deletion of n-terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in...

Deletion of N-terminus of HumanTyrosine Hydroxylase Type 1 EnhancesStability of the Enzyme in AtT-20 Cells

Akira Nakashima,1 Nobuhiro Hayashi,2 Yoko S. Kaneko,1 Keiji Mori,1

Hiromi Egusa,1 Toshiharu Nagatsu,3 and Akira Ota1*1Department of Physiology, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan2Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science,Fujita Health University, Toyoake, Aichi, Japan3Department of Pharmacology, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan

Wildtype human tyrosine hydroxylase (TH) type 1 and 4mutants (del-52, a form with the first 52 amino acid res-idues deleted; del-157, one with the first 157 aminoacid residues deleted; RR-EE, one in which Arg37-Arg38

was replaced by Glu37-Glu38; and S40D, one in whichSer40 was replaced by Asp40) were expressed in AtT-20mouse neuroendocrine cells in order to clarify howdeeply the N-terminus of TH is involved in the efficientproduction of dopamine (DA) in mammalian cells. Theamounts of DA that accumulated in AtT-20 cellsexpressing these human TH type 1 (hTH1) phenotypeswere in the following order: del-52 ¼ del-157 ¼ RR-EE >S40D > wildtype, although the enzyme activities of del-52 and del-157 were lower than those of wildtype, RR-EE, and S40D. The observation on immunoblot analy-ses that the N-terminus-deleted hTH1 mutants weremuch more stable than wildtype can reconcile the dis-crepant results. Computer-assisted analysis of the spa-tial configuration of hTH1 identified five newly recog-nized PEST motifs, one of which was located in the N-terminus sequence of Met1-Lys12 and predicted thatdeletion of the N-terminus region would alter the sec-ondary structure within the catalytic domain. Collec-tively, the high stability of the N-terminus-deleted hTH1mutants can be generated by the loss of a PEST motifin their N-termini and the structural change in the cata-lytic domain, which would promise an efficient produc-tion of DA in mammalian cells expressing N-terminusdeleted hTH1. VVC 2005 Wiley-Liss, Inc.

Key words: dopamine; PEST motif; stability

Tyrosine hydroxylase (TH; EC 1.14.16.2), whichcatalyzes the conversion of L-DOPA from L-tyrosine(Nagatsu et al., 1964), is the rate-limiting enzyme in thebiosynthesis of catecholamines (Levitt et al., 1965). Acatalytic domain is located in the C-terminal two-thirdsof the molecule and binds the substrates (L-tyrosine andmolecular oxygen) and the cofactor (6R-tetrahydrobiop-terin; 6RBPH4). The part of the enzyme controlling the

catalytic activity has been assigned to the N-terminalend, which serves as a regulatory domain.

The regulation of the catalytic activity of TH hasbeen discussed from several viewpoints. About 4 decadesago, it was reported that the catalytic activity of TH wasinhibited by the end-product catecholamines by Nagatsuet al. (1964). The phosphorylation of Ser residues resid-ing in the N-terminus of TH is one of the most impor-tant mechanisms for regulating the catalytic activity ofthe enzyme other than the control of 6RBPH4 biosyn-thesis, which is rate-limited by GTP cyclohydrolase I(Haycock and Wakade 1992; Sutherland et al., 1993;Hufton et al., 1995), because a number of studies havesuggested that phosphorylation of Ser40 disrupts the tightbinding between the catalytic domain of the TH mole-cule and catecholamine (Haavik et al., 1990; Daubneret al., 1992; Wu et al., 1992; McCulloch et al., 2001).Our laboratory also offered data suggesting that Arg37-Arg38 within the N-terminus is a key sequence for theinhibitory effect of dopamine (DA; Ota et al., 1997;Nakashima et al., 1999b, 2000). Collectively, thesereports led to the idea that the N-terminus of TH playsa critical role in regulating the catalytic activity of theenzyme.

Recently, TH was found to be a substrate for con-jugation to ubiquitin in a reconstituted in vitro systemand to be partially degraded by proteasomes in a reticu-locyte lysate system (Døskeland and Flatmark, 2002).The N-terminus of TH is supposed to be located on the

Contract grant sponsors: Ministry of Education, Science, Sports, and Cul-

ture of Japan (to A.N. and A.O.), Fujita Health University, Japan (to

A.N. and A.O.).

*Correspondence to: Akira Ota, M.D., Ph.D., Department of Physiol-

ogy, School of Medicine, Fujita Health University, Toyoake, Aichi 470-

1192, Japan. E-mail: [email protected]

Received 22 November 2004; Revised 10 March 2005; Accepted 16

March 2005

Published online 16 May 2005 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.20540

Journal of Neuroscience Research 81:110–120 (2005)

' 2005 Wiley-Liss, Inc.

surface of the molecule, because TH consists of homote-tramers whose subunits are gathered together by theinteraction of their C-termini (Goodwill et al., 1997).Moreover, amino acid residues 1–40 are presumed topossess flexible mobility to allow the proper conforma-tion of the regulatory site, as surmised from the suscepti-bility to proteolysis (McCulloch and Fitzpatrick, 1999;Nakashima et al., 1999a). Therefore, the N-terminus ofTH is thought to be a target region for intracellular pro-teolysis such as occurs in the ubiquitin–proteasome sys-tem. Taken together, these results suggest that the N-terminus of TH might play a critical role in the stabilityof the TH molecule, as well as in the regulation of THcatalytic activity in cells.

In this study, an experimental scheme was designedin order to prove the influence of the N-terminus onthe stability of the TH molecule. At first, the wildtypeenzyme and the N-terminus-deleted mutants of humanTH type 1 (hTH1) were expressed in AtT-20 cells tomeasure the hTH1 protein levels and the catalytic activ-ities and to examine the accumulation of catecholaminesin the cells. Then data were accumulated that indicatedthat the deletion of the N-terminus region augmentedthe stability of the hTH1 molecule. Collectively, ourdata suggest that the accumulation of DA in the cellsproducing N-terminus-deleted hTH1 can be attributedto augmented stabilization of the TH molecule.

MATERIALS AND METHODS

Materials

Restriction enzymes were obtained from New EnglandBioLabs (Beverly, MA). Substrates for the measurement ofluciferase activity, PicaGeneR, and PGV-C2 vector were pur-chased from TOYO B-Net (Tokyo, Japan). Cycloheximidewas purchased from Sigma-Aldrich (St. Louis, MO).

Mutation of hTH1

Deletion mutants of the N-terminal amino acid residuesof hTH1 were generated by conducting PCR reactions withthe pBluescript (pBS) vector containing hTH1 cDNA as atemplate according to the method previously described (Otaet al., 1996). The mutants missing the first 52 and 157 aminoacid residues are referred to herein as del-52 and del-157,respectively. Site-directed mutagenesis was performed withthe pKF18k vector containing hTH1 cDNA as a template bythe oligonucleotide-directed dual amber method according toNakashima et al. (2000, 2002). The mutant with Arg37-Arg38

replaced by Glu residues and that with Ser40 changed to Aspare referred to as RR-EE and S40D, respectively.

Construction of pcDNA3.1/HisA and pcDNA3.1(þ)Vectors Containing hTH1 cDNA

pcDNA3.1/HisA vector. The pBS vectors containingwildtype and del-52 hTH1 cDNAs described above weredigested with EcoRI, whereas the pBS vector containing del-157 hTH1 cDNA was digested with EcoRI and XbaI. Toconstruct the pcDNA3.1/HisA vectors containing hTH1

cDNA (hTH1-pcDNA3.1/HisA), we inserted the resultingEcoRI-EcoRI or EcoRI-XbaI fragment by ligation into thepcDNA3.1/HisA vector (Invitrogen, Carlsbad, CA).

The pKF18k vectors containing RR-EE and S40DhTH1 cDNAs described above were used as a template forPCR to obtain DNAs coding the N-terminal regions ofhTH1 mutants, RR-EE and S40D. The oligodeoxynucleotideprimers used were the following: sense primer, 50-GGGAATTCATATGCCCACCCCCGA-30; and anti-senseprimer, 50-GGCCGGGTCTCTAGAT-30. Sense primers weredesigned to introduce an NdeI site followed by an EcoRI site.The PCR procedure involved 25 cycles of denaturation at948C for 1 min, annealing at 558C for 1 min, and polymeriza-tion at 728C for 1 min. The amplified DNAs were digestedwith EcoRI and XbaI, then the resulting EcoRI-XbaI fragmentswere inserted by ligation into pcDNA3.1/HisA vectors. Toconstruct hTH1-pcDNA3.1/HisA, the pcDNA 3.1/HisA vec-tor containing the EcoRI-XbaI fragment of the PCR productwas linearized by XbaI digestion, then the XbaI-XbaI fragmentderived from the aforementioned pBS vector containing full-length wildtype hTH1 cDNA was inserted into the pcDNA3.1/HisA vector by ligation to introduce central and C-termi-nal regions.

pcDNA3.1(þ) vector. pcDNA3.1(þ) vectors contain-ing hTH1 cDNA (hTH1-pcDNA3.1[þ]) for wildtype, RR-EE, and S40D were generated according to the method previ-ously described (Nakashima et al., 2002). pET3c vectors con-taining del-52 and del-157 hTH1 cDNAs (Ota et al., 1996)were used as a template for PCR to produce DNAs codingthe N-terminal regions of the hTH1 mutants, del-52 anddel-157. The oligodeoxynucleotide primers used were asfollow: sense primers, 50-GCAAGCTTGCCACCATGG-CAGCAGCGGCCG-30 for del-52 and 50-GCAAGCTTG-CCACCATGGCGGGGCCCAAGGT-30 for del-157; andanti-sense primers, 50-GGCCGGGTCTCTAGAT-30 for del-52 and 50-GCTTGTTAGCAGCCGGATCC-30 for del-157.Sense primers were designed to introduce an HindIII site fol-lowed by the Kozac sequence. The PCR procedure was per-formed as described above. The DNA amplified for del-52was digested with HindIII and XbaI, and the resulting HindIII-XbaI fragment was inserted by ligation into the pcDNA3.1(þ)vector. To construct del-52 hTH1-pcDNA3.1(þ), pcDNA3.1(þ) vector containing the HindIII-XbaI fragment of thePCR product was linearized by XbaI digestion, then theXbaI-XbaI fragment derived from the aforementioned pBSvector containing full-length wildtype hTH1 cDNA wasinserted into the pcDNA 3.1(þ) vector by ligation to intro-duce central and C-terminal regions. To construct del-157 hTH1-pcDNA3.1(þ), the DNA amplified for del-157was digested with HindIII and BamHI, and the resultingHindIII-BamHI fragment was inserted by ligation into thepcDNA3.1(þ) vector.

Cell Culture

Mouse neuroendocrine cell line AtT-20/D16v-F2 cells(ATCC, Manassas, VA) and monkey kidney cell line COS-1cells were maintained in 60-mm dishes containing Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10%

Stability of Tyrosine Hydroxylase 111

fetal calf serum at 378C in humidified air containing 10% and5% CO2, respectively. Rat pheochromocytoma cell line PC12cells (Greene and Tischler, 1976) were maintained in 60-mmdishes containing DMEM supplemented with 10% fetal calfserum and 5% horse serum as well as 100 lg/ml streptomy-cin/penicillin at 378C in humidified air containing 5% CO2

(Menezes et al., 1996).

Expression of hTH1 Enzymes in Cells

Transfection of AtT-20/D16v-F2 cells was performedwith FuGENE6 Reagent (Roche Diagnostics, Mannheim,Germany) according to the supplier’s protocol. The ratio ofFuGENE6 reagent, hTH1-pcDNA3.1/HisA or hTH1-pcDNA3.1(þ) vector, and PGV-C2 vector were 12 ll:4 lg:0.4 lg. Transfections of COS-1 and PC12 cells were per-formed with Lipofectamine 2000 (Invitrogen) according tothe supplier’s protocol. The ratio of Lipofectamine 2000reagent, hTH1-pcDNA3.1/HisA vector, and PGV-C2 vectorwere 20 ll : 8 lg : 0.8 lg. The PGV-C2 vector, which is anSV40 promoter luciferase vector, was added to the mixture toassess the transfection efficiency. The transfection reagent con-taining the two vectors was added to the cells. The cells wereincubated for another 24 hr in the same culture and then har-vested. The cells were dissolved in Sigma CelLytic M (Sigma-Aldrich) or sonicated in 50 mM Tris-HCl buffer (pH 7.3)containing 8% sucrose, 1 mM dithiothreitol, and a proteaseinhibitor cocktail for mammalian tissue (Sigma-Aldrich).

Assay of Enzyme Activity and Measurement ofCatecholamine Contents

The catalytic activity of hTH1 was assayed using theHPLC apparatus as previously described (Nakashima et al.,2000). The assay was performed with 50 lM mM L-tyrosineand 1 mM 6RBPH4. The amounts of DA and its metabolite,3,4-dihydroxyphenylethylamine (DOPAC), in the cell lysatewere measured as previously described (Nakashima et al.,2002).

Immunoblotting Analysis

The cell lysate was mixed with one-fourth volume ofthe sample buffer consisting of 5% SDS, 40% glycerol, 5% 2-mercaptoetanol, and 0.02% BPB; then the mixture was boiledfor 3 min. Proteins in the mixture was separated on SDS-PAGE (10%) and electroblotted onto polyvinylidene difluor-ide membranes (Millipore, Bedford, MA). BioRad PrecisionPlus Protein Standard Dual Color (BioRad, Hercules, CA)was used as a standard. After having been blocked in 3% non-fat milk in phosphate-buffered saline (PBS), the membranewas incubated for 1 hr at room temperature with the primaryantibody diluted in PBS containing 0.05% Tween-20 (T-PBS)as follows: monoclonal anti-polyhistidine antibody mouseascites fluid (Sigma-Aldrich) or rabbit anti-ubiquitin antibody(Dako Cytomation, Glostrup, Denmark). After four washeswith T-PBS, the membrane was incubated for 1 hr at roomtemperature with T-PBS containing the appropriate secondaryantibody: rabbit antimouse IgG peroxidase conjugate IgG(Sigma-Aldrich) or goat antirabbit IgG peroxidase conjugateIgG (Cell Signaling, Beverly, MA). Immune complexes were

detected by chemiluminescence using ECL advance (Amer-sham Biosciences, Piscataway, NJ). The densitometry of thechemiluminescence signal was performed using a Fuji FilmLumino Image Analyzer LAS-1000 (Fuji Film, Tokyo, Japan).

Quantitative Real-Time PCR

Quantitative real-time PCR was performed accordingto the methods previously described (Kaneko et al., 2003).Total RNA was extracted from AtT-20 cells using RNA iso-lation reagent (Isogen, Nippon Gene, Tokyo, Japan). cDNAwas synthesized with 5 lg of total RNA by reverse transcrip-tion reaction (Life Technologies, Tokyo, Japan). The pair ofthe primers used in quantitative real-time PCR generated asingle band with the predicted size from cDNA on the con-ventional PCR (data not shown): forward primer of startingposition at 528, 50-TCATCACCTGGTCACCAAGTTC-30;and reverse primer of starting position at 633, 50-GAAGGC-GATCTCAGCAATCAG-30. Quantitative real-time PCR forthe samples comparative to 250 ng cDNA was performedwith SYBR Green (Perkin Elmer Biosystems, Boston, MA)on an ABI Prism 7000 PCR Instrument (Perkin Elmer Bio-systems) according to the manufacturer’s instructions.

Prediction of Secondary Structure

Secondary structures of the wildtype and mutants werepredicted from the amino acid sequences according to theNew Joint Method (Nishikawa and Noguchi 1991; Ito et al.,1997) on the PAPIA system (Akiyama et al., 1998). Theamino acid sequence of hTH1 (accession number X05290)was modified and used here after the correction of amino acidresidue 370 from Ser to Tyr (pers. commun. with the regis-trant. The registered data will be revised in the near future).Briefly, in the New Joint Method the following five methods,i.e., a method using neural network (Qian and Sejnowski1988), the one of Nagano and Ponnuswamy (1984), the oneof Ptitsyn and Finkelstein (1989), the one of Nishikawa andOoi (1980), and the one of Gibrat et al. (1987), were com-bined to predict the secondary structure of the proteins. Fivepredictions according to these five methods were performedseparately, and the final judgment was based on the five resultsas a whole.

Prediction of Proline, Glutamate/Aspartate, Serine,and Threonine (PEST) Motif

A search for PEST motifs, which consist of proline, glu-tamate/aspartate, serine, and threonine and confer rapid turn-over of many short-lived regulatory proteins, was conductedby using an algorithm developed by Rechsteiner and Rogers(1996) and Rogers et al. (1986). This algorithm produces ascore ranging from �50 to þ50, and a given score greaterthan þ5 denotes a significant PEST region.

Other Methods

Ten ll of the cell lysate was mixed with 50 ll of thesubstrate PicaGeneR and then the luciferase activity of themixture was measured with a Luminometer Lumat LB 9501(Berthold, Bad Wildbad, Germany). Protein concentration

112 Nakashima et al.

was measured with a Bio-Rad Protein Assay Kit with bovineserum albumin used as a standard.

Statistics

For data analysis, a two-tailed unpaired t-test orANOVA followed by Bonferroni’s multiple comparison testwas applied. These statistical analyses were performed usingSPSS for Windows v. 11.0J (SPSS, Chicago, IL).

RESULTS

Accumulation of DA in AtT-20 Cells

The luciferase activity derived from the controlvector PGV-C2 used along with the hTH1-pcDNA3.1/HisA vector to cotransfect AtT-20 cells was measured toassess the transfection efficiency. There were no signifi-cant differences in the luciferase activities among thecells transfected with wildtype cDNA or any of the fourmutants. The accumulation of DA and DOPAC withinthe AtT-20 cells reflects the ability of hTH1 to synthe-size L-DOPA because aromatic-L-amino acid decarboxy-lase (AADC) present in the cells converts L-DOPA toDA immediately. The quantities of DA and its metabo-lite DOPAC within AtT-20 cells expressing wildtype oreach of the mutants of hTH1 connected to the polyhisti-dine tag at their N-terminus (His-hTH1) are shown inFigure 1A. The levels of accumulated DA and DOPACwithin the cells expressing the mutants of His-hTH1were higher than those within the cells expressing thewildtype enzyme (DAþDOPAC, n ¼ 4, P < 0.05 orP < 0.01; Fig. 1A). However, there was no significantdifference in the DA and DOPAC accumulationbetween del-52 and del-157. There were very minimalamounts of L-DOPA, norepinephrine, and epinephrinewithin the cells expressing each enzyme. The resultsobtained in the experiment performed using AtT-20cells expressing hTH1 without the polyhistidine tag werealmost the same as those obtained with it (Fig. 1B).

Catalytic Activities of His-hTH1

For assessment of the catalytic activities of wildtypeand the mutants of His-hTH1, His-hTH1 should beexpressed in mammalian cells because the proteinsexpressed in E. coli cannot be phosphorylated. We usedCOS-1 cells to express His-hTH1 for the following rea-sons: the COS-1 cell line is able to express a large num-ber of proteins when exogenous cDNA is introduced. Inaddition, the inhibition of TH catalytic activity by DAcan be avoided in the reaction mixture containing thelysates of COS-1 cells because these cells lack TH andAADC. When the catalytic activities of His-hTH1 inthe cell lysates were measured, the values were correctedwith reference to the amount of His-hTH1 detected onimmunoblot analysis with mouse anti-polyhistidine anti-body (Table I). There were no significant differencesamong the activities of wildtype, RR-EE, and S40Denzymes. On the contrary, the activities of the N termi-nus-deleted mutants, del-52 and del-157, were signifi-cantly lower than the activity of the wildtype enzyme,

Fig. 1. DA and DOPAC accumulation in AtT-20 cells. His-hTH1 (A)and untagged hTH1 (B) were expressed in AtT-20 cells by transfectionwith hTH1-pcDNA3.1/HisA and hTH1-pcDNA3.1(þ) vectors, respec-tively. The amounts of DA and DOPAC were corrected according to thevalues of the luciferase activity and expressed as a percentage of theamount of DA and DOPAC in the cells transfected with wildtype hTH1cDNA, the value of which was 9.87 � 10�6 pmol/luciferase activity (A)and 5.85 � 10�6 pmol/luciferase activity (B). The measurements wereperformed in quadruplicate. The results shown are representatives ofthose obtained from three independent experiments. The significance ofthe difference among wildtype and the mutants was determined by usingANOVA followed by Bonferroni’s multiple comparison test. The resultsare displayed as mean6 SD. *P< 0.05, **P< 0.01 vs. that of wildtype.


although there was no significant difference betweenthese two mutants.

Quantities of His-hTH1 Proteins WithinAtT-20 Cells

Next, the quantities of His-hTH1 within the AtT-20 cells were determined using immunoblot analyseswith mouse anti-polyhistidine antibody (Fig. 2). Areas ofthe bands of His-hTH1 detected on immunoblots werecorrected by using the values of luciferase activities, andthen the corrected values of the areas were defined asthe quantities of the proteins expressed within the cells.

There were no significant differences among thequantities of histidine-tagged types of wildtype, RR-EE,and S40D within the cells (histidine-tagged (His-) wild-type, 100.00 6 39.50%; His-RREE, 128.57 6 23.14%;His-S40D, 180.76 6 29.14%; n ¼ 4; Fig. 2). On thecontrary, the quantities of His-del-52 and His-del-157were higher than that of His-wildtype (His-del-52,537.75 6 161.05%, n ¼ 4, P < 0.05 vs. His-wildtype;His-de157, 665.72 6 213.65%; n ¼ 4, P < 0.01 vs.His-wildtype).

Quantities of His-hTH1 mRNA WithinAtT-20 Cells

His-hTH1 mRNA expression levels were measuredto examine whether the amounts of the mRNAs encod-ing the N-terminus-deleted mutants molecules were ele-vated compared to the mRNAs encoding His-wildtypemolecules (Table II). However, quantitative real-timePCR did not detect any difference among the expressionlevels of hTH1 mRNA in these cells. Therefore, weconcluded that there were no differences in the amountsamong wildtype mRNAs and the N-terminus-deletedmutants mRNAs.

Degradation Rate of His-hTH1 Proteins WithinAtT-20 Cells

We next assessed whether the differences of thequantities of the His-hTH1 proteins detected in Figure 2

were determined by their degradation rate via the pro-teolytic pathway in the cells. AtT-20 cells expressingHis-wildtype or His-del-52 were treated with cyclohexi-mide for 2, 4, 8, and 24 hr to inhibit intracellular pro-tein synthesis. The bands of His-del-52 in the cells dissi-pated more slowly than those of His-wildtype on theimmunoblots using monoclonal anti-polyhistidine anti-body as the first antibody (Fig. 3). The data stronglyindicate that the degradation rate of His-del-52 is lowerthan that of His-wildtype, which means that His-del-52is more stable than His-wildtype.

Quantity of His-hTH1 in COS-1 and PC12 Cells

The quantities of His-hTH1 within COS-1 andPC12 cells transfected with hTH1-pcDNA3.1/HisAwere examined using immunoblot analysis with anti-mouse polyhistidine antibody (Fig. 4). The quantitieswere corrected with reference to the values of the luci-ferase activities. There were no significant differencesamong the quantities of His-wildtype, His-RR-EE, andHis-S40D (in COS-1 cells: 100.00 6 7.92% for His-wild-type, 93.35 6 6.93% for His-RR-EE, 116.46 6 8.64%

TABLE I. Catalytic Activity of His-hTH1

TH activity

(% of wildtype)

wildtype 100.00 6 26.39

RR-EE 112.73 6 25.91

S40D 65.95 6 15.25

del-52 19.20 6 15.25*

del-157 3.00 6 0.62*,**

The catalytic activities were corrected according to the areas of the bands

of His-hTH1 protein detected on an immunoblot by using mouse anti-

polyhistidine antibody. The measurements were performed in quadrupli-

cate. The results shown are representative of those obtained from three

independent experiments. The significance of the differences between

wildtype and the mutants were determined by ANOVA followed by

Bonferroni’s multiple comparison test as a post-hoc test. The results are

displayed as mean 6 SD.*P < 0.01 vs. that of the wildtype;**no significant difference vs. that of del-52 enzyme.

Fig. 2. Immunodetection of His-hTH1 expressed in AtT-20 cells.His-hTH1 was expressed in AtT-20 cells with hTH1-pcDNA3.1/HisA. The His-hTH1 was detected using mouse monoclonal anti-polyhistidine antibody. The measurements were performed in quad-ruplicate. The results shown are representative of those obtained fromthree independent experiments. The numeric data are described inthe Results section. The arrows indicate the bands of wildtype andthe N-terminus-deleted mutants of His-hTH1.

TABLE II. Quantification of His-hTH1 mRNA Expression

Levels in AtT-20 Cells

His-hTH1 mRNA (% of wildtype)

wildtype 100.00 6 5.98

RR-EE 136.75 6 8.18

S40D 107.81 6 6.45

del-52 89.57 6 5.36

del-157 101.75 6 6.08

The amounts of His-hTH1 mRNA within the cells transfected with

cDNAs encoding wildtype or any of the four mutants are presented as

the quotients divided by the luciferase activities. They are expressed as a

percentage of the mean value obtained from the cells transfected with

wildtype His-hTH1 cDNA. The measurements were performed in tripli-

cate. The results are displayed as mean 6 SD. ANOVA carried out

between wildtype and the mutants did not reject the null hypothesis.


for His-S40D, n ¼ 4; in PC12 cells: 100.00 6 14.49%for His-wildtype, 95.57 6 23.39% for His-RR-EE,122.82 6 12.86% for His-S40D, n ¼ 4). On the otherhand, the quantities of His-del-52 and His-del-157expressed in both cells were significantly higher than thatof the His-wildtype enzyme (in COS-1 cells: 568.31 624.10% for His-del-52, 1118.25 6 154.73% for His-del-157, n ¼ 4, P < 0.01; in PC12 cells: 256.91 6 18.16%for His-del-52, 347.35 6 88.46% for His-del-157, n ¼ 4,P < 0.01). Again, the quantities of the His-hTH1 should

reflect the degradation rate via the proteolytic pathwayin the cells.

Prediction of PEST Motifs of the Wildtype hTH1and Mutants del-52 and del-157

To consider the intracellular degradation, wesearched for the PEST motif in the amino acid sequen-ces. As shown in Figure 5, the presence of five PESTmotifs termed PEST-1 (Met1-Lys12), PEST-2 (Arg49-

Lys76), PEST-3 (Arg265-Arg277), PEST-4 (Arg410-

Lys441), and PEST-5 (Lys458-Arg476) was predicted.PEST-1, supposedly the most powerful PEST regiondue to its score of þ5.46 (the highest among the fivepredicted PEST motifs), was located in the N-terminalregion that was lost in the deletion mutants del-52 anddel-157. This result should be noted because the lack ofPEST-1 in these two mutants can be supposed to be thereason for the high stability of the mutants expressed inthe cells.

Prediction of Secondary Structures of theWildtype hTH1 and Mutants del-52 and del-157

To find some hints as to the influence of the N-terminal deletions, we predicted the secondary structuresof the wildtype hTH1 and del-52 and del-157 mutantsby using a combination of five different methods,

Fig. 3. Degradation rates of His-hTH1 proteins within AtT-20 cells.His-hTH1 proteins were expressed in the AtT-20 cells by incubationfor 20 hr after the addition of the transfection reagent and hTH1-pcDNA3.1/HisA vector to culture medium. In order to inhibit pro-tein synthesis, the cells were treated with 20 lg/ml cycloheximidefor 2, 4, 8, and 24 hr. His-hTH1 proteins were detected usingmonoclonal anti-polyhistidine antibody as the first antibody (A). Thelevels of His-hTH1 proteins determined by densitometric analysiswere expressed as a percentage of His-wildtype or His-del-52 at timezero (B). The measurements were performed in quadruplicate. Theresults shown are representative of those obtained from three inde-pendent experiments. The significance of the difference betweenwildtype and the mutant was determined using a two-tailed unpairedt-test. The results are displayed as mean 6 SD. **P < 0.01 vs. thatof wildtype.

Fig. 4. Immunodetection of His-hTH1 expressed in COS-1 andPC12 cells. His-hTH1 was expressed in COS-1 (A) and PC12 cells(B) with hTH1-pcDNA3.1/HisA and hTH1-pcDNA3.1(þ), respec-tively. The His-hTH1 proteins were detected using mouse monoclo-nal anti-polyhistidine antibody. The measurements were performedin quadruplicate. The results shown are representative of thoseobtained from three independent experiments. The numeric data aredescribed in the Results section. The arrows indicate the bands ofwildtype and the N-terminus-deleted mutants of His-hTH1.


termed the New Joint Method (Fig. 5). Interestingly,the N-terminal deletion supposedly had some effects onregions far from the N-terminus. We found threeregions where remarkable effects of the deletions on thesecondary structural properties were predicted: Region 1(Phe285-Pro289), Region 2 (Val385-His401), and Region 3(Phe455-Asp484), shown by the blue double-lined boxesin Figure 5. Region 1 is adjacent to PEST-3. PEST-4 islocated between Region 2 and Region 3. PEST-5 is inRegion 3. These results suggest that changes in thestructural properties induced by the N-terminal deletions

influenced the activities of PEST motifs negatively andraised enzyme stability.

Inhibition of Proteasome Pathway WithinAtT-20 Cells

Finally, we examined the possible role of the ubiq-uitin-proteasome pathway, a main pathway for the deg-radation of intracellular proteins, in degradation of His-hTH1 proteins in AtT-20 cells (Fig. 6). The proteasomeinhibitor lactacystin effectively inhibited the proteasome

Fig. 5. Prediction of secondary structures and PEST motifs of thewildtype hTH1 and mutants del-52 and del-157. Red characters ofthe sequence represent the predicted PEST motifs, i.e., PEST-1(Met1-Lys12), PEST-2 (Arg49-Lys76), PEST-3 (Arg265-Arg277), PEST-4 (Arg410-Lys441), and PEST-5 (Lys458-Arg476). Their scores for thepredictions are also shown with their labels. PEST-1 is emphasizedby a red double underline because the score of the prediction is

extremely high in comparison with those scores of the other pre-dicted motifs. Results of the secondary structure prediction are indi-cated under the amino acid sequence. H, E, and C represent a helix,b sheet, and coil, respectively. Red characters indicate inconsistenciesamong the results of the secondary structure prediction. Regions 1,2, and 3, in which the inconsistencies are concentrated, are alsoshown by blue double-lined boxes.


pathway in AtT-20 cells because the quantities of theubiquitin-immunoreactive bands indicating the ubiquiti-nation of the cytosolic proteins were increased (Fig. 6B).The quantity of His-wildtype protein showed a slightincrease in AtT-20 cells treated with lactacystin for 6 hr,although there was no significant difference (Fig. 6C).The quantities of His-del-52 and His-del-157 proteinsdid not increase in the cells treated with lactacystin for 3or 6 hr.

DISCUSSION

Several studies have indicated that under cell-freeconditions catecholamine-bound TH is more stable thancatecholamine-expelled TH due to phosphorylation ofthe N-terminus (Okuno and Fujisawa, 1991; Gahn and

Roskoski, 1995; Muga et al., 1998). hTH1 was alsofound to be a substrate for ubiquitin conjugation in areconstituted in vitro system because it was partiallydegraded by proteasomes in a reticulocyte lysate system(Døskeland and Flatmark, 2002). However, little infor-mation is available on the factors determining the stabil-ity of the TH molecule in mammalian cells from theviewpoint of protein degradation. In this study, we pre-sented data indicating that the N-terminus of hTH1could affect its stability of the enzyme, which affects theproduction rate of DA in mammalian cells to the samedegree as the electric charge intrinsic to Arg37-Arg38

amino acid residues (Nakashima et al., 2002).The level of DA accumulated in AtT-20 cells

expressing the mutant RR-EE (Arg37-Arg38 replaced by

Fig. 6. Immunodetection of His-hTH1 inthe AtT-20 cells treated with a proteasomeinhibitor. His-hTH1 proteins were expressedin the AtT-20 cells by incubation for 20 hrafter the addition of the transfection regentand hTH1-pcDNA3.1/HisA vector to cul-ture medium. In order to inhibit the protea-some pathway, we treated the cells with10 lM lactacystin for 3 or 6 hr. His-hTH1proteins were detected using monoclonalanti-polyhistidine antibody as the first anti-body (A). To ascertain the effective inhibi-tion of the proteasome pathway by lactacys-tin, the ubiquitinated proteins were detectedusing rabbit anti-cow ubiquitin antibody asthe first antibody (B). The levels of His-hTH1 proteins were determined by densito-metric analyses. The values at the 3 and6 hr time points for wildtype or eachmutant forms are expressed as a percentageof their respective counterparts as the incu-bation without lactacystin (C). The meas-urements were performed in quadruplicate.The results shown are representative ofthose obtained from three independentexperiments. The numeric data are describedin the Results section. The arrows indicatethe bands of wildtype and the N-terminus-deleted mutants of His-hTH1.


Glu37-Glu38) or the mutant S40D, mimicking the phos-phorylation of Ser40, was higher than that in cellsexpressing the wildtype enzyme because the mutantscould efficiently avoid DA inhibition (Nakashima et al.,2002). In our experiments performed on the AtT-20cells, the level of DA accumulated in the cells transfectedwith the N-terminus-deleted His-hTH1 mutants washigher than that accumulated in the cells transfected withthe wildtype cDNA and was expressed to the samedegree as found in the cells transfected with the mutantRR-EE (Fig. 1A). The levels of DA accumulated in thecells transfected with del-52 or del-157 were statisticallythe same. Next, untagged hTH1 protein was expressedin AtT-20 cells in order to exclude the influence of thepolyhistidine tag on the enzyme activity. The levels ofDA accumulated in the cells transfected with theuntagged form of the hTH1 mutants were almost thesame as those in the cells transfected with His-hTH1(Fig. 1B). These results strongly suggest that the N-ter-minus ranging from Met1 to Ala52 is critical for deter-mining the production rate of DA in AtT-20 cells.Therefore, we performed subsequent experiments toanswer the question as to why the N-terminus-deletedmutants had the capability of producing a large amountof DA.

We previously showed that deletion of the N-ter-minal 38-amino acid residues of hTH1 allowed theenzyme to escape from the inhibitory effect of DA onits catalytic activity (Ota et al., 1997; Nakashima et al.,1999b). On the other hand, the observation of thereduced catalytic activity of the N-terminus-deletedform of His-hTH1 was in good agreement with ourexpectation (Table I) because the Vmax of the mutanthTH1 devoid of its N-terminal 38 amino acid residues isabout one-fourth of that of the wildtype enzyme undercell-free conditions, contrary to no significant differencesamong the Km values (Ota et al., 1997; Nakashimaet al., 1999b). Recently, a very low level of immunor-eactive human phenylalanine hydroxylase (PAH) proteinpossessing a point mutation within its catalytic domainwas recovered in human embryonic kidney (A293) cellsin spite of the transient expression of a sufficient amountof the mutant PAH mRNA in the cells (Bjørgo et al.,1998). A change in the spatial configuration of the PAHprotein caused by the mutation was suggested to pro-mote susceptibility of the PAH enzyme to the limitedproteolysis, thus decreasing its intracellular stability. Onthe contrary, immunoblot analysis with anti-polyhistidineantibody revealed the higher level of both His-del-52and His-del-157 proteins than that of wildtype in theAtT-20 cells (Fig. 2). In addition, the higher level of theN-terminus-deleted His-hTH1 observed in COS-1 cellsappeared again in PC12 cells, which express TH proteinendogenously (Fig. 4). The intracellular level of His-hTH1 should be determined by the amount of the pro-tein flowing into the proteolysis pathway, because therewere no significant differences in the expression level ofthe His-hTH1 mRNA (Table II) and the turnover ofHis-wildtype protein was faster than that of His-del-52

protein (Fig. 3). Collectively, our data indicate thatAtT-20 cells transfected with N-terminus-deleted hTH1are able to produce a large amount of DA because ofthe high stability of the N-terminus-deleted hTH1 mol-ecule, i.e., resistance to degradation by proteases, whichis a more potent determinant for efficient DA produc-tion in these cells. Put another way, the stability of thehTH1 molecule is determined by the N-terminus regionof hTH1 up to Ala52.

Three aromatic amino acid hydroxylases, i.e., TH,PAH, and tryptophan hydroxylase, contain highly con-served central and C-terminal catalytic domains.Recently, they were reported to be substrates for theubiquitin-conjugating enzyme system that are degradedby proteasomes (Kojima et al., 2000; Døskeland andFlatmark, 1996, 2001, 2002). However, there is noinformation on which sites on the TH molecule couldbe targets for the proteasome system. Therefore, withthe assistance of several computer software programs, wescreened the motifs in the hTH1 molecule that shouldbe the target sites of the intracellular proteases involvedin the ubiquitin-conjugating enzyme system and alsosearched for the expected structural change in the hTH1molecule caused by the deletion of its N-terminus. Thecomputer-assisted search resulted in the following twopossibilities promising high stability of the N-terminus-deleted mutants of hTH1 in the cells: First, the hTH1molecule possessed a total of five PEST motifs, includingthe original PEST motif reported by Pasinetti et al.(1992; Fig. 5). The PEST motif confers rapid turnoverof many short-lived regulatory proteins (Rogers et al.,1986), although PEST motifs are not always the targetsfor the proteases. Especially, the following two PESTmotifs were newly identified as a result of our search:PEST-1, spanning from Met1 to Lys12, and PEST-2,spanning from Arg49 to Lys76 (Fig. 5). PEST-1 may be amain target of the intracellular proteases, because whenexpressed in AtT-20 cells the quantity of His-del-52lacking PEST-1 was higher than that of the His-wild-type enzyme and almost the same as that of His-del-157,which lacked both PEST-1 and PEST-2 (Fig. 2). Sec-ond, the deletion of the N-terminus region wasexpected to cause changes in the spatial configuration inthe following three regions of the enzyme: Region 1(Phe285-Pro289), Region 2 (Val385-His401), and Region 3(Phe455-Asp484). It should be noted that Region 1 andRegion 2 are adjacent to PEST-3 (Arg265-Ser277) andPEST-4 (Arg410-Lys441), respectively, and that Region 3covers PEST-5 (Lys458-Arg476, Fig. 5). Recently, thereduced level of TH protein observed in patients suffer-ing from dopa-responsive dystonia and in GTP cyclohy-droxylase gene knockout mice, both of which are inca-pable of de novo synthesis of 6RBPH4, was explainedby the reduced stability of the TH protein caused by the6RBPH4 deficiency. The reduction in the level of THprotein was readily reversed by exogenous administrationof 6RBPH4 (Furukawa et al., 1999; Sumi-Ichinoseet al., 2001). In addition, TH protein levels in the stria-tum of mice lacking the dopamine transporter were dra-


matically decreased (Jaber et al., 1999). These reportsmay be interpreted to indicate that the binding of THwith catecholamines synthesized by TH enzyme in thepresence of 6RBPH4 within the cells may prevent THprotein from reacting with the proteases. Similarly, it isreasonable to speculate that the structural changes occur-ring in the aforementioned three regions of the hTH1protein might reduce the accessibility of the proteases tothe PEST motifs, which would eventually result in anincreased level of hTH1 protein in the cells.

Finally, we examined whether the ubiquitin–pro-teasome pathway is deeply involved in determining theaugmented stability observed for the His-del-52 mutantexpressed in mammalian cells. The addition of lactacys-tin, a proteasomal inhibitor, to the culture mediumcaused a slight increase in the level of His-wildtype pro-tein in the cells, although the increase was just short ofstatistical significance (Fig. 6). On the contrary, treat-ment of the cells expressing His-del-52 and His-del-157by lactacystin did not affect the expression level of thesetwo proteins in the cells. We could not confirm thehypothesis that the high stability of the N-terminus-deleted mutants of hTH1 expressed in mammalian cellsis controlled by the ubiquitin-proteasome pathway.However, to our belief, the presence of the PEST motifsin the N-terminus of hTH1 protein and the structuralchange caused by the deletion of N-terminus providesvaluable information for understanding the mechanismunderlying the augmented stability of the N-terminus-deleted mutants within the cells.

In conclusion, our results indicate that the stabilityof the hTH1 molecule in the cells is determined by theN-terminus region up to Ala52 and that the high stabilitygenerated by the deletion of the N-terminus is a potentdeterminant of DA production in the cells. However,the precise reason for this high stability still remains tobe clarified. We believe that this study provides informa-tion showing the influence of stability of the hTH1 mol-ecule on DA production in the cells.

ACKNOWLEDGMENT

We thank Dr. Takahiro Suzuki (Tokyo Institute ofTechnology) for significant suggestions.

REFERENCES

Akiyama Y, Onizuka K, Noguchi T, Ando M. 1998. Parallel protein

information analysis (PAPIA) system running on a 64-node PC Cluster.

In: Miyano S, Takagi T, editors. Proceedings of the 9th Genome

Informatics Workshop (GIW’98). Tokyo: Universal Academy Press.

p 131–140.

Bjørgo E, Knappskog PM, Martınez A, Stevens RC, Flatmark T.

1998.Partial characterization and three-dimensional-structural localiza-

tion of eight mutations in exon 7 of the human phenylalanine hydroxy-

lase gene associated with phenylketonuria. Eur J Biochem 257:1–10.

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

directed mutagenesis of serine 40 of rat tyrosine hydroxylase. J Biol

Chem 267:12639–12646.

Døskeland AP, Flatmark T. 1996.Recombinant human phenylalanine

hydroxylase is a substrate for the ubiquitin-conjugating enzyme system.

Biochem J 319:941–945.

Døskeland AP, Flatmark T. 2001.Conjugation of phenylalanine hydroxy-

lase with polyubiquitin chains catalysed by rat liver enzymes. Biochim

Biophys Acta 1547:379–386.

Døskeland AP, Flatmark T. 2002.Ubiquitination of soluble and mem-

brane-bound tyrosine hydroxylase and degradation of the soluble form.

Eur J Biochem 269:1561–1569.

Furukawa Y, Nygaard TG, Gutlich M, Rajput AH, Pifl C, DiStefano L,

Chang LJ, Price K, Shimadzu M, Hornykiewicz O, Haycock JW, Kish

SJ. 1999.Striatal biopterin and tyrosine hydroxylase protein reduction in

dopa-responsive dystonia. Neurology 53:1032–1041.

Gahn LG, Roskoski R Jr. 1995.Thermal stability and CD analysis of rat

tyrosine hydroxylase. Biochemistry 34:252–256.

Gibrat JF, Garnier J, Robson B. 1987.Further developments of protein

secondary structure prediction using information theory: new parame-

ters and consideration of residue pairs. J Mol Biol 198:425–443.

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

4:578–585.

Greene LA, Tischler AS. 1976.Establishment of a noradrenergic clonal

line of rat adrenal pheochromocytoma cells which respond to nerve

growth factor. Proc Natl Acad Sci U S A 73:2424–2428.

Haavik J, Martınez A, Flatmark T. 1990.pH-dependent release of cate-

cholamines from tyrosine hydroxylase and the effect of phosphorylation

of ser-40. FEBS Lett 262:363–365.

Haycock JW, Wakade AR. 1992.Activation and multiple-site phosphory-

lation of tyrosine hydroxylase in perfused rat adrenal glands. J Neuro-

chem 58:57–64.

Hufton SE, Jennings IG, Cotton RG. 1995.Structure and function of the

aromatic amino acid hydroxylases. Biochem J 311:353–366.

Ito M, Matsuo Y, Nishikawa K. 1997.Prediction of protein secondary

structure using the 3D-1D compatibility algorithm. Comput Appl Bio-

sci 13:415–424.

Jaber M, Dumartin B, Sagne C, Haycock JW, Roubert C, Giros B,

Bloch B, Caron MG. 1999.Differential regulation of tyrosine hydroxy-

lase in the basal ganglia of mice lacking the dopamine transporter. Eur

J Neurosci 11:3499–3511.

Kaneko YS, Mori K, Nakashima A, Nagatsu I, Ota A. 2003.Peripheral

administration of lipopolysaccharide enhances the expression of guano-

sine triphosphate cyclohydrolase I mRNA in murine locus coeruleus.

Neuroscience 116:7–12.

Kojima M, Oguro K, Sawabe K, Iida Y, Ikeda R, Yamashita A, Naka-

nishi N, Hasegawa H. 2000.Rapid turnover of tryptophan hydroxylase

is driven by proteasomes in RBL2H3 cells, a serotonin producing mast

cell line. J Biochem 127:121–127.

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.

McCulloch RI, Fitzpatrick PF. 1999.Limited proteolysis of tyrosine

hydroxylase identifies residues 33-50 as conformationally sensitive to

phosphorylation state and dopamine binding. Arch Biochem Biophys

367:143–145.

McCulloch RI, Daubner SC, Fitzpatrick PF. 2001.Effects of substitution

at serine 40 of tyrosine hydroxylase on catecholamine binding. Bio-

chemistry 40:7273–7278.

Menezes A, Zeman R, Sabban EL. 1996.Involvement of intracellular or

extracellular calcium in activation of tyrosine hydroxylase gene expres-

sion in PC12 cells. J Neurochem 67:2316–2324.

Muga A, Arrondo JL, Martınez A, Flatmark T, Haavik J. 1998.The effect

of phosphorylation at Ser-40 on the structure and thermal stability of

tyrosine hydroxylase. Adv Pharmacol 42:15–18.


Nagano K, Ponnuswamy PK. 1984.Prediction of packing of secondary

structure. Adv Biophys 18:115–148.

Nagatsu T, Levitt M, Udenfriend S. 1964.Tyrosine hydroxylase: the ini-

tial step in norepinephrine biosynthesis. J Biol Chem 239:2910–2917.

Nakashima A, Mori K, Nagatsu T, Ota A. 1999a.Expression of human

tyrosine hydroxylase type 1 in Escherichia coli as a protease-cleavable

fusion protein. J Neural Transm 106:819–824.

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

A. 1999b.Dopamine inhibition of human tyrosine hydroxylase type 1 is

controlled by the specific portion in the N-terminus of the enzyme.

J Neurochem 72:2145–2153.

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

2000.Positive charge intrinsic to Arg37-Arg38 is critical for dopamine

inhibition of the catalytic activity of human tyrosine hydroxylase

type 1. FEBS Lett 465:59–63.

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 cata-

lytic activity in neuroendocrine AtT-20 cells. J Neurochem 82:202–206.

Nishikawa K, Noguchi T. 1991.Predicting protein secondary structure

based on amino acid sequence. Methods Enzymol 202:31–44.

Nishikawa K, Ooi T. 1980.Prediction of the surface-interior diagram of

globular proteins by an empirical method. Int J Pept Protein Res

16:19–32.

Okuno S, Fujisawa H. 1991.Conversion of tyrosine hydroxylase to stable

and inactive form by the end products. J Neurochem 57:53–60.

Ota A, Yoshida S, Nagatsu T. 1996.Regulation of N-terminus-deleted

human tyrosine hydroxylase type 1 by end products of catecholamine

biosynthetic pathway. J Neural Transm 103:1415–1428.

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 229:57–60.

Pasinetti GM, Osterburg HH, Kelly AB, Kohama S, Morgan DG, Rein-

hard JF Jr, Stellwagen RH, Finch CE. 1992.Slow changes of tyrosine

hydroxylase gene expression in dopaminergic brain neurons after neu-

rotoxin lesioning: a model for neuron aging. Mol Brain Res 13:63–73.

Ptitsyn OB, Finkelstein AV. 1989.Prediction of protein secondary struc-

ture based on physical theory: histones. Protein Eng 2:443–447.

Qian N, Sejnowski TJ. 1988.Predicting the secondary structure of globu-

lar proteins using neural network models. J Mol Biol 202:865–884.

Rechsteiner M, Rogers SW. 1996.PEST sequences and regulation by

proteolysis. Trends Biochem Sci 21:267–271.

Rogers S, Wells R, Rechsteiner M. 1986.Amino acid sequences com-

mon to rapidly degraded proteins: the PEST hypothesis. Science

234:364–368.

Sumi-Ichinose C, Urano F, Kuroda R, Ohye T, Kojima M, Tazawa M,

Shiraishi H, Hagino Y, Nagatsu T, Nomura T, Ichinose H. 2001.Cate-

cholamines and serotonin are differently regulated by tetrahydrobiop-

terin. A study from 6-pyruvoyltetrahydropterin synthase knockout

mice. J Biol Chem 276:41150–41160.

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 217:715–722.

Wu J, Filer D, Friedhoff AJ, Goldstein M. 1992.Site-directed mutagenesis

of tyrosine hydroxylase: role of serine 40 in catalysis. J Biol Chem

267:25754–25758.


deletion of n-terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in...

Documents