From the GSF-Institut für Klinische
Molekularbiologie und Tumorgenetik, the ¶ GSF-Institut für
Immunologie, and the
GSF-Institut für Experimentelle
Hämatologie, D-81377 München, Germany
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ABSTRACT |
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GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hültner, L., Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rödl, W. (1993) J. Biol. Chem. 268, 12544-12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivo labeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.
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INTRODUCTION |
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(6R)-H4biopterin1 serves as an electron donor for the hydroxylation of aromatic amino acids and, therefore, functions as the essential cofactor for tryptophan 5-monooxygenase (EC 1.14.16.4), for phenylalanine 4-monooxygenase (EC 1.14.16.1), and for tyrosine 3-monooxygenase (EC 1.14.16.2). Consequently, these oxygenases initiate serotonin and catecholamine biosynthesis, respectively (reviewed in Ref. 1). The cofactor H4biopterin is synthesized de novo from GTP, and the first and rate-limiting step in the biosynthetic pathway is catalyzed by GTP cyclohydrolase I (EC 3.5.4.16). The subsequent activity of 6-pyruvoyl H4pterin synthase (EC 4.6.1.10) and sepiapterin reductase (EC 1.1.1.153) yields the final product, H4biopterin (reviewed in Ref. 2).
The activity of mammalian GTP cyclohydrolase I is regulated by
cytokines in a cell line and tissue-specific manner. For example, in T
cells (3) and macrophages (4, 5) it is induced by interferon (3),
and in rat mesangial cells it is triggered by tumor necrosis factor
or interleukin 1
(6). Among a panel of cytokines that support the
growth of murine BMMC, KL (kit ligand; or stem cell factor) selectively
enhances the activity of GTP cyclohydrolase I about 6-fold (7). It was
demonstrated that either interferon
treatments of T cells and
macrophages (4) or cultivation of BMMC with KL (8) increases the steady
state mRNA levels of this enzyme; moreover, it was reported (6)
that increased GTP cyclohydrolase I protein levels occurred in rat mesangial cells exposed to interleukin 1
.
Evidence is accumulating, however, that GTP cyclohydrolase I activity
is not only subject to transcriptional or post-transcriptional regulation. For example, a conformational change may be induced by
binding of a feedback regulatory protein consisting of 9.5-kDa subunits
(9, 10). Moreover, the kinetics of cytokine-induced or of
cell-cycle-associated GTP cyclohydrolase I activity in T cells (4) and
in rat thymocytes (11), respectively, did not fully correlate with the
kinetics of steady state levels of mRNA specific for the enzyme. It
was suggested, therefore, that post-translational modification of GTP
cyclohydrolase I may contribute to the changes in its activity. The
combination of interleukin 1 with agents that elevate cellular cAMP
levels caused an additive increase in mRNA for GTP cyclohydrolase I
but also yielded a marked synergistic increase at the activity level in
rat mesangial cells; therefore, a prominent post-translational
modulation of the enzyme was postulated (6). Furthermore, the
observation that phorbol ester triggers a rapid but transient
accumulation of neopterin and biopterin in primed T cells and in
various cell lines (12) suggested that GTP cyclohydrolase I may become
phosphorylated. Finally, in PC12 cells exposed to high KCl
concentrations, Imazumi et al. (13) reported that a
rabphilin-3A antibody co-immunoprecipitated several phosphorylated
proteins, one of which was a 30-kDa protein with a peptide map and
amino acid analysis identical with GTP cyclohydrolase I. Nonetheless, a
direct and unequivocal proof for a post-translational modification of
this enzyme has been lacking.
Consequently, we examined phosphorylation of GTP cyclohydrolase I and
its modulation of activity in response to an external stimulus in
rodent mast cells. Among all primary cells of mammalian origin, the
expression level of GTP cyclohydrolase I activity in KL-induced BMMC is
best for a biochemical experimentation; the activity levels in these
cells are 5 to 50-fold higher than in liver, adrenal, brain (14), or in
primed T cells (3). On the other hand, the rat basophilic leukemia cell
line RBL-2H3 has been an extensively used model for stimulus protein
phosphorylation coupling in mast cells. Antigen binding to the
IgE-primed cells leads to a complex sequence of events that transduce
the signal. The sequence is initiated by tyrosine phosphorylation of
the and
chains in Fc
R1 by the protein-tyrosine kinase
p53/56lyn and subsequent activation
of p72syk(15-17). Activation of phospholipase
C-
1 by tyrosine phosphorylation possibly involves
p72syk and causes phosphoinositide breakdown.
This generates 1,2-sn-diacylglycerol as a potent activator
of all PKC isotypes (except PKC-
) that are reported to occur in
rodent mast cells (reviewed in Refs. 16 and 17). The
isoform of PKC
partly associates with and phosphorylates the receptor upon engagement
of Fc
R1 by antigen. Besides, PKC-
becomes phosphorylated on
tyrosine residues associated with the membrane and primed to
phosphorylate various substrates (Refs. 18 and 19; reviewed in Ref.
16).
Here we show that the activity of GTP cyclohydrolase I is rapidly and
transiently enhanced in response to antigen binding to FcR1 of
KL-induced BMMC and of RBL-2H3. Furthermore, we demonstrate that the
enzyme exists in the cell in a phosphorylated form and that it becomes
hyperphosphorylated upon antigen triggering. Evidence is provided that
PKC is linked to the modulation of enzyme phosphorylation and activity
and thus, to a rapid regulation of H4biopterin production of the mast cell.
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EXPERIMENTAL PROCEDURES |
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Reagents and Antibodies--
The sources of all reagents that
were used for cell cultivation (7, 20) as well as for determination of
enzymatic activities and for protein and biopterin determination (7,
21-24) have been described previously. The sources of the following
reagents appear in parentheses: phorbol ester (PMA) (Sigma); Ro-31-8220 and hemocyanin-DNP conjugate (Calbiochem);
5,6-dichloro-1--D-ribofuranosylbenzimidazole (Biomol);
protein G-Sepharose Fast Flow (Amersham Pharmacia Biotech ); ECL
detection system, ECL biotinylation system, Hybond nitrocellulose membrane (Amersham); CNBr-activated Sepharose (Amersham); alkaline phosphatase (Boehringer Mannheim). Antibodies were obtained from the
following sources: anti-rat Ig antibodies for immunoglobulin isotyping
(Zymed Laboratories Inc. and American Type Culture
Collection); peroxidase-conjugated anti-rat IgG and second antibodies
(Dianova); monoclonal IgE antibody to DNP (Sigma); Dulbecco's modified
Eagle's medium and supplements (Life Technologies); myelin basic
protein (Life Technologies, Inc.); green fluorescent protein vector
(CLONTECH).
Generation of Monoclonal Antibodies to Murine GTP Cyclohydrolase I-- Monoclonal antibodies were generated by immunizing Lou/C rats with a bacterially expressed murine GTP cyclohydrolase I fused in frame with Escherichia coli maltose binding protein (MGTP-maltose binding protein) (25). The procedure was essentially as described in Kremmer et al. (26). Screening of hybridoma supernatants was performed in a solid-phase immunoassay using bacterial extracts from E. coli expressing either the MGTP-maltose binding protein or a maltose binding protein control. The immunoglobulin type was determined with rat Ig class (anti-IgM) and IgG subclass-specific mouse mAbs. Antibody specificity of MGTP-6B6 (rat IgG2b) and MGTP-6H11 (rat IgG2a) was tested by immunoblotting against murine GTP cyclohydrolase I expressed in E. coli (see Fig. 1). The monoclonal antibodies were purified using Protein G-Sepharose columns. In immunoprecipitates obtained from extracts of KL-induced BMMC or from E. coli expressing recombinant GTP cyclohydrolase I, 90% of total GTP cyclohydrolase I activity was recovered. Moreover, pre-saturation of the mAb MGTP-6B6 with recombinant rat GTP cyclohydrolase I inhibited the subsequent immunoprecipitation as examined by activity determination in immunoprecipitates of KL-induced BMMC.
Cell Culture and Cell Stimulation-- Primary mouse BMMC were obtained from femoral bone marrow and were kept in interleukin 3-dependent growth. Their maturation was induced by KL for 40 h. Details of the culture conditions have been described previously (7, 20). P815 cells were obtained from the German Collection of Microorganisms and Cell Cultures, Braunschweig. The 2H3 subline of RBL cells was a gift of D. Arndt-Jovin, Goettingen, Germany. Cells were maintained in flasks with RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum in a 5% CO2 atmosphere. For experiments, they were seeded in 60-mm Petri dishes and grown to a density of 1-5 × 106 cells/dish. The cells were loaded with monoclonal anti-DNP IgE (0.5-1 µg/ml; 2-4 h). After washing in Hanks' solution, activation with DNP conjugated to hemocyanin (1-3 µg/ml) or with 60 nM PMA was performed at 37 °C for the indicated time according to standard procedures (27, 28). The reaction was terminated by freezing of the cell pellet (BMMC and P815) or the Petri dishes (RBL-2H3) in liquid nitrogen.
Overexpression of GTP Cyclohydrolase I in RBL-2H3 Cells-- An EcoRI/HindIII fragment of pNCO-GTP (25, 29), which comprises the entire rat GTP cyclohydrolase cDNA sequence, had been cloned into the pSBC1 vector (30), yielding pSBC1-GTP. The construct was cotransfected with pUHD15-1 (30), coding for the rtTA transactivator and a green fluorescent protein expressing vector (CLONTECH). The transfection was carried out according to the method of Alber et al. (31). Transfection efficiency was up to 50% as monitored by green fluorescent protein fluorescence.
Radiolabeling of Cells-- KL-induced BMMC (1 × 108 cells) were washed three times and then labeled with 32Pi (370 kBq/ml) in phosphate- and serum-free Dulbecco's modified Eagle's medium) for 2 h at 37 °C. After the addition of DNP-specific IgE (1 µg/ml), the incubation was continued for 1 h. The cells were centrifuged and resuspended in 10 ml of phosphate- and serum-free Dulbecco's modified Eagle's medium. Aliquots of 1.5 ml were activated with DNP-hemocyanin conjugate as described above for the indicated times. Identical numbers of RBL-2H3 cells were seeded in 60-mm plates, grown 36 h, and then treated as the BMMCs.
Cell Solubilization, Immunoprecipitation, and Immunoblotting-- Cells (0.8-1 × 107) were lysed, and GTP cyclohydrolase I was immunoprecipitated by MGTP-6B6 mAb and coupled with CNBr-activated Sepharose according to the manufacturer's instructions. The procedures of lysis and precipitation were essentially the same as described previously (32). Depending on the experiment, preincubations and precipitations with control antibodies were performed and are indicated in the legend. After solubilization of the immunoprecipitates, equal amounts of protein were resolved by 10% SDS-PAGE under reducing conditions and immediately transferred to nitrocellulose membranes. Membranes were blocked, probed with the biotinylated MGT-6H11 mAb, and visualized using peroxidase-conjugated streptavidin and enhanced chemiluminescence. 32Pi labeling of GTP cyclohydrolase I was documented by exposure of the blotting membrane to a Kodak X-Omat AR film. Relative quantitative estimations of radioactivity were performed with Fuji BAS1000 phosphoimaging. Nonlabeled extracts were blotted and then probed with purified MGTP-6H11 mAb, followed by second stage peroxidase-conjugated anti-rat IgG and ECL detection.
In Vitro Phosphorylation of GTP Cyclohydrolase I in Immune
Complex Kinase Assays--
Recombinant rat GTP cyclohydrolase I from
overexpressing E. coli and RBL-2H3 cells was
immunoprecipitated using Sepharose-coupled MGTP-6B6 mAb. The bead-bound
GTP cyclohydrolase I immune complexes were washed 2 times with lysis
buffer, resuspended in kinase buffer (33), and washed 3 times with 500 µl of the same buffer. The kinase reaction was performed for 30 min
at 30 °C essentially as described (33). Recombinant PKC isoform ,
, and
purified from baculo virus-infected insect cells (34) were
adjusted to equal activity using myelin basic protein as a substrate.
The reactions were stopped by the addition of SDS sample buffer, and the proteins were resolved on a 10% SDS-PAGE and blotted on
nitrocellulose membranes. The labeled substrates were visualized by
autoradiography after probing with MGTP-6H11 antibody as described
above.
Extraction of the Cells and HPLC Determination of Neopterin and Biopterin-- The cells were homogenized in Tris buffer (50 mM, pH 8.0) containing 2.5 mM EDTA. Aliquots of the centrifuged extracts (15 min at 14 000 × g) were used for determination of pterins and enzyme assays. H2neopterin and H4biopterin were determined after acidic iodine oxidation of the reduced forms. Deproteinization by trichloroacetic acid, prepurificaion by cation exchange chromatography, separation by reverse-phase HPLC, and fluorometric detection have been described previously (21). The modifications of the method were the same as detailed in (3, 21-24).
Enzyme Assays-- The activity of GTP cyclohydrolase I was determined in aliquots of the cell extract prepared as described above. The reaction product was oxidized to neopterin triphosphate by acidic iodine solution. After reduction of excess iodine by ascorbic acid, the sample was immediately separated by ion pair reverse-phase HPLC. The detailed assay conditions have been described previously (23, 24). For further verification, aliquots of the oxidized sample were neutralized and dephosphorylated by alkaline phosphatase (0.8 units/200 µl). Neopterin was then determined by reverse-phase HPLC as described above. For determination of 6-pyruvoyl-H4pterin synthase, dihydroneopterin triphosphate was first generated by incubation of GTP with recombinant murine GTP cyclohydrolase I. To determine the activity of the synthase, the cell extract was added, and the metastable intermediate 6-pyruvoyl-H4pterin was converted to H4biopterin by the addition of recombinant murine sepiapterin reductase and NADPH. After acidic iodine oxidation, biopterin was separated by HPLC. Detailed assay conditions have been described in Gütlich et al. (35). The activity of sepiapterin reductase was determined by reduction of sepiapterin to dihydrobiopterin with NADPH, acidic iodine oxidation, and HPLC separation of the product. The detailed conditions have been described in Kerler et al. (23, 24). Protein was estimated by the Coomassie dye binding reagent (Bio-Rad protein assay reagent) according to the manufacturer's instruction.
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RESULTS |
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Expression of GTP Cyclohydrolase I-- Cultivation of BMMC in the presence of KL increases the activity of GTP cyclohydrolase I more than 6-fold. Enzyme activities and H4biopterin production culminate after 40 h as described earlier (7). The cellular expression levels of the enzyme in extracts of interleukin 3-grown cells and of cells exposed to KL for 40 h were compared by immunoblotting. The blots were probed with either mAb MGTP-6H11 (Fig. 1) or mAb MGTP-6B6 (data not shown). The comparison with recombinant murine GTP cyclohydrolase I confirmed the specificity of both mAbs; the blots showed that cultivation with KL markedly enhances the expression of the GTP cyclohydrolase I (Fig. 1). The experiments, moreover, clearly demonstrate that such high expression levels of GTP cyclohydrolase I make KL-induced BMMC an appropriate tool to investigate regulation at the protein level.
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Modulation of GTP Cyclohydrolase I Activity by FcRI
Engagement--
To follow the activity of GTP cyclohydrolase I in
response to IgE and antigen stimulation of Fc
RI, we used KL-induced
BMMC and the rat tumor cell line RBL-2H3. With BMMC, the activities of
6-pyruvoyl-H4pterin synthase and sepiapterin reductase had earlier been found to largely exceed the activitiy of GTP
cyclohydrolase I (7). In RBL-2H3, the activity of GTP cyclohydrolase I
was much lower than in BMMC (for activity levels, see the Fig.
2 legend). Similarly, in RBL-2H3 cells,
the activities of the two subsequent enzymes in the de novo
pathway of H4biopterin synthesis were 16- to 20-fold higher
than GTP cyclohydrolase I activity (data not shown). This confirms that
GTP cyclohydrolase I represents the rate-limiting step in both BMMC and
the rat mast cell line.
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Hyperphosphorylation of GTP Cyclohydrolase I Mediated by FcRI
Signaling--
The expression of GTP cyclohydrolase I was induced in
BMMC by KL (see Fig. 1), and the cells were metabolically labeled with 32Pi. These cells were IgE-primed and
stimulated with antigen. GTP cyclohydrolase I was immunoprecipitated
from the lysate with the mAb MGTP-6B6. After separation by SDS-PAGE,
probing of the blots with the mAb MGTP-6H11 clearly identified the
major phosphorylated protein as GTP cyclohydrolase I (Fig.
3, A and B). It
initially exists in IgE primed and in unprimed cells in a
phosphorylated form. As expected, no changes in GTP cyclohydrolase I
protein levels occurred, whereas the phosphorylation transiently
increased (Fig. 3, A and C). This
hyperphosphorylation culminated 8-12 min after antigen stimulation. In
addition to GTP cyclohydrolase I, the mAb MGTP-6B6 co-precipitated
phosphorylated proteins that were not further characterized in this
study.
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Modulation of GTP Cyclohydrolase I Activity and Phosphorylation in Transiently Transfected RBL-2H3 Cells Overexpressing the Enzyme-- The result of co-transfecting pSBC1-GTP coding for GTP cyclohydrolase I and pUHD15-1 coding for rtTA transactivator protein was examined by immunoblotting, by determination of GTP cyclohydrolase I activity, and by determination of H4biopterin production by the cells; they demonstrated a 10- to 50-fold increase in all these parameter levels as compared with RBL-2H3.
After priming with IgE and stimulation with antigen, the transiently transfected cells showed a marked increase in GTP cyclohydrolase I activity (Fig. 4A). Likewise to BMMC, the enzyme, immunoprecipitated from cells that had been labeled in vivo with [32P]orthophosphate, was found to be present in the cells as a [32P]-labeled phosphorylated protein (Fig. 4, B and C). This confirms the results showing that GTP cyclohydrolase I initially exists as a phosphorylated enzyme in BMMC. Also, as in BMMC, antigen binding to Fc
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In Vitro Phosphorylation of GTP Cyclohydrolase I by PKC- and
Casein Kinase II--
A Prosite data base search revealed putative
phosphorylation sites for PKC and casein kinase II that are highly
conserved throughout eucaryotic GTP cyclohydrolases I including human,
rat, mouse, chicken, and fish ((38) see also Discussion)). To test the
ability of PKC isozymes and of casein kinase II to phosphorylate GTP
cyclohydrolase I, we carried out in vitro phosphorylation experiments using GTP cyclohydrolase I immunocomplexes as a substrate and the purified PKC isoforms
,
1, and
and casein kinase II. The PKC isoforms used in these experiments were adjusted to equal activity levels using myelin basic protein as a substrate. The substrate, GTP cyclohydrolase I, was overexpressed and
immunoprecipitated either from E. coli or from RBL-2H3
cells. The PKC isoforms
and
1 showed only a minor
phosphorylation capacity, but in contrast, PKC-
, under our
experimental conditions, catalyzed the phosphorylation of GTP
cyclohydrolase I. The potent PKC inhibitor Ro-31-8220 (IC50 = 10 nM (39)) also markedly reduced this in
vitro phosphorylation of the enzyme at concentrations of 10 nM (Fig. 5). Casein kinase II
also phosphorylated GTP cyclohydrolase I with an efficiency comparable
with the autophosphorylation of its own smaller subunit. The casein
kinase II inhibitor
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole, consistent
with its low inhibitory capacity (IC50 = 6 mM
(40, 41)), reduced the phosphorylation of GTP cyclohydrolase I by approximately 50% at concentrations of 10 mM (Fig. 5). The
data from these in vitro phosphorylation experiments
indicate that GTP cyclohydrolase I, expressed by both E. coli and RBL-2H3 cells, is likely to be phosphorylated by both
kinases.
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Enhancement of GTP Cyclohydrolase I Activity and Phosphorylation by
PMA and Inhibition by Ro-31-8220--
The putative involvement of PKC
in the activation of GTP cyclohydrolase I and in its
hyperphosphorylation was further examined by the use of PKC activators
and inhibitors. First, PMA, which mimics the generation of
diacylglycerol (42), effectively modulated GTP cyclohydrolase I
activity in RBL-2H3 cells (Fig.
6A). The kinetics as well as
the amplitude of PMA-induced enhancement was comparable with the
activation initiated by FcRI stimulation, which has been shown in
Fig. 2. Second, in vivo phosphorylation, subsequent
immunoprecipitation of GTP cyclohydrolase I, and separation of the cell
lysate by SDS-PAGE revealed that PMA induces similar kinetics and
extent of enzyme hyperphosphorylation in overexpressing RBL-2H3 cells
(Fig. 6, B and C) as previously found as a
consequence of Fc
RI activation. Finally, the hyperphosphorylation of
GTP cyclohydrolase was completely abolished by the addition of
Ro-31-8220, irrespective of treatment with PMA or by Fc
RI activation
(Fig. 6, B and C).
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DISCUSSION |
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Previous studies showed that KL regulates H4biopterin
synthesis through modulation of GTP cyclohydrolase I activity in BMMC (7). We now confirm that this cytokine-induced increase in activity,
culminating after 40 h, is due to increased levels of GTP
cyclohydrolase I. To the best of our knowledge, we also present for the
first time evidence that GTP cyclohydrolase I is additionally subject
to short term modifications at the post-translational level. By
development of monoclonal antibodies suitable for either immunoblotting
or for immunoprecipitation, respectively, we demonstrate that the
enzyme is present as a phosphorylated protein in the mammalian cell. In
both KL-induced BMMC and in RBL-2H3 cells overexpressing GTP
cyclohydrolase I, the enzyme undergoes additional phosphorylation when
FcRI aggregation has initiated a signal cascade resulting in a
stimulation of serine, threonine, and tyrosine phosphorylation of
cellular proteins. This hyperphosphorylation is transient and culminates after 8 min. Concomitantly, the activity of the enzyme is
modulated in response to Fc
RI aggregation and results in transiently increased cellular H4biopterin levels.
The amino acid sequence of GTP cyclohydrolase I is highly conserved;
the amino acid sequences that are essential for catalysis of the
E. coli enzyme (43) are identical in all of the 15 unrelated species compared (38). Furthermore, the mammalian enzyme possesses identical sequences at the proposed sites for phosphorylation by casein
kinase II ((S/T)XX(D/E) (44)) and by PKC
((S/T)X(R/K) (45). Prosite data base searches have revealed
the conserved casein kinase II sites at positions 14, 51, 82, 103, 131, and 231 and a PKC site at position 167 (numbering is according to mouse
GTP cyclohydrolase I sequence (38)). Under our experimental conditions,
GTP cyclohydrolase I appears to be a substrate for both kinases, and
among the PKC isoforms tested, PKC- is the most effective one.
Quantitative data will have to compare this substrate with classical
substrates for PKC-
and thus unequivocally prove its specificity.
In vitro studies, moreover, do not necessarily identify the
kinase being involved in the basal phosphorylation or the
hyperphosphorylation of GTP cyclohydrolase I under in vivo conditions.
To examine these reactions in vivo, our experiments did not
further investigate the basal phosphorylation but concentrated on the
hyperphosphorylation of GTP cyclohydrolase I, which is clearly part of
the signal cascade initiated by FcRI stimulation. PMA, which
activates PKC in a similar manner to diacylglycerol (42), can mimic the
antigen-triggered phosphorylation of the enzyme. This additional
(hyper)phosphorylation is almost completely abrogated by Ro-31-8220, a
selective PKC inhibitor (39). These data collectively indicate that PKC
is essentially involved in the in vivo process of
antigen-induced phosphorylation of GTP cyclohydrolase I and that the
isoform is involved in vivo. It is well established that
PKC-
is critical to the effector function of the mast cell and that
it associates with the Fc
RI
-chain and phosphorylates Fc
RI
-chain in response to antigen binding (28). In this way, PKC-
may
interact with p53/56lyn (19) and become
tyrosine-phosphorylated (18). The phosphorylation of PKC-
decreases
the activity toward the receptor
chain and modifies its specificity
to include new substrates (18). Our results are consistent with the
view that GTP cyclohydrolase I may represent one of these new
substrates.
Further studies are needed to verify the sites and pathways for the antigen-induced hyperphosphorylation of GTP cyclohydrolase I and to consider possible associations with other phosphorylated proteins that coprecipitated with GTP cyclohydrolase I in primary BMMC under our experimental conditions. The rapid reversal of hyperphosphorylation indicates that protein phosphatases are also involved that need to be identified as further important components in the process of GTP cyclohydrolase I modification.
The time course of GTP cyclohydrolase I hyperphosphorylation correlates closely with modulation of its activity. Similar to other cellular systems (3), this first enzyme specific to H4biopterin synthesis proved to be the rate-limiting step in mast cells, and consequently, cellular H4biopterin levels increased upon their antigen stimulation. Since these fluctuations in cellular H4biopterin levels take place around the Km value of tryptophan 5-monooxygenase for its cofactor, they occur in the most effective range. The rapid decline of the pterin levels following 8 min of stimulation (Fig. 2) appears to be caused by enzyme-catalyzed degradation rather than by secretion and/or auto-oxidation. Neither in the cells nor in the culture medium could the the typical products of H4biopterin auto-oxidation such as 6-carboxypterin (46) or 6-hydroxymethylpterin, be detected (data not shown). Pterin deaminase (EC 3.5.4.11), converting pterins to the corresponding lumazines, has been partially purified from both bacterial (47) and mammalian (48) sources. The activity of this enzyme is controlled by cAMP in Dictyostelium (49). However, this candidate enzyme for the additional control of H4biopterin levels during allergic mast cell response is currently only poorly characterized. In conclusion, these observations on post-translational modification of GTP cyclohydrolase I advance our knowledge of the short term regulation of H4biopterin production, but there is still much to be done to elucidate the inter-relationships existing between all the components of the system and their functional role and importance for the coupling of the biosynthesis, degradation, and release of serotonin or catecholamines in living cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Harald Mischak (Berlin) for the generous gift of PKC isozymes. We gratefully appreciate stimulating discussions and suggestions by Drs. Juan Rivera (Bethesda) and Walter Koelch (Glasgow). We thank Beatrix Scheffer for her participation in making the pSCB1-GTP expression vector, Hannelore Broszeit in the cultivation of BMMC, and Ursula Meincken and Ursula Ehrler in the development of antibodies and Lutz Weidner in the analytical work.
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FOOTNOTES |
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* The work was supported by Bundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie Grant 01GE9608.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dedicated to Professor Lothar Jaenicke, Köln, on occasion of his 75th birthday.
§ To whom correspondence should be addressed: GSF-Institut für Klinische Molekularbiologie und Tumorgenetik, Marchioninistr. 25, D-81377 München, Germany. Tel.: 49 89 7099 222; Fax: 49 89 7099 500; E-mail: hesslinger{at}gsf.de.
The abbreviations used are:
H4biopterin, 5,6,7,8-tetrahydrobiopterin6-pyruvoyl-H4pterin, (6R)-(1'2'-dioxopropyl)-5,6,7,8-tetrahydropterinbiopterin, L-erythro-1',2'-dihydroxypropylpterinBMMC, bone marrow-derived mast cells6-carboxypterin, 2-amino-4-hydroxypteridine-6-carboxylic acid6-hydroxymethylpterin, 2-amino-4-hydroxy-6-(hydroxymethyl) pteridineKL, kit ligandPMA, phorbol 12-myristate 13-acetateDNP, dinitrophenolFcR1, high
affinity receptor for IgEmAb, monoclonal antibodyPKC, protein
kinase CPAGE, polyacrylamide gel electrophoresisHPLC, high
performance liquid chromatographyMGTP, murine GTP
cyclohydrolase.
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REFERENCES |
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