Proteasome-driven turnover of tryptophan hydroxylase is triggered
by phosphorylation in RBL2H3 cells, a serotonin producing
mast cell line
Yoshiko Iida
1
, Keiko Sawabe
1
, Masayo Kojima
1
, Kazuya Oguro
1,2
, Nobuo Nakanishi
3
and
Hiroyuki Hasegawa
1,2
1
Department of Bioscience, and
2
Biotechnology Research Center, Teikyo University of Science and Technology, Yamanashi, Japan;
3
Departments of Biochemistry, Meikai University School of Dentistry, Sakado, Saitama, Japan
We previously demonstrated in mast cell lines RBL2H3 and
FMA3 that tryptophan hydroxylase (TPH) undergoes very
fast turnover driven by 26S-proteasomes [Kojima, M.,
Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A.,
Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo)
2000, 127, 121–127]. In the present study, we have examined
an involvement of TPH phosphorylation in the rapid turn-
over, using non-neural TPH. The proteasome-driven deg-
radation of TPH in living cells was accelerated by okadaic

Tryptophan hydroxylase (TPH, EC 1.14.16.4), a member of
a family of pterin-dependent aromatic amino acid hydroxy-
lases [1], catalyzes the conversion of
L
-tryptophan to
5-hydroxy-
L
-tryptophan. This reaction is the initial and
rate-limiting step in the biosynthesis of serotonin [2–5]. TPH
has been extensively purified from various sources such as
bovine pineal gland [6], mouse mastocytoma [7,8], and
mammalian brains [9–11]. Physicochemical, enzymic and
immunochemical properties differed between TPHs of
neural and non-neural tissue origin, and it is accepted that
neural TPH might be a different entity from the non-neural
enzyme [8,10,12,13]. Complimentary DNAs of TPH have
been cloned from various sources but no differences or only
trivial variation in amino acid sequences were found among
them [14–19]. The molecular basis of differences between the
neural and non-neural enzymes has not yet been explained.
Both types of cytosolic environment should be studied
further to detect differences in the control of gene expres-
sion, post-translational modification, and turnover of the
enzyme protein in a tissue-specific way.
We have demonstrated with RBL2H3, an established cell
line that expresses TPH in culture while retaining many of
the characteristics of mast cells, that: (a) cellular TPH
activity was seriously limited by insufficient supply with the
enzyme’s essential cofactor, ferrous iron, and the substrates
tryptophan and 6R-tetrahydrobiopterin [20]; (b) immune

(Received 10 March 2002, revised 11 June 2002,
accepted 19 August 2002)
Eur. J. Biochem. 269, 4780–4788 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03188.x
of TPH, a specific tag might be required for targeting by the
ligase. In many cases, phosphorylation of the target protein
provides the tag for the ubiquitinylation system, especially
of such families as the SCF-complex, Skp1/Cullin-1/F-box
protein (reviewed in [24,25]). Involvement of phosphoryla-
tion in TPH degradation was expected, however, phos-
phorylation of non-neural TPH has never been
demonstrated, although TPH of brain origin and recom-
binant TPH have been known to be phosphorylated by
PKA and by CaM kinase II [26–28]. On the other hand,
proteasome-driven turnover has only been demonstrated
with mast cell lines. The aim of this work was to elucidate
whether the phosphorylation of non-neural TPH takes
place and, if it does, whether it provides the tag for targeting
by the proteasomes involved in the rapid turnover of the
enzyme.
MATERIALS AND METHODS
Materials
MG132 (carbobenzoxy-Leu-Leu-Leu-H) and E-64-d [(
L
-
3-trans-ethoxycarbonyloxirane-2-carbonyl)-
L
-leucine(3-meth-
ylbutyl)amide] were purchased from Peptide Institute
(Osaka), lactacystin from Kyowa Medex (Tokyo), okadaic
acid and K252a from Alomone Labs (Jerusalem, Israel),

(500 mCiÆmL
)1
)and[c-
32
P]ATP (tetra-
triethylammonium salt; 4500 CiÆmmol
)1
) were purchased
from ICN Biochemicals.
Cell culture
RBL2H3, a mast cell line derived from rat basophilic
leukemia cells, was obtained from The Japanese Cancer
Research Resources Bank (Tokyo). RBL2H3 cells and
FMA3 (Furth’s mastocytoma) cells were maintained as
described [23]. One day before experiments, cells were plated
to well of a 96-well culture plate (Falcon, Cat. No. 35072) at
1 · 10
5
cells per well. Two hours before the experimental
treatment, cells were placed in serum-free medium buffered
with 25 m
M
Hepes/NaOH containing 100 UÆmL
)1
of
penicillin and 100 lgÆmL
)1
of streptomycin, then kept at
37 °C under 10% CO
2

4
)
2
,
and 4 mgÆmL
)1
catalase in a total volume of 100 lL.
Subsequently, 50 lL of another cocktail were added to
afford a final reaction mixture of 250 l
M
tryptophan,
400 l
M
6R-tetrahydrobiopterin, 500 l
M
NADH, 1 m
M
NSD-1015, 2 mgÆmL
)1
catalase, and 50 lgÆmL
)1
dihydrop-
teridine reductase in 0.1
M
potassium phosphate buffer
( pH 6.9). The enzyme reaction was allowed to proceed for
10 min at 30 °C, then was terminated by 1
M
perchloric
acid. The 5HTP formed was measured using an HPLC

2
,1m
M
CaCl
2
,2m
M
ATP,
10 lgÆmL
)1
creatine kinase, 10 m
M
phosphocreatine,
0.2 mgÆmL
)1
catalase, and 1 m
M
dithiothreitol in 50 m
M
Tris/HCl (pH 8.0). Purified TPH from P-815 cells with or
without in vitro phosphorylation was used as the sub-
strate. Inhibitors of proteasomes and protein kinases were
added prior to addition of the substrate TPH. Aliquots
were taken after various intervals of incubation (30 °C) for
the TPH enzyme activity assay and for Western blot
analysis.
Phosphorylation of TPH
In situ phosphorylation of TPH in FMA3 cells was
performed as follows. Cells (2 · 10
6

methanesulfonyl fluoride, 2 m
M
EDTA, 50 m
M
sodium
fluoride, and 1 m
M
sodium orthovanadate). The cell lysates
were mixed with anti-TPH serum (10 lL) and left overnight
at 4 °C with agitation. Total IgG was collected by the
addition of staphylococcal ghosts (Pansorbin; Calbiochem,
La Jolla, CA, USA) as a precipitant, solubilized in 1% SDS,
and subjected to SDS/PAGE. Cell-free phosphorylation by
PKA was carried out for 30 min at 37 °Cinareaction
mixture containing 3 lg of purified TPH as substrate, or rat
liver phenylalanine hydroxylase for comparison, 1 lgPKA
catalytic subunit and 2 lCi [c-
32
P]ATP in 50 m
M
Tris/HCl
( pH 7.4) containing 20 l
M
ATP and 10 m
M
MgCl
2
in a
total volume of 210 lL. For SDS/PAGE, proteins were
precipitated by the addition of trichloroacetic acid (5%) in

4
-Affigel-10 [8] for collecting TPH in the
presence of the inhibitor cocktail as above and 150 m
M
NaCl in 50 m
M
Tris/acetate (pH 8.0), then left overnight at
4 °C with agitation. The proteins obtained were subjected to
SDS/PAGE followed by immunoblotting and autoradio-
graphy.
SDS/PAGE, Western blot analysis, and autoradiography
Monolayer cultures washed with NaCl/P
i
or proteins
collected as a pellet as described above were solubilized in
1% SDS and subjected to SDS/PAGE according to
Laemmli [31]. Western blot analysis was performed as
described previously [23]. The protein signal was visualized
using an enhanced chemiluminescence detection system
(ECL; Amersham, Buckinghamshire, England). Protein
bands were exposed to an X-ray film (Konica, Medical Film
20287). For autoradiography with
32
P, gels following SDS/
PAGE were dried on filter paper, then subjected to exposure
either to an X-ray film (Konica) at )80 °Cfor3dayswith
an intensifying screen (Kodak, Bio Max MS) or to a fluoro-
image analyser (Fujifilm, FLA-3000) using an imaging plate
(Fujifilm, BSA-IP MS2040). Graphic images of Western
blot analysis or autoradiograms were analyzed using

and lactacystin but was not affected by a cystein protease
inhibitor, E-64-d; a representative finding showing that
the steady state level of TPH was determined by a
proteasome-driven degradation process. TPH degradation
in the cells was accelerated by okadaic acid (0.25 l
M
): the
half-life of TPH (T
1/2
) were estimated to be 29 min and
38 min in the presence and absence of okadaic acid,
respectively (Fig. 1A, OA), suggesting an involvement of
TPH phosphorylation in recognition of the enzyme by the
ubiquitinylation system. Based on this observation, we
examined whether TPH was phosphorylated in situ using
FMA3 cells in which cytosolic TPH was also rapidly
degradated by the proteasome-driven process while the
steady state TPH level was roughly 20-fold higher than
that of RBL2H3 cells [22,23]. Cellular proteins were
labeled by incubating the cells with [
32
P]orthophosphate,
and steady state phosphorylation levels of proteins were
performed in the presence and absence of okadaic acid
and/or MG132. By Western blot analysis of the whole
cell extracts, TPH of molecular mass 53 kDa was locali-
zed side-by-side with purified TPH and the anti-TPH
serum (Fig. 1B, WB). Addition of both okadaic acid and
MG132 caused the immunoreactive band to be twofold
thicker than the control band (see plot profiles of WB,

P-
incorporated protein with a molecular mass of 53 kDa
4782 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002
was obtained (Fig. 1B,
32
P, lane 4) and was coincident
with the TPH visualized by Western blot analysis
(Fig. 1B, WB, lane 3 and 4). This operation visualizes
32
P-incorporation into the specific proteins which were
protected from proteasome-driven digestion by MG132
among those
32
P-phosphorylated and protected from
dephosphorylation by okadaic acid, proteins which oth-
erwise would have been digested by proteasomes. Thus
the phosphorylated form of TPH was detectable only
when the proteasome action and phosphatase were
effectively blocked (lane 3 in both Fig. 1B,
32
P and
WB). Together with the fact that the blocking of protein
phosphatase by okadaic acid resulted in the acceleration
of TPH degradation (Fig. 1A, OA), the present result is
evidence that phosphorylation takes place on this protein
where it functions as the tag for the targeting of TPH by
the proteasomes. It was noteworthy that TPH detectable
under steady state conditions was unphosphorylated,
presumably because the phosphorylated TPH might have
been digested away in the absence of proteasome

)1
cycloheximide were added to each well and the culture
continued. TPH activities at 0, 10, 30, and 60 min after addition of cycloheximide were measured. Inhibitors used were: 10 l
M
MG132 (A-MG132),
30 l
M
lactacystin (A-Lact), 10 l
M
E-64-d (A-E64d), and 0.25 l
M
okadaic acid (A-OA). Values are means ± SD (n ¼ 4). (B) FMA3 cells (2 · 10
6
cellsÆmL
)1
) were placed in phosphate-free RPMI 1640 medium supplemented with 5 l
M
NaH
2
PO
4
for 1.5 h, and were exposed to 0.4 mCiÆmL
)1
32
P-phosphate. After 30 min exposure, the cells were further treated with okadaic acid and/or MG132 for the next 2 h. The cells were then disrupted
with 1% NP-40 in 50 m
M
Tris/HCl (pH 7.8) containing the protease inhibitor cocktail. (
32
P): In order to examine

tions higher than 100 l
M
, the inhibitor showed no further
effect (not shown). K252a and K252b (both 50 l
M
), protein
kinase inhibitors with relatively broad specificity, were also
effective as tested (Fig. 2B, K252a), but H-89 was not
significantly effective at 50 l
M
(Fig. 2B, H89). Staurospo-
rine and chelerythrine, potent inhibitors of protein kinase C,
were not significantly effective (not shown). These results
indicated that TPH digestion by proteasomes involved
phosphorylation of certain protein(s) as an essential step.
Taken together with the outcome of the experiment in
Fig. 1, it is likely that the TPH molecule was the protein to
be phosphorylated in the selective degradation. Further-
more, from the specificity of the protein kinase inhibitors
tested above, CaM kinase II seemed to function in the cell
extracts, at least in part.
Stimulation of TPH degradation in the cell free
proteasome system by the phosphorylation
of the enzyme by CaM kinase II
As described above (Fig. 1), the phosphorylation of non-
neuronal TPH was suggested for the first time using
RBL2H3 and FMA3 cells. We then examined TPH
phosphorylation in vitro in order to determine the type of
protein kinase responsible. TPH from brain is known to be
phosphorylated by PKA [26,28] and CaM kinase II [27].

32
P panel of Fig. 3A) was judged to be from contami-
nation of the PKA preparation because this band was not
detectable with Western blot analysis (WB in Fig. 3B) and
was seen with CBB staining only when PKA was added
(CBB panel in Fig. 3B; note that PKA alone had poor
recovery through trichloroacetic acid precipitation and
diethylether washing to remove trichloroacetic acid for
sampling). These results indicated that TPH protein incor-
porated virtually no
32
P or far less than the stoichiometric
amount of
32
P.
On the other hand, TPH was clearly
32
P-labeled by CaM
kinase II in vitro in the presence of both Ca
2+
and
calmodulin (Fig. 4). Besides TPH (molecular mass of
53 kDa), two diffuse bands appeared on autoradiography
(Fig. 4A, lanes 2 and 3). These were thought to be due to
autophosphorylation of CaM kinase II proteins of 54 kDa
and 63 kDa described by Yamauchi & Fujisawa [34]. This
Fig. 2. Inhibitory effect of protein kinase inhibitors on degradation of purified TPH in the cell-free proteasome system. TPH (1 lg) purified from
mastocytoma P-815 cells was subjected to a cell-free proteasome system composed of freshly prepared extracts (400 lg protein). The reaction
mixture (total volume of 210 lL) was incubated at 30 °C, and aliquots were taken at the indicated times for the TPH activity assay (A) and for
Western blot analysis (B). TPH activity was measured as described in Materials and methods after appropriate dilution (200-fold). Preparation of

We examined the effect of TPH phosphorylation by CaM
kinase II on the susceptibility of the enzyme to degradation
in our cell-free proteasome system. As shown in Fig. 4B,
degradation of TPH phosphorylated by CaM kinase II
prior to the proteasome reaction was much more rapid than
that of the nonphosphorylated TPH. This is presumably
because the nonphosphorylated TPH had to undergo prior
phosphorylation in situ to be targeted by the proteasomes in
the reconstituted cell-free system.
DISCUSSION
In the present study, using RBL2H3 and FMA3 cells as
representative non-neural cells, we examined whether TPH
is actually phosphorylated and whether phosphorylation is
the prerequisite step in the proteasome-driven TPH degra-
dation process. We presented evidence that: (a) TPH in
FMA3 cells was phosphorylated in vivo; (b) TPH purified
from mastocytoma P-815 cells was also phosphorylated
in vitro by CaM kinase II but not by PKA; (c) TPH thus
phosphorylated was degraded in vitro at a higher rate than
was the nonphosphorylated TPH; and (d) living RBL2H3
cells are furnished with a whole proteasome system inclu-
ding 26S-proteasomes and a specific ubiquitinylation system
that recognizes phosphorylated TPH. As to non-neural
TPH, rat pineal enzyme was reported to increase in enzymic
activity by treatment with cAMP [35]. The authors,
Fig. 3. Insignificant incorporation of
32
P into TPH by protein kinase A. TPH or rat liver phenylalanine hydroxylase (PAH), 3 lg each, were exposed
to 1 lg PKA-catalytic subunit in the presence of 2 lCi [c-
32

Ó FEBS 2002 TPH phosphorylation as proteasome targeting (Eur. J. Biochem. 269) 4785
however, did not observe phosphorylation of pineal TPH
protein, though they described PKA-dependent phosphory-
lation of brain TPH. The pineal gland is anatomically
classified as being outside the central nervous system and the
enzymic properties of pineal TPH were obviously peripheral
nature in every aspect we examined [6,36]. Careful investi-
gation of TPH from mouse mastocytoma P-815 failed to
uncover any activation of enzyme activity by cAMP-
dependent protein kinase action [37]. Thus far, no clear
evidence has appeared for phosphorylation of non-neural
TPH, including that of pineal or neoplastic mastocytoma
cells.
Evidence of TPH phosphorylation in living cells
We first observed that okadaic acid, a protein phosphatase
inhibitor, accelerated the degradation of the enzyme, which
was already quite rapid (Fig. 1A, OA). This fact suggested
that phosphorylation of certain proteins stimulates their
degradation. TPH was the protein most likely to be
phosphorylated, however, this meant that phosphorylated
TPH would be hardly detected unless the proteasome action
was blocked. Indeed, in the absence of proteasome
inhibitor, phosphorylated TPH was not detectable in the
steady state labeling experiment where numerous cellular
proteins were
32
P-labeled as shown in Fig. 1B,
32
P.Evenin
the presence of either okadaic acid (lane 2) or MG132 (not

sequently ubiquitinylated TPH, the presumable substrate of
the proteasomes. The experimental result was that MG132
administered to living cells somehow raised TPH activity
and increased the amount of TPH-like protein of molecular
mass of 53 kDa (Fig. 1A, MG132 and 1B, WB lane 3),
suggesting that de-ubiquinylating enzyme is considerably
active [23]. Based on this outcome, phosphorylation of non-
neural TPH and its role as an essential tag for protein
degradation were explored.
Phosphorylation of non-neural TPH
in vitro
Purified TPH from mastocytoma P-815 cells, i.e. TPH of
non-neural origin, was demonstrated to be phosphorylated
by CaM kinase II in vitro (Fig. 4). Although neural TPH
and recombinant enzymes were reportedly phosphorylated
by PKA (reviewed in [38]), in the present study, we could
not obtain positive evidence for PKA phosphorylation of
the TPH from P-815 (Fig. 3). A possibility remains that our
TPH preparation was fully phosphorylated at the PKA-
specific phosphorylation site before isolation and therefore
left no room for further phosphorylation. Indeed, phenyl-
alanine hydroxylase purified from rat liver contained
endogenously phosphorylated subunits with 1.3 mol of
phosphate per tetramer and was fully phosphorylated
in vitro to give 1 mol per subunit by the catalytic subunit
of PKA [39]. This possibility is difficult to rule out before
direct measurement of endogenous phosphate [40]. The
present observation that TPH protein incorporated
32
P-

sucrose to
minimize the dissociation of proteasomes and possible
disruption of lysosomes [41]. Based on the sensitivity to
protein kinase inhibitors and to proteasome inhibitors, it is
obvious that the cell-free proteasome system per se included
a relevant protein kinase system for TPH. Although the
endogenous protein kinase shares properties with CaM
kinase II in terms of sensitivity to inhibitors, the presence of
multiple kinase species was also possible since KN-62 alone
did not completely prevent the proteolysis, even at concen-
trations higher than 100 l
M
, while K252a of broad
specificity did. It was noteworthy that H-89, chelerythrine
and staurospoline were not effective in preventing TPH
digestion, suggesting little contribution from PKA or PKC
as far as the proteasome system in the RBL2H3 cells was
concerned.
Our observations supported the idea that phosphoryla-
tion is a prerequisite for proteasome digestion of non-
neural TPH. This phosphorylation enables the cell to
severely down-regulate the enzyme level by means of
stimulation of proteasome-driven degradation. This is a
new role for TPH phosphorylation, which does not
necessarily alter its enzyme activity. It will be interesting to
learn whether the phosphorylation of neural TPH also has
4786 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002
an association with proteasome-dependent degradation,
but there is as yet little information about TPH turnover
in the central nervous system. In addition, the question

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