catalytic

domains are nearly identical and they use the common co-

substrate tetrahydrobiopterin (BH4). Interestingly, genetic

defects in BH4 biosynthesis manifest as a neurodevelop-

mental disorder (Segawa disease) that can be managed, in

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 regula-

tion, 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.

Fig. 1 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

I. Tekin et al.

123

http://www.ncbi.nlm.nih.gov/SNP/

http://www.ncbi.nlm.nih.gov/SNP/

Tyrosine hydroxylase is able to catalyze the hydroxylation

of phenylalanine and tryptophan in addition to tyrosine.

The specificity constant (Vmax/Km) of tyrosine hydroxylase

for tyrosine is only 10-fold greater than that of phenylal-

anine (Daubner et al. 2000) and 30-fold greater than that of

tryptophan (Daubner et al. 2002). In contrast, the speci-

ficity constant of phenylalanine hydroxylase for phenylal-

anine is 105-fold greater than that for tyrosine (Daubner

et al. 2000). These data indicate that TH can be somewhat

promiscuous in its amino acid selection.

Tyrosine hydroxylase contains a mononuclear non-heme

iron atom. Ferrous iron, but not ferric iron, promotes

enzyme activity (Dix et al. 1987; Fitzpatrick 1989; Haavik

et al. 1988). The iron content of each enzyme subunit is

between 0.4 and 1 atom/subunit of protein (Almas et al.

1992; Haavik et al. 1991). The iron atom is coordinated by

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 oxy-

gen, 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, nor-

epinephrine, and epinephrine inhibit enzyme activity and it

has been postulated that such feedback inhibition is phys-

iologically 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 addi-

tion, 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 pro-

moter, 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 transcrip-

tion factors mediates tissue-specific expression of the gene

(Tinti et al. 1996).

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 con-

stitute 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).

The X-ray crystal structure of the catalytic and tetra-

merization 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 a-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 a-helix which forms the core of the tetramer

(Fig. 3). Each monomer contains 16 a-helices and 12 b-

strands including those of the regulatory segment (2 a-

helices and 4 b-strands). The catalytic domain has a 30-A

wide and 17-A deep active site within the basket-like

Fig. 2 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 polymor-

phisms (SNPs) exist in the population that produces variant mRNAs

and proteins. Once the transcript is translated, several post-transla-

tional modifications can occur to regulate catecholamine biosynthesis

Regulation of tyrosine hydroxylase activity

123

arrangement. Site-directed mutagenesis studies have iden-

tified 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 indi-

cate that specific residues in the loop fail to play a domi-

nant role in determining the amino acid substrate

specificity of either TH or PAH. Sura et al. (2006) sug-

gested 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 A 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-

Fig. 3 Molecular models prepared from the crystal structure of the

rat isoform of tyrosine hydroxylase. The catalytic domain of the

protein is about 50 % a-helix, 10 % b-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 Schrodinger, LLC

I. Tekin et al.

123

dihydrobiopterin (PDB ID 2TOH) reveals no conforma-

tional 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 pos-

sess 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 A 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 deter-

mine these structures and those of the unphosphorylated

and Ser40-phosphorylated holoenzymes. Thus far struc-

tural 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 b-

strands and a 40 A-long a-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-A long a-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 a-helices and 4 b-strands. The secondary

structure of the ACT domain consists of a b1-sheet, an a1-

helix, the b2- and b3-sheets, the a2-helix and finally the

b4-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 Fitz-

patrick 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 catechol-

amine 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 Zig-

mond 1994). These reactions occur at neutral pH, but not

under acidic conditions. Cells minimize the generation of

these toxic metabolites by maintaining production of cat-

echolamines at near-optimal levels, by providing on-

demand regulation of synthesis, and by storing them in

acidic synaptic vesicles. TH gene expression is strictly


123

controlled by numerous promoter sequences that are loca-

ted upstream of the 50 transcription initiation site (Lenar-

towski 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 physio-

logically, and several others are observed under patholog-

ical conditions. Expression can also be regulated in a cell

type-specific fashion by neuropeptides and by depolariza-

tion (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; Daub-

ner and Piper 1995; Nakashima et al. 2002). Phosphory-

lation occurs at four different serine residues (Campbell

et al. 1986), which activate the enzyme and alter its sta-

bility (Dunkley et al. 2004; Fujisawa and Okuno 2005;

Martinez et al. 1996; Zigmond et al. 1989). These regula-

tory 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-con-

taining 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 vari-

ants participate in the regulation of TH expression both

temporally and spatially. These modalities of TH regula-

tion 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 expres-

sion of TH. Transgenic mice containing the 9-kb 50-upstream segment of the rat TH promoter fused to the b-

galactosidase reporter gene, but not mice bearing 0.15 or

2.4 kb of 50 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-spe-

cific DNA elements reside between -9 and -2.4 kb of the

50 flanking region of the TH gene. Moreover, stimuli that

increase or reduce TH levels produce parallel changes in b-

galactosidase expression in various brain regions in trans-

genic mice bearing the 50 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 mainte-

nance of mouse midbrain dopaminergic neurons (Zetter-

strom 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 dopa-

minergic-specific TH gene expression. Satoh and Kuroda

(2002) reported that Nurr1 interaction with the TH pro-

moter 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 tran-

scription 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 neuro-

blastoma cells (SK-N-AS). Following activation, they

found that ERK1/2 proteins enhance transcriptional acti-

vation 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)

I. Tekin et al.

123

(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.

Lazaroff et al. (1998) compared the expression of a

transiently transfected chloramphenicol acetyltransferase

(CAT) reporter gene under the transcriptional control of the

TH 50 flanking DNA in undifferentiated and differentiated

mouse CNS CAD (Cath.a-differentiated) cells and found

that TH expression varied as a function of their differen-

tiated 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 contribu-

tions 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)

Fig. 4 Schematic

representation and overview of

the regulation of the rat TH

promoter. Some of the well-

known stimuli and their

corresponding sites on the

promoter are depicted with the

black arrows (GRE

glucocorticoid response

element, HRE hypoxia response

element, E-box enhancer box,

AP-1 activator protein-1, Egr/

Sp1 early growth response

protein 1/steroidogenic factor 1,

CRE1/2 cAMP response

element 1/2, ERE estrogen-

response element, CaRE

calcium response element,

pDSE partial dyad symmetry

element)


123

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 immobili-

zation 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 immo-

bilization increases CREB phosphorylation in the LC. In

studies from the same laboratory, Hebert et al. (2005)

observed an increase in Fos levels and CREB phosphory-

lation 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 tran-

scription factor binding to the promoter region in TH is an

important regulatory mechanism that determines cate-

cholamine 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 glu-

cocorticoid 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 dis-

cussed 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 bac-

terial CAT reporter fused to wild-type or mutant 50 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 con-

cluded 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 phos-

phorylation, 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 reg-

ulates 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

I. Tekin et al.

123

for 1–2 days increased both TH and dopamine b-hydrox-

ylase 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 (Nank-

ova 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 multi-

ple 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 ele-

ments by DNAse I footprint analysis from extracts pre-

pared 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 (Papa-

nikolaou 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 lucif-

erase 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 partici-

pate in regulating TH expression. AP-1 activation occurs

via stimulation of the upstream ERK1/2 pathway in neu-

rons of the developing rat brain striatum (Guo et al. 1998).

Cazorla et al. (2000) reported that CRE in mouse neuro-

blastoma Neuro2A cells does not interact with the previ-

ously 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 investigat-

ing 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 con-

struct. 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 dexameth-

asone, 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


123

tumors of the CNS, renal cell carcinomas, and pheochro-

mocytomas (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 ubiq-

uitination and degradation of hypoxia-inducible transcrip-

tion 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 (pre-

sumably 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 con-

struct 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-a/b(ER-a/b) through an estrogen-response element (ERE) that

overlaps with the CRE/CaRE site (Maharjan et al. 2005)

(Fig. 4). The action of 17 b-estradiol can also be mediated

at the plasma membrane as demonstrated by experiments

performed with an impermeable estradiol derivative (Ma-

harjan et al. 2010). An estradiol/ER-a complex can acti-

vate 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 neuroendo-

crine 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 neuro-

blastoma BE(2)-C-16 cells. The addition of a 1.2-kb frag-

ment 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 immunoprecipita-

tion, 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 pro-

moter 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 buty-

rate, a histone deacetylase inhibitor (Aranyi et al. 2007).

Detailed discussion of these mechanisms and their physi-

ological 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 tran-

scription initiation site and 976 bp downstream from the 30

end are required for TH expression (Gardaneh et al. 2000).

However, transgenic mice that contain the 50 11 kb region

of the human TH promoter express a reporter in TH posi-

tive cells in vivo (Kessler et al. 2003). Sequence analysis of

this region led to the identification of five common con-

sensus 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.

I. Tekin et al.

123

Constructs prepared with minimal promoters that con-

tain the five conserved consensus sites were able to drive

TH expression in a commercially available human neuro-

nal 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 pre-

sents 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 catechol-

amines in both the central and peripheral nervous systems.

In addition, such knowledge may also contribute to a fur-

ther 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; Wil-

lemsen et al. 2010).

Several factors that control TH promoter activity regu-

late 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 30-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 30 UTR (Roe et al. 2004). Long-term

stress also induces protein binding to the poly-pyrimidine

rich region in the 30-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 30-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 30-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 reg-

ulatory, 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


123

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.

All of the human TH isoforms are found in adrenal

chromaffin cells (Haycock 1991) and in brain catechol-

aminergic 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 pheochro-

mocytomas (Haycock 2002b) and neuroblastomas (Hay-

cock 1993). Among the major four isoforms, hTH-1 and

hTH-2 mRNA transcripts are detected in highest abun-

dance (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, investiga-

tors 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

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)

I. Tekin et al.

123

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 investi-

gators 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 Ki 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 cate-

cholamines can be harmful, as these substances have been

shown to form toxic quinones as noted earlier. For exam-

ple, dopamine forms reactive quinone metabolites (Has-

tings 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 con-

centrations that are in the nanomolar range. This low Ki 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 depen-

dent, 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 Km

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-sub-

strate 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 Ki

value for DA and decreases the Km for BH4 (Daubner et al.

1992; Ramsey and Fitzpatrick 1998; Ribeiro et al. 1992).

Note that an increase in Ki and a decrease in Km 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 phosphoryla-

tion as catalyzed by PKA have been shown in vitro (An-

dersson et al. 1992). Inhibition of TH also occurs in vivo,

as the enzyme isolated from brain is found in a complex

with DA. However, this may result from oxidation of fer-

rous 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 con-

formations for DA, whose binding is favored by neutral

pH, while DOPA has only one conformation that is ener-

getically 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 Ki 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 phosphoryla-

tion 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


123

and showed that this site also resides in the catalytic

domain (Gordon et al. 2009b). Using site-directed muta-

genesis of selected residues in the carboxyl-terminal cata-

lytic 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 dis-

similar dissociation constants, rather than two different

binding sites. These differing KD 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 shal-

low. 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 conse-

quences of phosphorylation on enzyme activity, but note

that there is a considerable recent literature on the physi-

ological 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; Hay-

cock 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). Phosphoryla-

tion 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 gen-

erated by alternative splicing). Four sites have been iden-

tified, 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 produc-

tion. 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 phospha-

tases. 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 iso-

forms. 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 Km for BH4 and an increase in the Ki for

I. Tekin et al.

123

dopamine both in vitro (Daubner et al. 1992; Lovenberg

et al. 1975; Okuno and Fujisawa 1985; Ramsey and Fitz-

patrick 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 hypothe-

sized to interact with DA, which is reversed upon phos-

phorylation (McCulloch et al. 2001). The PKA-mediated

decrease in the Km for BH4 and the resulting increase in

activity have also been demonstrated in bovine adrenal

chromaffin cells in situ (Meligeni et al. 1982). Phosphor-

ylation 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 Km of PKA for rat TH and for peptides

corresponding to the TH Ser40 phosphorylation site has

been shown to be about 100 lM, which is rather high

(Roskoski and Ritchie 1991). Almas et al. (1992) reported

that the Km of PKA for bovine TH was about 150 lM

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

Km values of only 5 lM. However, the addition of Fe2?

and DA increased the Km about threefold. This suggests

that the presence of tightly bound iron and DA in the

enzyme isolated from mammalian cells contributes to the

higher Km observed for the rat pheochromocytoma and

bovine adrenal enzymes.

Funakoshi et al. (1991) reported that TH phosphoryla-

tion 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 sub-

unit, 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 Km for BH4 and

increases the Ki 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 dif-

ferent 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 acetyl-

choline, 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 (Hay-

cock 2002a). Royo and Colette Daubner (2006) reported

that the phosphorylation of recombinant rat tyrosine

Table 1 Tyrosine hydroxylase phosphorylation sites and protein

kinases

Site Protein kinase References

Ser40 PKA Edelman et al. (1978), Joh et al.

(1978), Campbell et al. (1986)

PKG Roskoski et al. (1987)

PKC Albert et al. (1984)

CaMPKII Vulliet et al. (1984)

MSK1 Toska et al. (2002a)

MAPKAPK1 Sutherland et al. (1993)


MAPKAPK5/PRAK Toska et al. (2002a)

Ser19 CaMPKII Schworer and Soderling (1983),

Campbell et al. (1986)


MAPKAPK5/PRAK Toska et al. (2002a, b)

Cdk11 Sachs and Vaillancourt (2004)

Ser31 ERK1/2 Haycock et al. (1992)

Cdk5 Kansy et al. (2004)

The three serine residues are numbered as per the rat enzyme and

human TH-1 (see Fig. 5)

CaMPKII calcium–calmodulin-dependent protein kinase II, Cdk

cyclin-dependent kinase, ERK1/2 extracellular signal-regulated

kinase 1 and 2, or microtubule-associated protein kinases 1 and 2,

MAPKAPK microtubule-associated protein kinase-activated protein

kinase, MSK1 mitogen and stress-activated kinase 1, PKA protein

kinase A, or cyclic AMP-dependent protein kinase, PKC protein

kinase C, where C refers to calcium ion, PKG protein kinase G, or

cyclic GMP-dependent protein kinase, PRAK p38-regulated/activated

protein kinase


123

hydroxylase at Ser40 by purified bovine heart PKA was

diminished in the presence of dopamine. Ser40 phosphor-

ylation 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 confor-

mation 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 phys-

iological insults and influences mediate Ser40 phosphory-

lation and TH activation. Such effectors include

depolarization, hormones, receptor stimulation, and path-

ological 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 characteris-

tically 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 pro-

tein 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 c-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 phos-

phorylated 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 phosphor-

ylate 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 phos-

phorylation 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 phos-

phorylation 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 recombi-

nant mouse ERK2 at Ser31 occurs at a much higher rate

(Sutherland et al. 1993). Activation by phosphorylation of

I. Tekin et al.

123

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 phosphoryla-

tion by ERK1/2 was investigated in neuronal cells (Gordon

et al. 2009a). Human neuroblastoma SH-SY5Y dopami-

nergic cells were transfected with hTH-1 or hTH-2. hTH-1

displayed variable levels of phosphorylation upon stimu-

lation 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 cat-

alyzed 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 con-

sidered 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 physi-

ological 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 incorpo-

ration of phosphate under in vitro assay conditions sug-

gesting 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 pep-

tides 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 (Rodri-

guez-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 dis-

tinct 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 phos-

pho-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 phos-

phorylated at either residue. Moreover, different subpopu-

lations 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 phos-

phorylation increased in rat striatal slices upon depolar-

ization, 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 phosphor-

ylation-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,


123

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 phos-

phorylation 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 neu-

ronal, 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 depolar-

ization 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 depolariza-

tion-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 phosphory-

lation. This proved impractical because its inactive con-

gener had the same inhibitory effects. They reported that

PKA is responsible for the increase in Ser40 phosphory-

lation in PC12 cells and they concluded that Ser31 phos-

phorylation is necessary and sufficient for depolarization-

dependent increases in catecholamine synthesis in PC12

and A126-B1 cells. They concluded that Ser40 phosphor-

ylation 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 con-

cluded 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 produc-

tion. Increases in calcium concentration at the nerve ter-

minal to induce synaptic vesicle fusion to the presynaptic

cell membrane will also simultaneously activate CaMPKII,

causing an increase in DA production. Ser40 phosphoryla-

tion (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 Km for BH4 and higher Ki 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 phos-

phorylation and decreases feedback inhibition.

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). Sur-

prisingly, 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.

Table 2 Phosphorylation stoichiometry of tyrosine hydroxylase

SN Striatum VTA NA Hypothalamus PC12 cells A126-B1

Ser19 0.25 0.10 0.25 0.15 0.26 0.05 0.05

Ser31 0.08 0.30 0.10 0.20 0.13 0.09 0.08

Ser40 0.03 0.03 0.04 0.04 0.05 0.035 0.01

Number of moles of phosphate/mole of tyrosine hydroxylase subunit; data from Salvatore et al. (2000, 2001)

SN substantia nigra, VTA ventral tegmental area, NA nucleus accumbens

I. Tekin et al.

123

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 dephospho-

rylation 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, a-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 a-synuc-

lein on PP2A did not provide a similar observation in vivo

(Drolet et al. 2006). Interestingly, a-synuclein overex-

pression 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 func-

tion 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/B0b) 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) (Rag-

huraman et al. 2009).

Fig. 6 Schematic depiction of

the summary of the proposed

physiological effect of TH

phosphorylation. a The relative

amount of TH phosphorylated at

different sites is depicted in

accordance with their

subcellular locations. b The role

of Ser40 phosphorylation and its

regulation in maintaining

dopamine in the nerve terminal

are shown for stimuli

originating from autoreceptors,

reuptake transporters, cAMP,

and Ca2?


123

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; Yama-

uchi et al. 1981). Binding requires stimuli such as depo-

larization or an increase in calcium to activate CaMPKII.

Itagaki et al. (1999) studied the interaction between

recombinant hTH-1 and 14-3-3g. Phosphorylation of TH

by purified rat brain CaMPKII resulted in Ser19 phos-

phorylation and binding of 14-3-3 with a Kd of 3 nM.

Phosphorylation by recombinant PKA leads to phosphor-

ylation 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 iso-

forms occurs following phosphorylation of Ser40, but

binding of bovine brain 14-3-3-f 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-phos-

phorylated 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 cir-

cular dichroism.

Many studies have been conducted to identify the spe-

cific 14-3-3 subtype(s) interacting with TH. The 14-3-3 cisoform 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

14-3-3c forms a triplet complex with bovine adrenal

chromaffin membrane lipids and Ser19-phosphorylated

recombinant hTH-1, suggesting a role for cellular locali-

zation and hTH-1 activation by this protein. These inves-

tigators 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 a-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 f 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 spe-

cific subcellular localization for TH interaction with dif-

ferent 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 res-

idues 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 g 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 neurodegen-

eration. These include a-synuclein (a-Syn), which is a

major protein in Lewy bodies, the intraneuronal protein

aggregates characteristic of Parkinson’s disease (Spillantini

et al. 1998). a-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). a-Syn co-immunoprecipi-

tates with TH in rat brain and in mouse MN9D dopami-

nergic cells (Perez et al. 2002). a-Syn overexpression not

only reduces TH activity, but it also causes a decrease in

mouse MN9D cellular dopamine content. a-Syn is over-

expressed upon increased dopamine oxidation; hence pro-

ducing 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 a-syn

expression in mouse MN9D cells reduces endogenous TH

Ser19 phosphorylation. They also reported that either wild-

type or mutant human Ser129Ala a-syn enhances PP2A

activity in these cells with the Ser129Ala mutant producing

greater activation. Phosphorylation of a-syn at Ser129

decreases its PP2A stimulatory activity. They also found

that overexpression of wild-type human a-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 a-syn depletes cellular

dopamine content (Perez et al. 2002). Regulation of a-

synuclein, therefore, provides an additional control point in

catecholamine homeostasis.

I. Tekin et al.

123

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-immu-

noprecipitation, and complex formation involving these

components provides a mechanism that links DA syn-

thesis with synaptic vesicle filling (Cartier et al. 2010).

This linkage may maintain low cytosolic dopamine levels

and decreases the generation of reactive neurotoxic cate-

chol-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 sta-

bility 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 dopa-

mine 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 phos-

phorylation 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 exis-

tence 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 catechol-

amine 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 dif-

ferent 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 condi-

tions. TH dysfunction has been implicated in several dis-

eases 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 polymor-

phisms indicates that there are more than 50 genetic vari-

ants in human TH coding region that alter amino acids


123

(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 lab-

oratory for enzyme structure–function relationships. There

is surprisingly little information on the functional conse-

quences 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 asso-

ciated pathology. Indeed, given that TH deletion is asso-

ciated with embryonic lethality, we anticipate that there

may be severe consequences for a few genetic variants.

Moreover, genetically mediated changes in protein struc-

ture may influence the regulation of this pivotal enzyme

and so modulate catecholamine levels. For this reason,

future work in personalized medicine will need to incor-

porate knowledge of the regulatory effects of TH SNPs (as

well as other enzymes) on catecholamine levels in health

and disease.

Acknowledgments 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.

References

Abate C, Smith JA, Joh TH (1988) Characterization of the catalytic

domain of bovine adrenal tyrosine hydroxylase. Biochem

Biophys Res Commun 151(3):1446–1453

Ahn JH, Kim Y, Kim HS, Greengard P, Nairn AC (2011) Protein

kinase C-dependent dephosphorylation of tyrosine hydroxylase

requires the B56delta heterotrimeric form of protein phosphatase

2A. PLoS One 6(10):e26292. doi:10.1371/journal.pone.0026292

Albert KA, Helmer-Matyjek E, Nairn AC, Muller TH, Haycock JW,

Greene LA, Goldstein M, Greengard P (1984) Calcium/

phospholipid-dependent protein kinase (protein kinase C) phos-

phorylates and activates tyrosine hydroxylase. Proc Natl Acad

Sci USA 81(24):7713–7717

Alerte TN, Akinfolarin AA, Friedrich EE, Mader SA, Hong CS, Perez

RG (2008) a-Synuclein aggregation alters tyrosine hydroxylase

phosphorylation and immunoreactivity: lessons from viral

transduction of knockout mice. Neurosci Lett 435(1):24–29.

doi:10.1016/j.neulet.2008.02.014

Almas B, Le Bourdelles B, Flatmark T, Mallet J, Haavik J (1992)

Regulation of recombinant human tyrosine hydroxylase

isozymes by catecholamine binding and phosphorylation. Struc-

ture/activity studies and mechanistic implications. Eur J Bio-

chem 209(1):249–255

Alterio J, Ravassard P, Haavik J, Le Caer JP, Biguet NF, Waksman G,

Mallet J (1998) Human tyrosine hydroxylase isoforms. Inhibition

by excess tetrahydropterin and unusual behavior of isoform 3

after camp-dependent protein kinase phosphorylation. J Biol

Chem 273(17):10196–10201

Andersson KK, Cox DD, Que L Jr, Flatmark T, Haavik J (1988)

Resonance Raman studies on the blue-green-colored bovine

adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evi-

dence that the feedback inhibitors adrenaline and noradrenaline

are coordinated to iron. J Biol Chem 263(35):18621–18626

Andersson KK, Vassort C, Brennan BA, Que L Jr, Haavik J, Flatmark

T, Gros F, Thibault J (1992) Purification and characterization of

the blue-green rat phaeochromocytoma (PC12) tyrosine hydrox-

ylase with a dopamine-Fe(III) complex. Reversal of the endog-

enous feedback inhibition by phosphorylation of serine-40.

Biochem J 284(Pt 3):687–695

Apostolova G, Dechant G (2009) Development of neurotransmitter

phenotypes in sympathetic neurons. Auton Neurosci

151(1):30–38. doi:10.1016/j.autneu.2009.08.012

Aranyi T, Sarkis C, Berrard S, Sardin K, Siron V, Khalfallah O,

Mallet J (2007) Sodium butyrate modifies the stabilizing

complexes of tyrosine hydroxylase mRNA. Biochem Biophys

Res Commun 359(1):15–19. doi:10.1016/j.bbrc.2007.05.025

Bademci G, Vance JM, Wang L (2012) Tyrosine hydroxylase gene:

another piece of the genetic puzzle of Parkinson’s disease. CNS

Neurol Disord Drug Targets 11(4):469–481

Bailey SW, Dillard SB, Thomas KB, Ayling JE (1989) Changes in the

cofactor binding domain of bovine striatal tyrosine hydroxylase

at physiological pH upon cAMP-dependent phosphorylation

mapped with tetrahydrobiopterin analogues. Biochemistry

28(2):494–504

Bauer AL, Paulding WR, Striet JB, Schnell PO, Czyzyk-Krzeska MF

(2002) Endogenous von Hippel–Lindau tumor suppressor protein

regulates catecholaminergic phenotype in PC12 cells. Cancer

Res 62(6):1682–1687

Beitner-Johnson D, Millhorn DE (1998) Hypoxia induces phosphor-

ylation of the cyclic AMP response element-binding protein by a

novel signaling mechanism. J Biol Chem 273(31):19834–19839

Bellivier F (2005) Schizophrenia, antipsychotics and diabetes: genetic

aspects. Eur Psychiatry 20(Suppl 4):S335–S339

Berresheim U, Kuhn DM (1994) Dephosphorylation of tyrosine

hydroxylase by brain protein phosphatases: a predominant role

for type 2A. Brain Res 637(1–2):273–276

Best JA, Tank AW (1998) The THCRE2 site in the rat tyrosine

hydroxylase gene promoter is responsive to phorbol ester.

Neurosci Lett 258(3):131–134

Bevilaqua LR, Graham ME, Dunkley PR, von Nagy-Felsobuki EI,

Dickson PW (2001) Phosphorylation of Ser(19) alters the

conformation of tyrosine hydroxylase to increase the rate of

phosphorylation of Ser(40). J Biol Chem 276(44):40411–40416.

doi:10.1074/jbc.M105280200

Fig. 7 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

I. Tekin et al.

123

http://dx.doi.org/10.1371/journal.pone.0026292

http://dx.doi.org/10.1016/j.neulet.2008.02.014

http://dx.doi.org/10.1016/j.autneu.2009.08.012

http://dx.doi.org/10.1016/j.bbrc.2007.05.025

http://dx.doi.org/10.1074/jbc.M105280200

Bevilaqua LR, Cammarota M, Dickson PW, Sim AT, Dunkley PR

(2003) Role of protein phosphatase 2C from bovine adrenal

chromaffin cells in the dephosphorylation of phospho-serine 40

tyrosine hydroxylase. J Neurochem 85(6):1368–1373

Birman S, Morgan B, Anzivino M, Hirsh J (1994) A novel and major

isoform of tyrosine hydroxylase in Drosophila is generated by

alternative RNA processing. J Biol Chem 269(42):26559–26567

Bobrovskaya L, Dunkley PR, Dickson PW (2004) Phosphorylation of

Ser19 increases both Ser40 phosphorylation and enzyme activity

of tyrosine hydroxylase in intact cells. J Neurochem

90(4):857–864. doi:10.1111/j.1471-4159.2004.02550.x

Bodeau-Pean S, Ravassard P, Neuner-Jehle M, Faucheux B, Mallet J,

Dumas S (1999) A human tyrosine hydroxylase isoform

associated with progressive supranuclear palsy shows altered

enzymatic activity. J Biol Chem 274(6):3469–3475

Bowling KM, Huang Z, Xu D, Ferdousy F, Funderburk CD, Karnik

N, Neckameyer W, O’Donnell JM (2008) Direct binding of GTP

cyclohydrolase and tyrosine hydroxylase: regulatory interactions

between key enzymes in dopamine biosynthesis. J Biol Chem

283(46):31449–31459. doi:10.1074/jbc.M802552200

Briggs GD, Gordon SL, Dickson PW (2011) Mutational analysis of

catecholamine binding in tyrosine hydroxylase. Biochemistry

50(9):1545–1555. doi:10.1021/bi101455b

Cahill AL, Horwitz J, Perlman RL (1989) Phosphorylation of tyrosine

hydroxylase in protein kinase C-deficient PC12 cells. Neurosci-

ence 30(3):811–818

Campbell DG, Hardie DG, Vulliet PR (1986) Identification of four

phosphorylation sites in the N-terminal region of tyrosine

hydroxylase. J Biol Chem 261(23):10489–10492

Cartier EA, Parra LA, Baust TB, Quiroz M, Salazar G, Faundez V,

Egana L, Torres GE (2010) A biochemical and functional protein

complex involving dopamine synthesis and transport into

synaptic vesicles. J Biol Chem 285(3):1957–1966. doi:10.1074/

jbc.M109.054510

Cazorla P, Smidt MP, O’Malley KL, Burbach JP (2000) A response

element for the homeodomain transcription factor Ptx3 in the

tyrosine hydroxylase gene promoter. J Neurochem

74(5):1829–1837

Chen X, Xu L, Radcliffe P, Sun B, Tank AW (2008) Activation of

tyrosine hydroxylase mRNA translation by cAMP in midbrain

dopaminergic neurons. Mol Pharmacol 73(6):1816–1828. doi:10.

1124/mol.107.043968

Chou A, Toon C, Pickett J, Gill AJ (2013) von Hippel–Lindau

syndrome. Front Horm Res 41:30–49. doi:10.1159/000345668

Chow MS, Eser BE, Wilson SA, Hodgson KO, Hedman B, Fitzpatrick

PF, Solomon EI (2009) Spectroscopy and kinetics of wild-type

and mutant tyrosine hydroxylase: mechanistic insight into O2

activation. J Am Chem Soc 131(22):7685–7698. doi:10.1021/

ja810080c

Cohen P (2002) The origins of protein phosphorylation. Nat Cell Biol

4(5):E127–E130. doi:10.1038/ncb0502-e127

Coker GT 3rd, Studelska D, Harmon S, Burke W, O’Malley KL

(1990) Analysis of tyrosine hydroxylase and insulin transcripts

in human neuroendocrine tissues. Brain Res Mol Brain Res

8(2):93–98

Czyzyk-Krzeska MF, Beresh JE (1996) Characterization of the

hypoxia-inducible protein binding site within the pyrimidine-rich

tract in the 30-untranslated region of the tyrosine hydroxylase

mRNA. J Biol Chem 271(6):3293–3299

Daubner SC, Piper MM (1995) Deletion mutants of tyrosine

hydroxylase identify a region critical for heparin binding.

Protein Sci 4(3):538–541. doi:10.1002/pro.5560040320

Daubner SC, Lauriano C, Haycock JW, Fitzpatrick PF (1992) Site-

directed mutagenesis of serine 40 of rat tyrosine hydroxylase.

Effects of dopamine and cAMP-dependent phosphorylation on

enzyme activity. J Biol Chem 267(18):12639–12646

Daubner SC, Melendez J, Fitzpatrick PF (2000) Reversing the

substrate specificities of phenylalanine and tyrosine hydroxylase:

aspartate 425 of tyrosine hydroxylase is essential for L-DOPA

formation. Biochemistry 39(32):9652–9661

Daubner SC, Moran GR, Fitzpatrick PF (2002) Role of tryptophan

hydroxylase phe313 in determining substrate specificity. Bio-

chem Biophys Res Commun 292(3):639–641. doi:10.1006/bbrc.

2002.6719

Daubner SC, McGinnis JT, Gardner M, Kroboth SL, Morris AR,

Fitzpatrick PF (2006) A flexible loop in tyrosine hydroxylase

controls coupling of amino acid hydroxylation to tetrahydrop-

terin oxidation. J Mol Biol 359(2):299–307. doi:10.1016/j.jmb.

2006.03.016

Daubner SC, Le T, Wang S (2011) Tyrosine hydroxylase and

regulation of dopamine synthesis. Arch Biochem Biophys

508(1):1–12. doi:10.1016/j.abb.2010.12.017

Dickson PW, Briggs GD (2013) Tyrosine hydroxylase: regulation by

feedback inhibition and phosphorylation. Adv Pharmacol

68:13–21. doi:10.1016/B978-0-12-411512-5.00002-6

Diliberto EJ Jr, Daniels AJ, Viveros OH (1991) Multicompartmental

secretion of ascorbate and its dual role in dopamine beta-

hydroxylation. Am J Clin Nutr 54(6 Suppl):1163S–1172S

Dix TA, Kuhn DM, Benkovic SJ (1987) Mechanism of oxygen

activation by tyrosine hydroxylase. Biochemistry

26(12):3354–3361

Doskeland AP, Flatmark T (2002) Ubiquitination of soluble and

membrane-bound tyrosine hydroxylase and degradation of the

soluble form. Eur J Biochem 269(5):1561–1569

Drolet RE, Behrouz B, Lookingland KJ, Goudreau JL (2006)

Substrate-mediated enhancement of phosphorylated tyrosine

hydroxylase in nigrostriatal dopamine neurons: evidence for a

role of a-synuclein. J Neurochem 96(4):950–959. doi:10.1111/j.

1471-4159.2005.03606.x

Dumas S, Le Hir H, Bodeau-Pean S, Hirsch E, Thermes C, Mallet J

(1996) New species of human tyrosine hydroxylase mRNA are

produced in variable amounts in adrenal medulla and are

overexpressed in progressive supranuclear palsy. J Neurochem

67(1):19–25

Dunkley PR, Bobrovskaya L, Graham ME, von Nagy-Felsobuki EI,

Dickson PW (2004) Tyrosine hydroxylase phosphorylation:

regulation and consequences. J Neurochem 91(5):1025–1043.

doi:10.1111/j.1471-4159.2004.02797.x

Edelman AM, Raese JD, Lazar MA, Barchas JD (1978) In vitro

phosphorylation of a purified preparation of bovine corpus

striatal tyrosine hydroxylase. Commun Psychopharmacol

2(6):461–465

Ellis HR, Daubner SC, Fitzpatrick PF (2000) Mutation of serine 395

of tyrosine hydroxylase decouples oxygen–oxygen bond cleav-

age and tyrosine hydroxylation. Biochemistry 39(14):4174–4181

Ernsberger U (2001) The development of postganglionic sympathetic

neurons: coordinating neuronal differentiation and diversifica-

tion. Auton Neurosci 94(1–2):1–13. doi:10.1016/S1566-

0702(01)00336-8

Fitzpatrick PF (1988) The pH dependence of binding of inhibitors to

bovine adrenal tyrosine hydroxylase. J Biol Chem

263(31):16058–16062

Fitzpatrick PF (1989) The metal requirement of rat tyrosine hydrox-

ylase. Biochem Biophys Res Commun 161(1):211–215

Fitzpatrick PF (1991) Steady-state kinetic mechanism of rat tyrosine

hydroxylase. Biochemistry 30(15):3658–3662

Fitzpatrick PF (1999) Tetrahydropterin-dependent amino acid

hydroxylases. Annu Rev Biochem 68:355–381. doi:10.1146/

annurev.biochem.68.1.355

Fitzpatrick PF (2003) Mechanism of aromatic amino acid hydroxyl-

ation. Biochemistry 42(48):14083–14091. doi:10.1021/

bi035656u


123

http://dx.doi.org/10.1111/j.1471-4159.2004.02550.x


http://dx.doi.org/10.1021/bi101455b

http://dx.doi.org/10.1074/jbc.M109.054510


http://dx.doi.org/10.1124/mol.107.043968


http://dx.doi.org/10.1159/000345668

http://dx.doi.org/10.1021/ja810080c

http://dx.doi.org/10.1021/ja810080c

http://dx.doi.org/10.1038/ncb0502-e127

http://dx.doi.org/10.1002/pro.5560040320

http://dx.doi.org/10.1006/bbrc.2002.6719


http://dx.doi.org/10.1016/j.jmb.2006.03.016


http://dx.doi.org/10.1016/j.abb.2010.12.017

http://dx.doi.org/10.1016/B978-0-12-411512-5.00002-6

http://dx.doi.org/10.1111/j.1471-4159.2005.03606.x

http://dx.doi.org/10.1111/j.1471-4159.2005.03606.x

http://dx.doi.org/10.1111/j.1471-4159.2004.02797.x

http://dx.doi.org/10.1016/S1566-0702(01)00336-8

http://dx.doi.org/10.1016/S1566-0702(01)00336-8

http://dx.doi.org/10.1146/annurev.biochem.68.1.355

http://dx.doi.org/10.1146/annurev.biochem.68.1.355

http://dx.doi.org/10.1021/bi035656u

http://dx.doi.org/10.1021/bi035656u

Fujisawa H, Okuno S (2005) Regulatory mechanism of tyrosine

hydroxylase activity. Biochem Biophys Res Commun

338(1):271–276. doi:10.1016/j.bbrc.2005.07.183

Fukuda T, Ishii K, Nanmoku T, Isobe K, Kawakami Y, Takekoshi K

(2007) 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofurano-

side stimulates tyrosine hydroxylase activity and catecholamine

secretion by activation of AMP-activated protein kinase in PC12

cells. J Neuroendocrinol 19(8):621–631. doi:10.1111/j.1365-

2826.2007.01570.x

Funakoshi H, Okuno S, Fujisawa H (1991) Different effects on

activity caused by phosphorylation of tyrosine hydroxylase at

serine 40 by three multifunctional protein kinases. J Biol Chem

266(24):15614–15620

Gahn LG, Roskoski R Jr (1995) Thermal stability and CD analysis of

rat tyrosine hydroxylase. Biochemistry 34(1):252–256

Gardaneh M, Gilbert J, Haber M, Norris MD, Cohn SL, Schmidt ML,

Marshall GM (2000) Synergy between 50 and 30 flanking regions

of the human tyrosine hydroxylase gene ensures specific, high-

level expression in neuroblastoma cells. Neurosci Lett

292(3):147–150

Ghee M, Baker H, Miller JC, Ziff EB (1998) AP-1, CREB and CBP

transcription factors differentially regulate the tyrosine hydrox-

ylase gene. Brain Res Mol Brain Res 55(1):101–114

Goncalves CA, Hall A, Sim AT, Bunn SJ, Marley PD, Cheah TB,

Dunkley PR (1997) Tyrosine hydroxylase phosphorylation in

digitonin-permeabilized bovine adrenal chromaffin cells: the

effect of protein kinase and phosphatase inhibitors on Ser19 and

Ser40 phosphorylation. J Neurochem 69(6):2387–2396

Gonzalez-Flores O, Gomora-Arrati P, Garcia-Juarez M, Miranda-

Martinez A, Armengual-Villegas A, Camacho-Arroyo I, Guerra-

Araiza C (2011) Progesterone receptor isoforms differentially

regulate the expression of tryptophan and tyrosine hydroxylase

and glutamic acid decarboxylase in the rat hypothalamus.

Neurochem Int 59(5):671–676. doi:10.1016/j.neuint.2011.06.013

Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens

RC (1997) Crystal structure of tyrosine hydroxylase at 2.3 A and

its implications for inherited neurodegenerative diseases. Nat

Struct Biol 4(7):578–585

Goodwill KE, Sabatier C, Stevens RC (1998) Crystal structure of

tyrosine hydroxylase with bound cofactor analogue and iron at

2.3 A resolution: self-hydroxylation of Phe300 and the pterin-

binding site. Biochemistry 37(39):13437–13445. doi:10.1021/

bi981462g

Gordon SL, Quinsey NS, Dunkley PR, Dickson PW (2008) Tyrosine

hydroxylase activity is regulated by two distinct dopamine-

binding sites. J Neurochem 106(4):1614–1623. doi:10.1111/j.

1471-4159.2008.05509.x

Gordon SL, Bobrovskaya L, Dunkley PR, Dickson PW (2009a)

Differential regulation of human tyrosine hydroxylase isoforms 1

and 2 in situ: Isoform 2 is not phosphorylated at Ser35. Biochim

Biophys Acta 1793(12):1860–1867. doi:10.1016/j.bbamcr.2009.

10.001

Gordon SL, Webb JK, Shehadeh J, Dunkley PR, Dickson PW (2009b)

The low affinity dopamine binding site on tyrosine hydroxylase:

the role of the N-terminus and in situ regulation of enzyme

activity. Neurochem Res 34(10):1830–1837. doi:10.1007/

s11064-009-9989-5

Gozal E, Shah ZA, Pequignot JM, Pequignot J, Sachleben LR,

Czyzyk-Krzeska MF, Li RC, Guo SZ, Gozal D (2005) Tyrosine

hydroxylase expression and activity in the rat brain: differential

regulation after long-term intermittent or sustained hypoxia. J Appl

Physiol 99(2):642–649. doi:10.1152/japplphysiol.00880.2004

Grenett HE, Ledley FD, Reed LL, Woo SL (1987) Full-length cDNA

for rabbit tryptophan hydroxylase: functional domains and

evolution of aromatic amino acid hydroxylases. Proc Natl Acad

Sci USA 84(16):5530–5534

Griffith LC, Schulman H (1988) The multifunctional Ca2?/calmod-

ulin-dependent protein kinase mediates Ca2?-dependent phos-

phorylation of tyrosine hydroxylase. J Biol Chem

263(19):9542–9549

Grima B, Lamouroux A, Blanot F, Biguet NF, Mallet J (1985)

Complete coding sequence of rat tyrosine hydroxylase mRNA.

Proc Natl Acad Sci USA 82(2):617–621

Grima B, Lamouroux A, Boni C, Julien JF, Javoy-Agid F, Mallet J

(1987) A single human gene encoding multiple tyrosine

hydroxylases with different predicted functional characteristics.

Nature 326(6114):707–711. doi:10.1038/326707a0

Guo Z, Du X, Iacovitti L (1998) Regulation of tyrosine hydroxylase

gene expression during transdifferentiation of striatal neurons:

changes in transcription factors binding the AP-1 site. J Neurosci

18(20):8163–8174

Haavik J, Andersson KK, Petersson L, Flatmark T (1988) Soluble

tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine

adrenal medulla: large-scale purification and physicochemical

properties. Biochim Biophys Acta 953(2):142–156

Haavik J, Schelling DL, Campbell DG, Andersson KK, Flatmark T,

Cohen P (1989) Identification of protein phosphatase 2A as the

major tyrosine hydroxylase phosphatase in adrenal medulla and

corpus striatum: evidence from the effects of okadaic acid. FEBS

Lett 251(1–2):36–42

Haavik J, Martinez A, Flatmark T (1990) pH-dependent release of

catecholamines from tyrosine hydroxylase and the effect of

phosphorylation of Ser-40. FEBS Lett 262(2):363–365

Haavik J, Le Bourdelles B, Martinez A, Flatmark T, Mallet J (1991)

Recombinant human tyrosine hydroxylase isozymes. Reconsti-

tution with iron and inhibitory effect of other metal ions. Eur J

Biochem 199(2):371–378

Haavik J, Blau N, Thony B (2008) Mutations in human monoamine-

related neurotransmitter pathway genes. Hum Mutat

29(7):891–902. doi:10.1002/humu.20700

Halloran SM, Vulliet PR (1994) Microtubule-associated protein

kinase-2 phosphorylates and activates tyrosine hydroxylase

following depolarization of bovine adrenal chromaffin cells.

J Biol Chem 269(49):30960–30965

Halskau O Jr, Ying M, Baumann A, Kleppe R, Rodriguez-Larrea D,

Almas B, Haavik J, Martinez A (2009) Three-way interaction

between 14-3-3 proteins, the N-terminal region of tyrosine

hydroxylase, and negatively charged membranes. J Biol Chem

284(47):32758–32769. doi:10.1074/jbc.M109.027706

Harada WuJ, Haycock JW, Goldstein M (1996) Regulation of L-

DOPA biosynthesis by site-specific phosphorylation of tyrosine

hydroxylase in AtT-20 cells expressing wild-type and serine

40-substituted enzyme. J Neurochem 67(2):629–635

Hastings TG, Zigmond MJ (1994) Identification of catechol-protein

conjugates in neostriatal slices incubated with [3H]dopamine: impact

of ascorbic acid and glutathione. J Neurochem 63(3):1126–1132

Haycock JW (1990) Phosphorylation of tyrosine hydroxylase in situ

at serine 8, 19, 31, and 40. J Biol Chem 265(20):11682–11691

Haycock JW (1991) Four forms of tyrosine hydroxylase are present in

human adrenal medulla. J Neurochem 56(6):2139–2142

Haycock JW (1993) Multiple forms of tyrosine hydroxylase in human

neuroblastoma cells: quantitation with isoform-specific antibod-

ies. J Neurochem 60(2):493–502

Haycock JW (2002a) Peptide substrates for ERK1/2: structure-

function studies of serine 31 in tyrosine hydroxylase. J Neurosci

Methods 116(1):29–34

Haycock JW (2002b) Species differences in the expression of

multiple tyrosine hydroxylase protein isoforms. J Neurochem

81(5):947–953

Haycock JW, Haycock DA (1991) Tyrosine hydroxylase in rat brain

dopaminergic nerve terminals. Multiple-site phosphorylation

in vivo and in synaptosomes. J Biol Chem 266(9):5650–5657

I. Tekin et al.

123


http://dx.doi.org/10.1111/j.1365-2826.2007.01570.x

http://dx.doi.org/10.1111/j.1365-2826.2007.01570.x

http://dx.doi.org/10.1016/j.neuint.2011.06.013

http://dx.doi.org/10.1021/bi981462g

http://dx.doi.org/10.1021/bi981462g

http://dx.doi.org/10.1111/j.1471-4159.2008.05509.x

http://dx.doi.org/10.1111/j.1471-4159.2008.05509.x

http://dx.doi.org/10.1016/j.bbamcr.2009.10.001

http://dx.doi.org/10.1016/j.bbamcr.2009.10.001

http://dx.doi.org/10.1007/s11064-009-9989-5

http://dx.doi.org/10.1007/s11064-009-9989-5

http://dx.doi.org/10.1152/japplphysiol.00880.2004

http://dx.doi.org/10.1038/326707a0

http://dx.doi.org/10.1002/humu.20700


Haycock JW, Wakade AR (1992) Activation and multiple-site

phosphorylation of tyrosine hydroxylase in perfused rat adrenal

glands. J Neurochem 58(1):57–64

Haycock JW, Ahn NG, Cobb MH, Krebs EG (1992) ERK1 and

ERK2, two microtubule-associated protein 2 kinases, mediate

the phosphorylation of tyrosine hydroxylase at serine-31 in situ.

Proc Natl Acad Sci USA 89(6):2365–2369

Haycock JW, Lew JY, Garcia-Espana A, Lee KY, Harada K, Meller

E, Goldstein M (1998) Role of serine-19 phosphorylation in

regulating tyrosine hydroxylase studied with site- and phospho-

specific antibodies and site-directed mutagenesis. J Neurochem

71(4):1670–1675

He X, Lee KY, Li LhL, Meller E, Goldstein M (1996) Relationship

between enzymatic activity and oligomerization state of tyrosine

hydroxylase. J Biomed Sci 3(5):332–337

Hebert MA, Serova LI, Sabban EL (2005) Single and repeated

immobilization stress differentially trigger induction and phos-

phorylation of several transcription factors and mitogen-acti-

vated protein kinases in the rat locus coeruleus. J Neurochem

95(2):484–498. doi:10.1111/j.1471-4159.2005.03386.x

Hiremagalur B, Nankova B, Nitahara J, Zeman R, Sabban EL (1993)

Nicotine increases expression of tyrosine hydroxylase gene.

Involvement of protein kinase A-mediated pathway. J Biol Chem

268(31):23704–23711

Horellou P, Le Bourdelles B, Clot-Humbert J, Guibert B, Leviel V,

Mallet J (1988) Multiple human tyrosine hydroxylase enzymes,

generated through alternative splicing, have different specific

activities in Xenopus oocytes. J Neurochem 51(2):652–655

Ichikawa S, Ichinose H, Nagatsu T (1990) Multiple mRNAs of

monkey tyrosine hydroxylase. Biochem Biophys Res Commun

173(3):1331–1336

Ichimura T, Isobe T, Okuyama T, Yamauchi T, Fujisawa H (1987)

Brain 14-3-3 protein is an activator protein that activates

tryptophan 5-monooxygenase and tyrosine 3-monooxygenase

in the presence of Ca2?, calmodulin-dependent protein kinase II.

FEBS Lett 219(1):79–82

Ichinose H, Ohye T, Fujita K, Yoshida M, Ueda S, Nagatsu T (1993)

Increased heterogeneity of tyrosine hydroxylase in humans.

Biochem Biophys Res Commun 195(1):158–165. doi:10.1006/

bbrc.1993.2024

Ichinose H, Ohye T, Fujita K, Pantucek F, Lange K, Riederer P,

Nagatsu T (1994) Quantification of mRNA of tyrosine hydrox-

ylase and aromatic L-amino acid decarboxylase in the substantia

nigra in Parkinson’s disease and schizophrenia. J Neural Transm

8(1–2):149–158

Ichinose H, Suzuki T, Inagaki H, Ohye T, Nagatsu T (1999)

Molecular genetics of dopa-responsive dystonia. Biol Chem

380(12):1355–1364. doi:10.1515/BC.1999.175

Isobe T, Ichimura T, Sunaya T, Okuyama T, Takahashi N, Kuwano R,

Takahashi Y (1991) Distinct forms of the protein kinase-

dependent activator of tyrosine and tryptophan hydroxylases.

J Mol Biol 217(1):125–132

Itagaki C, Isobe T, Taoka M, Natsume T, Nomura N, Horigome T,

Omata S, Ichinose H, Nagatsu T, Greene LA, Ichimura T (1999)

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-

3 proteins. Biochemistry 38(47):15673–15680

Iwawaki T, Kohno K, Kobayashi K (2000) Identification of a

potential nurr1 response element that activates the tyrosine

hydroxylase gene promoter in cultured cells. Biochem Biophys

Res Commun 274(3):590–595. doi:10.1006/bbrc.2000.3204

Jacobs FM, van Erp S, van der Linden AJ, von Oerthel L, Burbach JP,

Smidt MP (2009) Pitx3 potentiates Nurr1 in dopamine neuron

terminal differentiation through release of SMRT-mediated repres-

sion. Development 136(4):531–540. doi:10.1242/dev.029769

Jacobsen KX, MacDonald H, Lemonde S, Daigle M, Grimes DA,

Bulman DE, Albert PR (2008) A Nurr1 point mutant, implicated

in Parkinson’s disease, uncouples ERK1/2-dependent regulation

of tyrosine hydroxylase transcription. Neurobiol Dis

29(1):117–122. doi:10.1016/j.nbd.2007.08.003

Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL Jr (2013)

A new model for allosteric regulation of phenylalanine hydrox-

ylase: implications for disease and therapeutics. Arch Biochem

Biophys 530(2):73–82. doi:10.1016/j.abb.2012.12.017

Jensik PJ, Arbogast LA (2011) Differential and interactive effects of

ligand-bound progesterone receptor A and B isoforms on

tyrosine hydroxylase promoter activity. J Neuroendocrinol

23(10):915–925. doi:10.1111/j.1365-2826.2011.02197.x

Jin H, Romano G, Marshall C, Donaldson AE, Suon S, Iacovitti L

(2006) Tyrosine hydroxylase gene regulation in human neuronal

progenitor cells does not depend on Nurr1 as in the murine and

rat systems. J Cell Physiol 207(1):49–57. doi:10.1002/jcp.20534

Joh TH, Park DH, Reis DJ (1978) Direct phosphorylation of brain

tyrosine hydroxylase by cyclic AMP-dependent protein kinase:

mechanism of enzyme activation. Proc Natl Acad Sci USA

75(10):4744–4748

Kaneda N, Kobayashi K, Ichinose H, Kishi F, Nakazawa A,

Kurosawa Y, Fujita K, Nagatsu T (1987) Isolation of a novel

cDNA clone for human tyrosine hydroxylase: alternative RNA

splicing produces four kinds of mRNA from a single gene.

Biochem Biophys Res Commun 146(3):971–975

Kansy JW, Daubner SC, Nishi A, Sotogaku N, Lloyd MD, Nguyen C,

Lu L, Haycock JW, Hope BT, Fitzpatrick PF, Bibb JA (2004)

Identification of tyrosine hydroxylase as a physiological sub-

strate for Cdk5. J Neurochem 91(2):374–384. doi:10.1111/j.

1471-4159.2004.02723.x

Kawahata I, Tokuoka H, Parvez H, Ichinose H (2009) Accumulation

of phosphorylated tyrosine hydroxylase into insoluble protein

aggregates by inhibition of an ubiquitin-proteasome system in

PC12D cells. J Neural Transm 116(12):1571–1578. doi:10.1007/

s00702-009-0304-z

Kelly BB, Hedlund E, Kim C, Ishiguro H, Isacson O, Chikaraishi

DM, Kim KS, Feng G (2006) A tyrosine hydroxylase-yellow

fluorescent protein knock-in reporter system labeling dopami-

nergic neurons reveals potential regulatory role for the first

intron of the rodent tyrosine hydroxylase gene. Neuroscience

142(2):343–354. doi:10.1016/j.neuroscience.2006.06.032

Kessler MA, Yang M, Gollomp KL, Jin H, Iacovitti L (2003) The

human tyrosine hydroxylase gene promoter. Brain Res Mol

Brain Res 112(1–2):8–23

Kim KS, Kim CH, Hwang DY, Seo H, Chung S, Hong SJ, Lim JK,

Anderson T, Isacson O (2003) Orphan nuclear receptor Nurr1

directly transactivates the promoter activity of the tyrosine

hydroxylase gene in a cell-specific manner. J Neurochem

85(3):622–634

Kim SM, Yang JW, Park MJ, Lee JK, Kim SU, Lee YS, Lee MA

(2006) Regulation of human tyrosine hydroxylase gene by

neuron-restrictive silencer factor. Biochem Biophys Res Com-

mun 346(2):426–435. doi:10.1016/j.bbrc.2006.05.142

Kleppe R, Toska K, Haavik J (2001) Interaction of phosphorylated

tyrosine hydroxylase with 14-3-3 proteins: evidence for a phospho-

serine 40-dependent association. J Neurochem 77(4):1097–1107

Kobayashi K, Nagatsu T (2005) Molecular genetics of tyrosine

3-monooxygenase and inherited diseases. Biochem Biophys Res

Commun 338(1):267–270. doi:10.1016/j.bbrc.2005.07.186

Kobayashi K, Kiuchi K, Ishii A, Kaneda N, Kurosawa Y, Fujita K,

Nagatsu T (1988) Expression of four types of human tyrosine

hydroxylase in COS cells. FEBS Lett 238(2):431–434

Kobayashi K, Morita S, Sawada H, Mizuguchi T, Yamada K, Nagatsu

I, Hata T, Watanabe Y, Fujita K, Nagatsu T (1995) Targeted

disruption of the tyrosine hydroxylase locus results in severe

catecholamine depletion and perinatal lethality in mice. J Biol

Chem 270(45):27235–27243


123

http://dx.doi.org/10.1111/j.1471-4159.2005.03386.x



http://dx.doi.org/10.1515/BC.1999.175


http://dx.doi.org/10.1242/dev.029769

http://dx.doi.org/10.1016/j.nbd.2007.08.003


http://dx.doi.org/10.1111/j.1365-2826.2011.02197.x

http://dx.doi.org/10.1002/jcp.20534

http://dx.doi.org/10.1111/j.1471-4159.2004.02723.x

http://dx.doi.org/10.1111/j.1471-4159.2004.02723.x

http://dx.doi.org/10.1007/s00702-009-0304-z

http://dx.doi.org/10.1007/s00702-009-0304-z

http://dx.doi.org/10.1016/j.neuroscience.2006.06.032



Kroll SL, Paulding WR, Schnell PO, Barton MC, Conaway JW,

Conaway RC, Czyzyk-Krzeska MF (1999) von Hippel–Lindau

protein induces hypoxia-regulated arrest of tyrosine hydroxylase

transcript elongation in pheochromocytoma cells. J Biol Chem

274(42):30109–30114

Kuczenski R (1973) Soluble, membrane-bound, and detergent-

solubilized rat striatal tyrosine hydroxylase. pH-dependent

cofactor binding. J Biol Chem 248(14):5074–5080

Kuczenski R (1983) Effects of phospholipases on the kinetic

properties of rat striatal membrane-bound tyrosine hydroxylase.

J Neurochem 40(3):821–829

Kuhn DM, Arthur RE Jr, Thomas DM, Elferink LA (1999) Tyrosine

hydroxylase is inactivated by catechol-quinones and converted to

a redox-cycling quinoprotein: possible relevance to Parkinson’s

disease. J Neurochem 73(3):1309–1317

Kumer SC, Vrana KE (1996) Intricate regulation of tyrosine

hydroxylase activity and gene expression. J Neurochem

67(2):443–462

Laniece P, Le Hir H, Bodeau-Pean S, Charon Y, Valentin L, Thermes

C, Mallet J, Dumas S (1996) A novel rat tyrosine hydroxylase

mRNA species generated by alternative splicing. J Neurochem

66(5):1819–1825

Lazar MA, Truscott RJ, Raese JD, Barchas JD (1981) Thermal

denaturation of native striatal tyrosine hydroxylase: increased

thermolability of the phosphorylated form of the enzyme.


Lazaroff M, Qi Y, Chikaraishi DM (1998) Differentiation of a

catecholaminergic CNS cell line modifies tyrosine hydroxylase

transcriptional regulation. J Neurochem 71(1):51–59

Le Bourdelles B, Boularand S, Boni C, Horellou P, Dumas S, Grima

B, Mallet J (1988) Analysis of the 50 region of the human

tyrosine hydroxylase gene: combinatorial patterns of exon

splicing generate multiple regulated tyrosine hydroxylase iso-

forms. J Neurochem 50(3):988–991

Leal RB, Sim AT, Goncalves CA, Dunkley PR (2002) Tyrosine

hydroxylase dephosphorylation by protein phosphatase 2A in

bovine adrenal chromaffin cells. Neurochem Res 27(3):207–213

Lebel M, Gauthier Y, Moreau A, Drouin J (2001) Pitx3 activates

mouse tyrosine hydroxylase promoter via a high-affinity binding

site. J Neurochem 77(2):558–567

Lehmann IT, Bobrovskaya L, Gordon SL, Dunkley PR, Dickson PW

(2006) Differential regulation of the human tyrosine hydroxylase

isoforms via hierarchical phosphorylation. J Biol Chem

281(26):17644–17651. doi:10.1074/jbc.M512194200

Lenartowski R, Goc A (2011) Epigenetic, transcriptional and

posttranscriptional regulation of the tyrosine hydroxylase gene.

Int J Dev Neurosci 29(8):873–883. doi:10.1016/j.ijdevneu.2011.

07.006

Lenartowski R, Grzybowski T, Miscicka-Sliwka D, Wojciechowski

W, Goc A (2003) The bovine tyrosine hydroxylase gene

associates in vitro with the nuclear matrix by its first intron

sequence. Acta Biochim Pol 50(3):865–873

Leong SL, Cappai R, Barnham KJ, Pham CL (2009) Modulation of a-

synuclein aggregation by dopamine: a review. Neurochem Res

34(10):1838–1846. doi:10.1007/s11064-009-9986-8

Levitt M, Spector S, Sjoerdsma A, Udenfriend S (1965) Elucidation

of the rate-limiting step in norepinephrine biosynthesis in the

perfused guinea-pig heart. J Pharmacol Exp Ther 148:1–8

Lewis DA, Melchitzky DS, Haycock JW (1993) Four isoforms of

tyrosine hydroxylase are expressed in human brain. Neurosci-

ence 54(2):477–492

Lewis DA, Melchitzky DS, Haycock JW (1994) Expression and

distribution of two isoforms of tyrosine hydroxylase in macaque

monkey brain. Brain Res 656(1):1–13

Lewis-Tuffin LJ, Quinn PG, Chikaraishi DM (2004) Tyrosine

hydroxylase transcription depends primarily on cAMP response

element activity, regardless of the type of inducing stimulus. Mol

Cell Neurosci 25(3):536–547. doi:10.1016/j.mcn.2003.10.010

Lindgren N, Goiny M, Herrera-Marschitz M, Haycock JW, Hokfelt T,

Fisone G (2002) Activation of extracellular signal-regulated

kinases 1 and 2 by depolarization stimulates tyrosine hydroxy-

lase phosphorylation and dopamine synthesis in rat brain. Eur J

Neurosci 15(4):769–773

Liu B, Arbogast LA (2008) Phosphorylation state of tyrosine

hydroxylase in the stalk-median eminence is decreased by

progesterone in cycling female rats. Endocrinology

149(4):1462–1469. doi:10.1210/en.2007-1345

Liu X, Vrana KE (1991) Leucine zippers and coiled-coils in the

aromatic amino acid hydroxylases. Neurochem Int 18(1):27–31

Lohse DL, Fitzpatrick PF (1993) Identification of the intersubunit

binding region in rat tyrosine hydroxylase. Biochem Biophys


Lopez Verrilli MA, Pirola CJ, Pascual MM, Dominici FP, Turyn D,

Gironacci MM (2009) Angiotensin-(1-7) through AT receptors

mediates tyrosine hydroxylase degradation via the ubiquitin–

proteasome pathway. J Neurochem 109(2):326–335. doi:10.

1111/j.1471-4159.2009.05912.x

Lou H, Montoya SE, Alerte TN, Wang J, Wu J, Peng X, Hong CS,

Friedrich EE, Mader SA, Pedersen CJ, Marcus BS, McCormack

AL, Di Monte DA, Daubner SC, Perez RG (2010) Serine 129

phosphorylation reduces the ability of a-synuclein to regulate

tyrosine hydroxylase and protein phosphatase 2A in vitro and

in vivo. J Biol Chem 285(23):17648–17661. doi:10.1074/jbc.

M110.100867

Lovenberg W, Bruckwick EA, Hanbauer I (1975) ATP, cyclic AMP,

and magnesium increase the affinity of rat striatal tyrosine

hydroxylase for its cofactor. Proc Natl Acad Sci USA

72(8):2955–2958

Luke TM, Hexum TD (2008) Tyrosine hydroxylase phosphorylation

increases in response to ATP and neuropeptide Y co-stimulation

of ERK2 phosphorylation. Pharmacol Res 58(1):52–57. doi:10.

1016/j.phrs.2008.06.010

Maass A, Scholz J, Moser A (2003) Modeled ligand-protein

complexes elucidate the origin of substrate specificity and

provide insight into catalytic mechanisms of phenylalanine

hydroxylase and tyrosine hydroxylase. Eur J Biochem

270(6):1065–1075

Maharjan S, Serova L, Sabban EL (2005) Transcriptional regulation

of tyrosine hydroxylase by estrogen: opposite effects with

estrogen receptors a and beta and interactions with cyclic AMP.

J Neurochem 93(6):1502–1514. doi:10.1111/j.1471-4159.2005.

03142.x

Maharjan S, Serova LI, Sabban EL (2010) Membrane-initiated

estradiol signaling increases tyrosine hydroxylase promoter

activity with ER a in PC12 cells. J Neurochem 112(1):42–55.

doi:10.1111/j.1471-4159.2009.06430.x

Martinat C, Bacci JJ, Leete T, Kim J, Vanti WB, Newman AH, Cha

JH, Gether U, Wang H, Abeliovich A (2006) Cooperative

transcription activation by Nurr1 and Pitx3 induces embryonic

stem cell maturation to the midbrain dopamine neuron pheno-

type. Proc Natl Acad Sci USA 103(8):2874–2879. doi:10.1073/

pnas.0511153103

Martinez A, Haavik J, Flatmark T, Arrondo JL, Muga A (1996)

Conformational properties and stability of tyrosine hydroxylase

studied by infrared spectroscopy. Effect of iron/catecholamine

binding and phosphorylation. J Biol Chem 271(33):

19737–19742

Maxwell SL, Ho HY, Kuehner E, Zhao S, Li M (2005) Pitx3 regulates

tyrosine hydroxylase expression in the substantia nigra and

identifies a subgroup of mesencephalic dopaminergic progenitor

neurons during mouse development. Dev Biol 282(2):467–479.

doi:10.1016/j.ydbio.2005.03.028

I. Tekin et al.

123


http://dx.doi.org/10.1016/j.ijdevneu.2011.07.006


http://dx.doi.org/10.1007/s11064-009-9986-8

http://dx.doi.org/10.1016/j.mcn.2003.10.010

http://dx.doi.org/10.1210/en.2007-1345


http://dx.doi.org/10.1111/j.1471-4159.2009.05912.x

http://dx.doi.org/10.1111/j.1471-4159.2009.05912.x



http://dx.doi.org/10.1016/j.phrs.2008.06.010


http://dx.doi.org/10.1111/j.1471-4159.2005.03142.x

http://dx.doi.org/10.1111/j.1471-4159.2005.03142.x

http://dx.doi.org/10.1111/j.1471-4159.2009.06430.x

http://dx.doi.org/10.1073/pnas.0511153103

http://dx.doi.org/10.1073/pnas.0511153103

http://dx.doi.org/10.1016/j.ydbio.2005.03.028

McCulloch RI, Fitzpatrick PF (1999) Limited proteolysis of tyrosine

hydroxylase identifies residues 33-50 as conformationally sen-

sitive to phosphorylation state and dopamine binding. Arch

Biochem Biophys 367(1):143–145. doi:10.1006/abbi.1999.1259

McCulloch RI, Daubner SC, Fitzpatrick PF (2001) Effects of

substitution at serine 40 of tyrosine hydroxylase on catechol-

amine binding. Biochemistry 40(24):7273–7278

McTigue M, Cremins J, Halegoua S (1985) Nerve growth factor and

other agents mediate phosphorylation and activation of tyrosine

hydroxylase. A convergence of multiple kinase activities. J Biol

Chem 260(15):9047–9056

Meligeni JA, Haycock JW, Bennett WF, Waymire JC (1982)

Phosphorylation and activation of tyrosine hydroxylase mediate

the cAMP-induced increase in catecholamine biosynthesis in

adrenal chromaffin cells. J Biol Chem 257(21):12632–12640

Messmer K, Remington MP, Skidmore F, Fishman PS (2007)

Induction of tyrosine hydroxylase expression by the transcription

factor Pitx3. Int J Dev Neurosci 25(1):29–37. doi:10.1016/j.

ijdevneu.2006.11.003

Meyer-Klaucke W, Winkler H, Schunemann V, Trautwein AX,

Nolting HF, Haavik J (1996) Mossbauer, electron-paramagnetic-

resonance and X-ray-absorption fine-structure studies of the iron

environment in recombinant human tyrosine hydroxylase. Eur J

Biochem 241(2):432–439

Milsted A, Serova L, Sabban EL, Dunphy G, Turner ME, Ely DL

(2004) Regulation of tyrosine hydroxylase gene transcription by

Sry. Neurosci Lett 369(3):203–207. doi:10.1016/j.neulet.2004.

07.052

Min N, Joh TH, Kim KS, Peng C, Son JH (1994) 50 upstream DNA

sequence of the rat tyrosine hydroxylase gene directs high-level

and tissue-specific expression to catecholaminergic neurons in

the central nervous system of transgenic mice. Brain Res Mol

Brain Res 27(2):281–289

Min N, Joh TH, Corp ES, Baker H, Cubells JF, Son JH (1996) A

transgenic mouse model to study transsynaptic regulation of

tyrosine hydroxylase gene expression. J Neurochem 67(1):11–18

Mishra RR, Adhikary G, Simonson MS, Cherniack NS, Prabhakar NR

(1998) Role of c-fos in hypoxia-induced AP-1 cis-element

activity and tyrosine hydroxylase gene expression. Brain Res

Mol Brain Res 59(1):74–83

Mogi M, Kojima K, Nagatsu T (1984) Detection of inactive or less

active forms of tyrosine hydroxylase in human adrenals by a

sandwich enzyme immunoassay. Anal Biochem 138(1):125–132

Morgenroth VH 3rd, Hegstrand LR, Roth RH, Greengard P (1975)

Evidence for involvement of protein kinase in the activation by

adenosine 30:50-monophosphate of brain tyrosine 3-monooxy-

genase. J Biol Chem 250(5):1946–1948

Moy LY, Tsai LH (2004) Cyclin-dependent kinase 5 phosphorylates

serine 31 of tyrosine hydroxylase and regulates its stability.

J Biol Chem 279(52):54487–54493. doi:10.1074/jbc.

M406636200

Nagamoto-Combs K, Piech KM, Best JA, Sun B, Tank AW (1997)

Tyrosine hydroxylase gene promoter activity is regulated by

both cyclic AMP-responsive element and AP1 sites following

calcium influx. Evidence for cyclic amp-responsive element

binding protein-independent regulation. J Biol Chem

272(9):6051–6058

Nagatsu T, Levitt M, Udenfriend S (1964) Tyrosine hydroxylase. The

initial step in norepinephrine biosynthesis. J Biol Chem

239:2910–2917

Nakashima A, Mori K, Suzuki T, Kurita H, Otani M, Nagatsu T, Ota

A (1999) Dopamine inhibition of human tyrosine hydroxylase

type 1 is controlled by the specific portion in the N-terminus of

the enzyme. J Neurochem 72(5):2145–2153

Nakashima A, Hayashi N, Mori K, Kaneko YS, Nagatsu T, Ota A

(2000) Positive charge intrinsic to Arg(37)–Arg(38) is critical for

dopamine inhibition of the catalytic activity of human tyrosine

hydroxylase type 1. FEBS Lett 465(1):59–63

Nakashima A, Kaneko YS, Mori K, Fujiwara K, Tsugu T, Suzuki T,

Nagatsu T, Ota A (2002) The mutation of two amino acid

residues in the N-terminus of tyrosine hydroxylase (TH)

dramatically enhances the catalytic activity in neuroendocrine

AtT-20 cells. J Neurochem 82(1):202–206

Nakashima A, Ota A, Sabban EL (2003) Interactions between Egr1

and AP1 factors in regulation of tyrosine hydroxylase transcrip-

tion. Brain Res Mol Brain Res 112(1–2):61–69

Nakashima A, Hayashi N, Kaneko YS, Mori K, Egusa H, Nagatsu T,

Ota A (2005) Deletion of N-terminus of human tyrosine

hydroxylase type 1 enhances stability of the enzyme in AtT-20

cells. J Neurosci Res 81(1):110–120. doi:10.1002/jnr.20540

Nakashima A, Hayashi N, Kaneko YS, Mori K, Sabban EL, Nagatsu

T, Ota A (2007) RNAi of 14-3-3eta protein increases intracel-

lular stability of tyrosine hydroxylase. Biochem Biophys Res

Commun 363(3):817–821. doi:10.1016/j.bbrc.2007.09.042

Nakashima A, Hayashi N, Kaneko YS, Mori K, Sabban EL, Nagatsu

T, Ota A (2009) Role of N-terminus of tyrosine hydroxylase in

the biosynthesis of catecholamines. J Neural Transm

116(11):1355–1362. doi:10.1007/s00702-009-0227-8

Nakashima A, Mori K, Kaneko YS, Hayashi N, Nagatsu T, Ota A

(2011) Phosphorylation of the N-terminal portion of tyrosine

hydroxylase triggers proteasomal digestion of the enzyme.

Biochem Biophys Res Commun 407(2):343–347. doi:10.1016/

j.bbrc.2011.03.020

Nakashima A, Kaneko YS, Kodani Y, Mori K, Nagasaki H, Nagatsu

T, Ota A (2013) Intracellular stability of tyrosine hydroxylase:

phosphorylation and proteasomal digestion of the enzyme. Adv

Pharmacol 68:3–11. doi:10.1016/B978-0-12-411512-5.00001-4

Nankova B, Hiremagalur B, Menezes A, Zeman R, Sabban E (1996)

Promoter elements and second messenger pathways involved in

transcriptional activation of tyrosine hydroxylase by ionomycin.

Brain Res Mol Brain Res 35(1–2):164–172

Nasrin S, Ichinose H, Hidaka H, Nagatsu T (1994) Recombinant

human tyrosine hydroxylase types 1–4 show regulatory kinetic

properties for the natural (6R)-tetrahydrobiopterin cofactor.

J Biochem 116(2):393–398

Obsilova V, Nedbalkova E, Silhan J, Boura E, Herman P, Vecer J,

Sulc M, Teisinger J, Dyda F, Obsil T (2008) The 14-3-3 protein

affects the conformation of the regulatory domain of human

tyrosine hydroxylase. Biochemistry 47(6):1768–1777. doi:10.

1021/bi7019468

Ohye T, Ichinose H, Yoshizawa T, Kanazawa I, Nagatsu T (2001) A

new splicing variant for human tyrosine hydroxylase in the

adrenal medulla. Neurosci Lett 312(3):157–160

Okuno S, Fujisawa H (1985) A new mechanism for regulation of

tyrosine 3-monooxygenase by end product and cyclic AMP-

dependent protein kinase. J Biol Chem 260(5):2633–2635

Osaka H, Sabban EL (1997) Requirement for cAMP/calcium response

element but not AP-1 site in fibroblast growth factor-2-elicited

activation of tyrosine hydroxylase gene expression in PC12 cells.

Brain Res Mol Brain Res 49(1–2):222–228

Ota A, Yoshida S, Nagatsu T (1995) Deletion mutagenesis of human

tyrosine hydroxylase type 1 regulatory domain. Biochem Biophys


Ota A, Nakashima A, Mori K, Nagatsu T (1997) Effects of dopamine

on N-terminus-deleted human tyrosine hydroxylase type 1

expressed in Escherichia coli. Neurosci Lett 229(1):57–60

Papanikolaou NA, Sabban EL (1999) Sp1/Egr1 motif: a new

candidate in the regulation of rat tyrosine hydroxylase gene

transcription by immobilization stress. J Neurochem

73(1):433–436

Papanikolaou NA, Sabban EL (2000) Ability of Egr1 to activate

tyrosine hydroxylase transcription in PC12 cells. Cross-talk with


123

http://dx.doi.org/10.1006/abbi.1999.1259







http://dx.doi.org/10.1002/jnr.20540


http://dx.doi.org/10.1007/s00702-009-0227-8



http://dx.doi.org/10.1016/B978-0-12-411512-5.00001-4

http://dx.doi.org/10.1021/bi7019468

http://dx.doi.org/10.1021/bi7019468


AP-1 factors. J Biol Chem 275(35):26683–26689. doi:10.1074/

jbc.M000049200

Parareda A, Villaescusa JC, Sanchez de Toledo J, Gallego S (2003)

New splicing variants for human Tyrosine Hydroxylase gene

with possible implications for the detection of minimal residual

disease in patients with neuroblastoma. Neurosci Lett

336(1):29–32

Patankar S, Lazaroff M, Yoon SO, Chikaraishi DM (1997) A novel

basal promoter element is required for expression of the rat

tyrosine hydroxylase gene. J Neurosci 17(11):4076–4086

Paulding WR, Czyzyk-Krzeska MF (1999) Regulation of tyrosine

hydroxylase mRNA stability by protein-binding, pyrimidine-rich

sequence in the 30-untranslated region. J Biol Chem

274(4):2532–2538

Peng X, Tehranian R, Dietrich P, Stefanis L, Perez RG (2005) a-

Synuclein activation of protein phosphatase 2A reduces tyrosine

hydroxylase phosphorylation in dopaminergic cells. J Cell Sci

118(Pt 15):3523–3530. doi:10.1242/jcs.02481

Perez RG, Waymire JC, Lin E, Liu JJ, Guo F, Zigmond MJ (2002) A

role for a-synuclein in the regulation of dopamine biosynthesis.

J Neurosci 22(8):3090–3099

Piech-Dumas KM, Best JA, Chen Y, Nagamoto-Combs K, Osterhout

CA, Tank AW (2001) The cAMP responsive element and CREB

partially mediate the response of the tyrosine hydroxylase gene

to phorbol ester. J Neurochem 76(5):1376–1385

Que L Jr (2000) One motif—many different reactions. Nat Struct Biol

7(3):182–184. doi:10.1038/73270

Quinsey NS, Lenaghan CM, Dickson PW (1996) Identification of

Gln313 and Pro327 as residues critical for substrate inhibition in

tyrosine hydroxylase. J Neurochem 66(3):908–914

Raghuraman G, Rai V, Peng YJ, Prabhakar NR, Kumar GK (2009)

Pattern-specific sustained activation of tyrosine hydroxylase by

intermittent hypoxia: role of reactive oxygen species-dependent

downregulation of protein phosphatase 2A and upregulation of

protein kinases. Antioxid Redox Signal 11(8):1777–1789.

doi:10.1089/ARS.2008.2368

Ramsey AJ, Fitzpatrick PF (1998) Effects of phosphorylation of

serine 40 of tyrosine hydroxylase on binding of catecholamines:

evidence for a novel regulatory mechanism. Biochemistry

37(25):8980–8986. doi:10.1021/bi980582l

Ramsey AJ, Fitzpatrick PF (2000) Effects of phosphorylation on

binding of catecholamines to tyrosine hydroxylase: specificity

and thermodynamics. Biochemistry 39(4):773–778

Ramsey AJ, Daubner SC, Ehrlich JI, Fitzpatrick PF (1995) Identi-

fication of iron ligands in tyrosine hydroxylase by mutagenesis

of conserved histidinyl residues. Protein Sci 4(10):2082–2086.

doi:10.1002/pro.5560041013

Rani CS, Elango N, Wang SS, Kobayashi K, Strong R (2009)

Identification of an activator protein-1-like sequence as the

glucocorticoid response element in the rat tyrosine hydroxylase

gene. Mol Pharmacol 75(3):589–598. doi:10.1124/mol.108.

051219

Rao F, Zhang L, Wessel J, Zhang K, Wen G, Kennedy BP, Rana BK,

Das M, Rodriguez-Flores JL, Smith DW, Cadman PE, Salem

RM, Mahata SK, Schork NJ, Taupenot L, Ziegler MG, O’Connor

DT (2007) Tyrosine hydroxylase, the rate-limiting enzyme in

catecholamine biosynthesis: discovery of common human

genetic variants governing transcription, autonomic activity,

and blood pressure in vivo. Circulation 116(9):993–1006. doi:10.

1161/CIRCULATIONAHA.106.682302

Ribeiro P, Kaufman S (1994) The effect of tetrahydrobiopterin on the

in situ phosphorylation of tyrosine hydroxylase in rat striatal

synaptosomes. Neurochem Res 19(5):541–548

Ribeiro P, Wang Y, Citron BA, Kaufman S (1992) Regulation of

recombinant rat tyrosine hydroxylase by dopamine. Proc Natl

Acad Sci USA 89(20):9593–9597

Ribeiro P, Wang Y, Citron BA, Kaufman S (1993) Deletion

mutagenesis of rat PC12 tyrosine hydroxylase regulatory and

catalytic domains. J Mol Neurosci 4(2):125–139. doi:10.1007/

BF02782125

Roberts KM, Fitzpatrick PF (2013) Mechanisms of tryptophan and

tyrosine hydroxylase. IUBMB Life 65(4):350–357. doi:10.1002/

iub.1144

Rodriguez-Pascual F, Ferrero R, Miras-Portugal MT, Torres M (1999)

Phosphorylation of tyrosine hydroxylase by cGMP-dependent

protein kinase in intact bovine chromaffin cells. Arch Biochem

Biophys 366(2):207–214. doi:10.1006/abbi.1999.1199

Roe DF, Craviso GL, Waymire JC (2004) Nicotinic stimulation

modulates tyrosine hydroxylase mRNA half-life and protein

binding to the 30UTR in a manner that requires transcription.

Brain Res Mol Brain Res 120(2):91–102

Roma J, Saus E, Cuadros M, Reventos J, Sanchez de Toledo J,

Gallego S (2007) Characterisation of novel splicing variants of

the tyrosine hydroxylase C-terminal domain in human neurob-

lastic tumours. Biol Chem 388(4):419–426. doi:10.1515/BC.

2007.041

Romano G, Suon S, Jin H, Donaldson AE, Iacovitti L (2005)

Characterization of five evolutionary conserved regions of the

human tyrosine hydroxylase (TH) promoter: implications for the

engineering of a human TH minimal promoter assembled in a

self-inactivating lentiviral vector system. J Cell Physiol

204(2):666–677. doi:10.1002/jcp.20319

Romano G, Macaluso M, Lucchetti C, Iacovitti L (2007) Transcrip-

tion and epigenetic profile of the promoter, first exon and first

intron of the human tyrosine hydroxylase gene. J Cell Physiol

211(2):431–438. doi:10.1002/jcp.20949

Roskoski R Jr (2012) ERK1/2 MAP kinases: structure, function, and

regulation. Pharmacol Res 66(2):105–143. doi:10.1016/j.phrs.

2012.04.005

Roskoski R Jr, Ritchie P (1991) Phosphorylation of rat tyrosine

hydroxylase and its model peptides in vitro by cyclic AMP-

dependent protein kinase. J Neurochem 56(3):1019–1023

Roskoski R Jr, Roskoski LM (1987) Activation of tyrosine hydrox-

ylase in PC12 cells by the cyclic GMP and cyclic AMP second

messenger systems. J Neurochem 48(1):236–242

Roskoski R Jr, Vulliet PR, Glass DB (1987) Phosphorylation of

tyrosine hydroxylase by cyclic GMP-dependent protein kinase.


Roskoski R Jr, Wilgus H, Vrana KE (1990) Inactivation of tyrosine

hydroxylase by pterin substrates following phosphorylation by

cyclic AMP-dependent protein kinase. Mol Pharmacol

38(4):541–546

Roskoski R Jr, Gahn LG, Roskoski LM (1993) Inactivation of

phosphorylated rat tyrosine hydroxylase by ascorbate in vitro.

Eur J Biochem 218(2):363–370

Royo M, Colette Daubner S (2006) Kinetics of regulatory serine

variants of tyrosine hydroxylase with cyclic AMP-dependent

protein kinase and extracellular signal-regulated protein kinase

2. Biochim Biophys Acta 1764(4):786–792. doi:10.1016/j.

bbapap.2006.01.019

Royo M, Fitzpatrick PF, Daubner SC (2005) Mutation of regulatory

serines of rat tyrosine hydroxylase to glutamate: effects on

enzyme stability and activity. Arch Biochem Biophys

434(2):266–274. doi:10.1016/j.abb.2004.11.007

Sabban EL, Hebert MA, Liu X, Nankova B, Serova L (2004)

Differential effects of stress on gene transcription factors in

catecholaminergic systems. Ann N Y Acad Sci 1032:130–140.

doi:10.1196/annals.1314.010

Sachs NA, Vaillancourt RR (2004) Cyclin-dependent kinase

11p110 and casein kinase 2 (CK2) inhibit the interaction

between tyrosine hydroxylase and 14-3-3. J Neurochem

88(1):51–62

I. Tekin et al.

123



http://dx.doi.org/10.1242/jcs.02481

http://dx.doi.org/10.1038/73270

http://dx.doi.org/10.1089/ARS.2008.2368

http://dx.doi.org/10.1021/bi980582l

http://dx.doi.org/10.1002/pro.5560041013



http://dx.doi.org/10.1161/CIRCULATIONAHA.106.682302

http://dx.doi.org/10.1161/CIRCULATIONAHA.106.682302

http://dx.doi.org/10.1007/BF02782125

http://dx.doi.org/10.1007/BF02782125

http://dx.doi.org/10.1002/iub.1144

http://dx.doi.org/10.1002/iub.1144

http://dx.doi.org/10.1006/abbi.1999.1199

http://dx.doi.org/10.1515/BC.2007.041

http://dx.doi.org/10.1515/BC.2007.041





http://dx.doi.org/10.1016/j.bbapap.2006.01.019

http://dx.doi.org/10.1016/j.bbapap.2006.01.019


http://dx.doi.org/10.1196/annals.1314.010

Sakurada K, Ohshima-Sakurada M, Palmer TD, Gage FH (1999)

Nurr1, an orphan nuclear receptor, is a transcriptional activator

of endogenous tyrosine hydroxylase in neural progenitor cells

derived from the adult brain. Development 126(18):4017–4026

Salvatore MF, Garcia-Espana A, Goldstein M, Deutch AY, Haycock

JW (2000) Stoichiometry of tyrosine hydroxylase phosphoryla-

tion in the nigrostriatal and mesolimbic systems in vivo: effects

of acute haloperidol and related compounds. J Neurochem

75(1):225–232

Salvatore MF, Waymire JC, Haycock JW (2001) Depolarization-

stimulated catecholamine biosynthesis: involvement of protein

kinases and tyrosine hydroxylase phosphorylation sites in situ.


Saraf A, Virshup DM, Strack S (2007) Differential expression of the

B’beta regulatory subunit of protein phosphatase 2A modulates

tyrosine hydroxylase phosphorylation and catecholamine syn-

thesis. J Biol Chem 282(1):573–580. doi:10.1074/jbc.

M607407200

Saraf A, Oberg EA, Strack S (2010) Molecular determinants for PP2A

substrate specificity: charged residues mediate dephosphoryla-

tion of tyrosine hydroxylase by the PP2A/B’ regulatory subunit.

Biochemistry 49(5):986–995. doi:10.1021/bi902160t

Satoh J, Kuroda Y (2002) The constitutive and inducible expression

of Nurr1, a key regulator of dopaminergic neuronal differenti-

ation, in human neural and non-neural cell lines. Neuropathology

22(4):219–232

Schimmel JJ, Crews L, Roffler-Tarlov S, Chikaraishi DM (1999)

4.5 kb of the rat tyrosine hydroxylase 50 flanking sequence

directs tissue specific expression during development and

contains consensus sites for multiple transcription factors. Brain

Res Mol Brain Res 74(1–2):1–14

Schnell PO, Ignacak ML, Bauer AL, Striet JB, Paulding WR, Czyzyk-

Krzeska MF (2003) Regulation of tyrosine hydroxylase promoter

activity by the von Hippel–Lindau tumor suppressor protein and

hypoxia-inducible transcription factors. J Neurochem 85(2):483–491

Schworer CM, Soderling TR (1983) Substrate specificity of liver

calmodulin-dependent glycogen synthase kinase. Biochem Bio-

phys Res Commun 116(2):412–416

Segawa M (2011) Hereditary progressive dystonia with marked

diurnal fluctuation. Brain Dev 33(3):195–201. doi:10.1016/j.

braindev.2010.10.015

Segawa M, Nomura Y, Nishiyama N (2003) Autosomal dominant

guanosine triphosphate cyclohydrolase I deficiency (Segawa

disease). Ann Neurol 54(Suppl 6):S32–S45. doi:10.1002/ana.

10630

Seta KA, Millhorn DE (2004) Functional genomics approach to

hypoxia signaling. J Appl Physiol 96(2):765–773. doi:10.1152/

japplphysiol.00836.2003

Shi X, Habecker BA (2012) gp130 cytokines stimulate proteasomal

degradation of tyrosine hydroxylase via extracellular signal

regulated kinases 1 and 2. J Neurochem 120(2):239–247. doi:10.

1111/j.1471-4159.2011.07539.x

Skjevik AA, Mileni M, Baumann A, Halskau O, Teigen K, Stevens

RC, Martinez A (2014) The N-terminal sequence of tyrosine

hydroxylase is a conformationally versatile motif that binds

14-3-3 proteins and membranes. J Mol Biol 426(1):150–168.

doi:10.1016/j.jmb.2013.09.012

Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M

(1998) a-Synuclein in filamentous inclusions of Lewy bodies

from Parkinson’s disease and dementia with Lewy bodies. Proc

Natl Acad Sci USA 95(11):6469–6473

Stefano L, Al Sarraj J, Rossler OG, Vinson C, Thiel G (2006) Up-

regulation of tyrosine hydroxylase gene transcription by tetra-

decanoylphorbol acetate is mediated by the transcription factors

Ets-like protein-1 (Elk-1) and Egr-1. J Neurochem 97(1):92–104.

doi:10.1111/j.1471-4159.2006.03749.x

Stokes AH, Hastings TG, Vrana KE (1999) Cytotoxic and genotoxic

potential of dopamine. J Neurosci Res 55(6):659–665

Stott SR, Metzakopian E, Lin W, Kaestner KH, Hen R, Ang SL

(2013) Foxa1 and foxa2 are required for the maintenance of

dopaminergic properties in ventral midbrain neurons at late

embryonic stages. J Neurosci 33(18):8022–8034. doi:10.1523/

JNEUROSCI.4774-12.2013

Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T,

Hidaka H (1991) The newly synthesized selective Ca2?/

calmodulin dependent protein kinase II inhibitor KN-93 reduces

dopamine contents in PC12h cells. Biochem Biophys Res

Commun 181(3):968–975

Sumi-Ichinose C, Ichinose H, Ikemoto K, Nomura T, Kondo K (2010)

Advanced research on dopamine signaling to develop drugs for

the treatment of mental disorders: regulation of dopaminergic

neural transmission by tyrosine hydroxylase protein at nerve

terminals. J Pharmacol Sci 114(1):17–24

Sun B, Tank AW (2003) c-Fos is essential for the response of the

tyrosine hydroxylase gene to depolarization or phorbol ester.

J Neurochem 85(6):1421–1430

Sun B, Sterling CR, Tank AW (2003) Chronic nicotine treatment

leads to sustained stimulation of tyrosine hydroxylase gene

transcription rate in rat adrenal medulla. J Pharmacol Exp Ther

304(2):575–588. doi:10.1124/jpet.102.043596

Sun B, Chen X, Xu L, Sterling C, Tank AW (2004) Chronic nicotine

treatment leads to induction of tyrosine hydroxylase in locus

ceruleus neurons: the role of transcriptional activation. Mol

Pharmacol 66(4):1011–1021. doi:10.1124/mol.104.001974

Sura GR, Daubner SC, Fitzpatrick PF (2004) Effects of phosphor-

ylation by protein kinase A on binding of catecholamines to the

human tyrosine hydroxylase isoforms. J Neurochem

90(4):970–978. doi:10.1111/j.1471-4159.2004.02566.x

Sura GR, Lasagna M, Gawandi V, Reinhart GD, Fitzpatrick PF

(2006) Effects of ligands on the mobility of an active-site loop in

tyrosine hydroxylase as monitored by fluorescence anisotropy.

Biochemistry 45(31):9632–9638. doi:10.1021/bi060754b

Sutherland C, Alterio J, Campbell DG, Le Bourdelles B, Mallet J,

Haavik J, Cohen P (1993) Phosphorylation and activation of

human tyrosine hydroxylase in vitro by mitogen-activated

protein (MAP) kinase and MAP-kinase-activated kinases 1 and

2. Eur J Biochem 217(2):715–722

Suzuki T, Yamakuni T, Hagiwara M, Ichinose H (2002) Identification

of ATF-2 as a transcriptional regulator for the tyrosine hydrox-

ylase gene. J Biol Chem 277(43):40768–40774. doi:10.1074/jbc.

M206043200

Suzuki T, Kurahashi H, Ichinose H (2004) Ras/MEK pathway is

required for NGF-induced expression of tyrosine hydroxylase

gene. Biochem Biophys Res Commun 315(2):389–396. doi:10.

1016/j.bbrc.2004.01.068

Tachikawa E, Tank AW, Weiner DH, Mosimann WF, Yanagihara N,

Weiner N (1987) Tyrosine hydroxylase is activated and phosphor-

ylated on different sites in rat pheochromocytoma PC12 cells treated

with phorbol ester and forskolin. J Neurochem 48(5):1366–1376

Tan XF, Jin GH, Tian ML, Qin JB, Zhang L, Zhu HX, Li HM (2011)

The co-transduction of Nurr1 and Brn4 genes induces the

differentiation of neural stem cells into dopaminergic neurons.

Cell Biol Int 35(12):1217–1223. doi:10.1042/CBI20110028

Tank AW, Xu L, Chen X, Radcliffe P, Sterling CR (2008) Post-

transcriptional regulation of tyrosine hydroxylase expression in

adrenal medulla and brain. Ann N Y Acad Sci 1148:238–248.

doi:10.1196/annals.1410.054

Tekin I, Vrana KE (2013) Caveat emptor: single nucleotide

polymorphism reporting in pharmacogenomics. Pharmacology

92(5–6):319–323. doi:10.1159/000356324

Thomas G, Haavik J, Cohen P (1997) Participation of a stress-

activated protein kinase cascade in the activation of tyrosine


123



http://dx.doi.org/10.1021/bi902160t

http://dx.doi.org/10.1016/j.braindev.2010.10.015

http://dx.doi.org/10.1016/j.braindev.2010.10.015

http://dx.doi.org/10.1002/ana.10630

http://dx.doi.org/10.1002/ana.10630



http://dx.doi.org/10.1111/j.1471-4159.2011.07539.x

http://dx.doi.org/10.1111/j.1471-4159.2011.07539.x


http://dx.doi.org/10.1111/j.1471-4159.2006.03749.x

http://dx.doi.org/10.1523/JNEUROSCI.4774-12.2013


http://dx.doi.org/10.1124/jpet.102.043596


http://dx.doi.org/10.1111/j.1471-4159.2004.02566.x

http://dx.doi.org/10.1021/bi060754b





http://dx.doi.org/10.1042/CBI20110028

http://dx.doi.org/10.1196/annals.1410.054

http://dx.doi.org/10.1159/000356324

hydroxylase in chromaffin cells. Eur J Biochem

247(3):1180–1189

Thorolfsson M, Doskeland AP, Muga A, Martinez A (2002) The

binding of tyrosine hydroxylase to negatively charged lipid

bilayers involves the N-terminal region of the enzyme. FEBS

Lett 519(1–3):221–226

Tinti C, Conti B, Cubells JF, Kim KS, Baker H, Joh TH (1996)

Inducible cAMP early repressor can modulate tyrosine hydrox-

ylase gene expression after stimulation of cAMP synthesis.

J Biol Chem 271(41):25375–25381

Tinti C, Yang C, Seo H, Conti B, Kim C, Joh TH, Kim KS (1997)

Structure/function relationship of the cAMP response element in

tyrosine hydroxylase gene transcription. J Biol Chem

272(31):19158–19164

Toska K, Kleppe R, Armstrong CG, Morrice NA, Cohen P, Haavik J

(2002a) Regulation of tyrosine hydroxylase by stress-activated

protein kinases. J Neurochem 83(4):775–783

Toska K, Kleppe R, Cohen P, Haavik J (2002b) Phosphorylation of

tyrosine hydroxylase in isolated mice adrenal glands. Ann N Y

Acad Sci 971:66–68

Vrana KE, Roskoski R Jr (1983) Tyrosine hydroxylase inactivation

following cAMP-dependent phosphorylation activation. J Neuro-

chem 40(6):1692–1700

Vrana KE, Allhiser CL, Roskoski R Jr (1981) Tyrosine hydroxylase

activation and inactivation by protein phosphorylation condi-

tions. J Neurochem 36(1):92–100

Vrana KE, Walker SJ, Rucker P, Liu X (1994) A carboxyl terminal

leucine zipper is required for tyrosine hydroxylase tetramer

formation. J Neurochem 63(6):2014–2020

Vulliet PR, Langan TA, Weiner N (1980) Tyrosine hydroxylase: a

substrate of cyclic AMP-dependent protein kinase. Proc Natl

Acad Sci USA 77(1):92–96

Vulliet PR, Woodgett JR, Cohen P (1984) Phosphorylation of tyrosine

hydroxylase by calmodulin-dependent multiprotein kinase.

J Biol Chem 259(22):13680–13683

Vulliet PR, Woodgett JR, Ferrari S, Hardie DG (1985) Characteriza-

tion of the sites phosphorylated on tyrosine hydroxylase by Ca2?

and phospholipid-dependent protein kinase, calmodulin-depen-

dent multiprotein kinase and cyclic AMP-dependent protein

kinase. FEBS Lett 182(2):335–339

Walker SJ, Liu X, Roskoski R, Vrana KE (1994) Catalytic core of rat

tyrosine hydroxylase: terminal deletion analysis of bacterially

expressed enzyme. Biochim Biophys Acta 1206(1):113–119

Wang J, Lou H, Pedersen CJ, Smith AD, Perez RG (2009) 14-3-3zeta

contributes to tyrosine hydroxylase activity in MN9D cells:

localization of dopamine regulatory proteins to mitochondria.

J Biol Chem 284(21):14011–14019. doi:10.1074/jbc.

M901310200

Wang S, Lasagna M, Daubner SC, Reinhart GD, Fitzpatrick PF

(2011) Fluorescence spectroscopy as a probe of the effect of

phosphorylation at serine 40 of tyrosine hydroxylase on the

conformation of its regulatory domain. Biochemistry

50(12):2364–2370. doi:10.1021/bi101844p

Waymire JC, Johnston JP, Hummer-Lickteig K, Lloyd A, Vigny A,

Craviso GL (1988) Phosphorylation of bovine adrenal chromaf-

fin cell tyrosine hydroxylase. Temporal correlation of acetyl-

choline’s effect on site phosphorylation, enzyme activation, and

catecholamine synthesis. J Biol Chem 263(25):12439–12447

Wilgus H, Roskoski R Jr (1988) Inactivation of tyrosine hydroxylase

activity by ascorbate in vitro and in rat PC12 cells. J Neurochem

51(4):1232–1239

Willemsen MA, Verbeek MM, Kamsteeg EJ, de Rijk-van Andel JF,

Aeby A, Blau N, Burlina A, Donati MA, Geurtz B, Grattan-

Smith PJ, Haeussler M, Hoffmann GF, Jung H, de Klerk JB, van

der Knaap MS, Kok F, Leuzzi V, de Lonlay P, Megarbane A,

Monaghan H, Renier WO, Rondot P, Ryan MM, Seeger J,

Smeitink JA, Steenbergen-Spanjers GC, Wassmer E, Weschke

B, Wijburg FA, Wilcken B, Zafeiriou DI, Wevers RA (2010)

Tyrosine hydroxylase deficiency: a treatable disorder of brain

catecholamine biosynthesis. Brain 133(Pt 6):1810–1822. doi:10.

1093/brain/awq087

Witkovsky P, Veisenberger E, Haycock JW, Akopian A, Garcia-

Espana A, Meller E (2004) Activity-dependent phosphorylation

of tyrosine hydroxylase in dopaminergic neurons of the rat

retina. J Neurosci 24(17):4242–4249. doi:10.1523/JNEUROSCI.

5436-03.2004

Xu Y, Stokes AH, Roskoski R Jr, Vrana KE (1998a) Dopamine, in the

presence of tyrosinase, covalently modifies and inactivates

tyrosine hydroxylase. J Neurosci Res 54(5):691–697

Xu ZQ, Lew JY, Harada K, Aman K, Goldstein M, Deutch A,

Haycock JW, Hokfelt T (1998b) Immunohistochemical studies

on phosphorylation of tyrosine hydroxylase in central catechol-

amine neurons using site- and phosphorylation state-specific

antibodies. Neuroscience 82(3):727–738

Xu L, Sterling CR, Tank AW (2009) cAMP-mediated stimulation of

tyrosine hydroxylase mRNA translation is mediated by polypy-

rimidine-rich sequences within its 30-untranslated region and

poly(C)-binding protein 2. Mol Pharmacol 76(4):872–883.

doi:10.1124/mol.109.057596

Yamauchi T, Fujisawa H (1979) In vitro phosphorylation of bovine

adrenal tyrosine hydroxylase by adenosine 30:50-monophosphate-

dependent protein kinase. J Biol Chem 254(2):503–507

Yamauchi T, Fujisawa H (1981) Tyrosine 3-monoxygenase is

phosphorylated by Ca2?-, calmodulin-dependent protein kinase,

followed by activation by activator protein. Biochem Biophys

Res Commun 100(2):807–813

Yamauchi T, Nakata H, Fujisawa H (1981) A new activator protein

that activates tryptophan 5-monooxygenase and tyrosine

3-monooxygenase in the presence of Ca2?-, calmodulin-depen-

dent protein kinase. Purification and characterization. J Biol

Chem 256(11):5404–5409

Yang C, Kim HS, Seo H, Kim KS (1998) Identification and

characterization of potential cis-regulatory elements governing

transcriptional activation of the rat tyrosine hydroxylase gene.

J Neurochem 71(4):1358–1368

Yohrling GJ IV, Jiang GC, Mockus SM, Vrana KE (2000) Intersub-

unit binding domains within tyrosine hydroxylase and trypto-

phan hydroxylase. J Neurosci Res 61(3):313–320

Yu HS, Kim SH, Park HG, Kim YS, Ahn YM (2011) Intracerebro-

ventricular administration of ouabain, a Na/K-ATPase inhibitor,

activates tyrosine hydroxylase through extracellular signal-

regulated kinase in rat striatum. Neurochem Int 59(6):779–786.

doi:10.1016/j.neuint.2011.08.011

Zetterstrom RH, Solomin L, Jansson L, Hoffer BJ, Olson L, Perlmann

T (1997) Dopamine neuron agenesis in Nurr1-deficient mice.

Science 276(5310):248–250

Zhang D, Kanthasamy A, Yang Y, Anantharam V, Kanthasamy A

(2007) Protein kinase C delta negatively regulates tyrosine

hydroxylase activity and dopamine synthesis by enhancing

protein phosphatase-2A activity in dopaminergic neurons.

J Neurosci 27(20):5349–5362. doi:10.1523/JNEUROSCI.4107-

06.2007

Zhang S, Huang T, Ilangovan U, Hinck AP, Fitzpatrick PF (2014) The

solution structure of the regulatory domain of tyrosine hydrox-

ylase. J Mol Biol 426(7):1483–1497. doi:10.1016/j.jmb.2013.12.

015

Zhou QY, Quaife CJ, Palmiter RD (1995) Targeted disruption of the

tyrosine hydroxylase gene reveals that catecholamines are

I. Tekin et al.

123



http://dx.doi.org/10.1021/bi101844p

http://dx.doi.org/10.1093/brain/awq087

http://dx.doi.org/10.1093/brain/awq087




http://dx.doi.org/10.1016/j.neuint.2011.08.011





required for mouse fetal development. Nature

374(6523):640–643. doi:10.1038/374640a0

Zhu Y, Zhang J, Zeng Y (2012) Overview of tyrosine hydroxylase in

Parkinson’s disease. CNS Neurol Disord Drug Targets

11(4):350–358

Zigmond RE (1998) Regulation of tyrosine hydroxylase by neuro-

peptides. Adv Pharmacol 42:21–25

Zigmond RE, Schwarzschild MA, Rittenhouse AR (1989) Acute

regulation of tyrosine hydroxylase by nerve activity and by

neurotransmitters via phosphorylation. Annu Rev Neurosci

12:415–461. doi:10.1146/annurev.ne.12.030189.002215


123

http://dx.doi.org/10.1038/374640a0

http://dx.doi.org/10.1146/annurev.ne.12.030189.002215

complex molecular regulation of tyrosine hydroxylase

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