Báo cáo Y học: Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line pptx

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

acid, a protein phosphatase inhibitor. Incorporation of
32
P
into a 53-kDa protein, which was judged to be TPH based on
autoradiography and Western blot analysis using anti-TPH
serum and purified TPH as the size marker, was observed in
FMA3 cells only in the presence of both okadaic acid and
MG132, inhibitors of protein phosphatase and proteasome,
respectively. In a cell-free proteasome system constituted
mainly of RBL2H3 cell extracts, degradation of exogenous
TPH isolated from mastocytoma P-815 cells was inhibited
by protein kinase inhibitors KN-62 and K252a but not by
H89. Consistent with the inhibitor specificity, the same TPH
was phosphorylated by exogenous Ca
2+
/calmodulin-
dependent protein kinase II in the presence of Ca
2+
and
calmodulin but not by protein kinase A (catalytic subunit).
TPH protein thus phosphorylated by Ca
2+
/calmodulin-
dependent protein kinase II was digested more rapidly in the
cell-free proteasome system than was the nonphosphoryl-
ated enzyme. These results indicated that the phosphoryla-
tion of TPH was a prerequisite for proteasome-driven TPH
degradation.
Keywords: tetrahydrobiopterin; CaM kinase II; proteasome
target; ubiquitin ligase; enzyme turnover.

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

stimulation lead to a marked increase in TPH level by
means of enhanced expression of the TPH gene [21]; and
(c) the steady state TPH level of this cell was maintained
at extremely low levels by rapid degradation of the enzyme
(T
1/2
, 15–60 min) [22,23]. In the latter report, the turnover of
TPH protein was shown to be driven by ATP-dependent
action of 26S-proteasomes including, at least in part,
ubiquitinylation of TPH. Furthermore, it was noted that
this rapid turnover was suppressed by a protein kinase
inhibitor. Since proteasomes might, in general, be ubiquit-
ous in the cell, recognition of the specific target is crucial in
terms of the specific protein to be digested. Poly-ubiquiti-
nylation represents a major tag for proteasomes. The
ubiquitinylation of a specific protein is determined by
the ubiquitin ligase complex E3. The molecular basis of the
structure–function relationship enabling E3 to specifically
recognize a wide variety of substrates is one of the major
subjects of investigation in this field. In the ubiquitinylation
Correspondence to H. Hasegawa, Department of Bioscience,
Teikyo University of Science and Technology, Uenohara,
Yamanashi 409–0193, Japan. Fax: + 81 554 63 4431,
E-mail:
Abbreviations: TPH, tryptophan hydroxylase; CaM kinase II,
calcium/calmodulin-dependent protein kinase II; PKA, cyclic
AMP-dependent protein kinase; 5HTP, 5-hydroxy-
L
-tryptophan.
Enzyme: Tryptophan hydroxylase (EC 1.14.16.4).

(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),

and KN-62 from LC Laboratories (La
¨
ufelfingen, Switzer-
land). Cycloheximide, creatine kinase, cyclic AMP-depend-
ent protein kinase catalytic subunit from beef heart (Cat.
No. P2645), rat liver phenylalanine hydroxylase (Cat. No.
P6268), phosphocreatine, and sodium orthovanadate were
purchased from Sigma. Sodium fluoride was obtained from
Nacalai Tesque (Kyoto, Japan). The concentrations of
inhibitors used in this study, MG132, E-64-d, lactacystin,
okadaic acid, K252a, K252b, KN-62, and cycloheximide,
were those that gave the maximum effect on evaluation.
TPH was purified from P-815, a mouse mastocytoma,
essentially according to Nakata and Fujisawa [8]. Rabbit
polyclonal anti-TPH serum was raised against the purified
TPH [13]. Bovine liver dihydropteridine reductase was
purified up to the second ammonium sulfate fractionation
step [29]. (6R)-
L
-erythro-5,6,7,8-Tetrahydrobiopterin was
donated by Suntory (Tokyo, Japan). CaM kinase II and
calmodulin, both purified from rat brain, were donated by
T. Yamauchi (Department of Biochemistry, Faculty of
Pharmaceutical Science, The University of Tokushima,
Japan). [
32
P]H
3
PO
4

(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

/90% air throughout the experiments
except at the time of manipulation. Agents of low
solubility in water were dissolved in dimethylsulfoxide at
a concentration 100-fold greater than final one used, unless
otherwise stated, so that dimethylsulfoxide would be at an
equivalent level in each experimental culture with no
vehicle effect.
Tryptophan hydroxylase assay
TPH activity was determined essentially as described
previously [13,23]. Cells in monolayer culture in wells of
the96-wellplatewereplacedin20lLofNaCl/P
i
(–), then
subjected twice to freezing in liquid nitrogen and thawing in
water. Reaction mixtures for the cell-free treatment of
purified TPH (phosphorylation and proteolysis as described
below) were prepared just prior to measuring the enzyme
activity. The disrupted cells or TPH mixture were preincu-
bated for 15 min at 30 °Cin0.1
M
Tris/HCl ( pH 8.0)
containing 30 m
M
dithiothreitol, 50 l
M
Fe(NH
4
)
2
(SO

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

system equipped with a fluorescence monitor (JASCO
model, FP920) set at 302 nm and 350 nm for excitation and
emission, respectively. The solid phase was ODS
(4.6 · 250 mm, JASCO, Finepak SIL-C18T5), the mobile
phase was a 90 : 7 : 5 mixture of 40 m
M
sodium acetate
(adjusted to pH 3.5 with formic acid), acetonitrile and
methanol and the flow rate was 1 mLÆmin
)1
[30].
Cell-free proteolysis of TPH
Extracts from RBL2H3 cells as the source of proteasomes
were prepared essentially as described [23]. The cells were
homogenized in 5 volumes of 50 m
M
Tris/HCl (pH 7.5)
containing 1 m
M
dithiothreitol, 2 m
M
ATP, and 0.25
M
sucrose using an Ultra-disperser (model T25; IKA Labor-
technik, Staufen, Germany). The homogenate was centri-
fuged at 18 000 g for 5 min. In vitro proteolysis was
performed in a reaction mixture containing the RBL2H3
cell extracts, 5 m
M
MgCl

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

cells) were adapted to
phosphate-free RPMI 1640 (Gibco, Cat. No. 11877–032)
supplemented with 5 l
M
NaH
2
PO
4
for 90 min.
Ó FEBS 2002 TPH phosphorylation as proteasome targeting (Eur. J. Biochem. 269) 4781
Subsequently, cells were fed 0.4 mCiÆmL
)1
[
32
P]NaH
2
PO
4
for 30 min.
32
P-Loading was further continued for 120 min
in the presence of protein-kinase inhibitors or protein-
phosphatase inhibitors. Cells were then rinsed with NaCl/P
i
and disrupted with 1% NP-40 in 50 m
M
Tris/HCl
( pH 7.8) containing an inhibitor cocktail (1 m
M
phenyl-

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

the cold and centrifuged. The pellets were then washed twice
with 400 lL of diethylether, dried and dissolved in 50 lLof
the lysis buffer for SDS/PAGE. Ca
2+
/calmodulin-depend-
ent phosphorylation was carried out with 1 lgTPHas
substrate and 0.1 lg CaM kinase II for 30 min at 37 °Cin
the presence of 0.1 l
M
calmodulin, in 210 lLof50m
M
Tris/HCl ( pH 7.4) containing 10 l
M
ATP (2 lCi
[c-
32
P]ATP), 5 m
M
MgCl
2
,120l
M
CaCl
2
,and100l
M
EGTA. Aliquots were taken for the assay of TPH activity
or for subjecting to the cell-free proteolysis described above.
The remaining reaction mixture was mixed with affinity gel
beads DMPH

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

NIH
IMAGE
ver 1.62 software, Wayne Rasband, National
Institute of Health, USA.
Other methods
Proteins were determined by Bradford’s method [32] using
bovine serum albumin as the standard. Data were expressed
as means ± SD (n ¼ 4) unless otherwise stated.
RESULTS
Involvement of protein phosphorylation in TPH
degradation in living cells
In previous works [22,23], we demonstrated in mast cell
lines RBL2H3 and FMA3 that de novo biosynthesis of
TPH enzyme protein was accompanied by rapid degra-
dation with 26S-proteasomes and that ubiquitinylation of
TPH protein was involved in the process, presumably by
providing the targeting tag. In search for any connection
between protein phosphorylation and TPH turnover, we
examined the effect of okadaic acid, a protein phospha-
tase inhibitor, on TPH degradation in the living cell
system and compared it with those of protease inhibitors
(Fig. 1). When protein synthesis was arrested by cycloh-
eximide (10 lgÆmL
)1
), a rapid decrease in TPH activity
was observed in the absence of the inhibitors (T
1/2
:
around 30 min, Fig. 1A). This decrease was much slower
or was virtually stopped by proteasome inhibitors MG132

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,

right-most patterns), however, no discrete bands of
32
P-incorporation were recognized over the dense back-
ground by autoradiography of this blot membrane. In
order to concentrate the proteins of interest, immunopre-
cipitation of the same cell extracts with the anti-TPH
serum was performed before SDS/PAGE as described in
Materials and methods. Even after the immunoprecipita-
tion,
32
P-labeled TPH-like protein was not detected (lane
1inFig.1B,
32
P), indicating a very low steady state level
of phosphorylated TPH or none at all. Addition of either
okadaic acid (lane 2 in Fig. 1B,
32
P)orMG132(not
shown) made little difference. By simultaneous addition
of okadaic acid (0.5 l
M
) and MG132 (3 l
M
), a protein
band of 53 kDa became detectable among several inten-
sified proteins (lane 3). We conducted an Ôimage-mathÕ
operation to obtain clearer difference by subtracting the
image of lane 2 (okadaic acid alone) from the image of
lane 3 (okadaic acid plus MG132). A clear band of
32

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

inhibitors (lane 1 in Fig. 1B,
32
P vs. WB).
Inhibition of TPH degradation in the cell-free
proteasome system by protein kinase inhibitors
We examined the involvement of TPH phosphorylation in
proteasome-driven degradation of the enzyme in vitro.Our
system contained extracts of RBL2H3 cells as the source of
proteasomes and ubiquitinylating enzymes [23], and purified
TPH from mouse mastocytoma P-815 cells as the substrate
for proteolysis. When incubated under complete conditions,
the amount of TPH protein of 53 kDa decreased progres-
sively concomitantly with the enzyme activity (Fig. 2A,B).
Along with the decrease in TPH protein, other TPH-like
immunoreactive polypeptide bands of smaller molecular
mass were not present; this was in contrast to TPH digestion
in cell extracts without ATP, which presumably occurred by
the action of lysozomal enzymes [33]. Taken together, our
system was representative of a TPH-proteasome system
with regard to the sensitivity to specific proteasome
inhibitors (Fig. 2A). Degradation of TPH in this cell-free
system was inhibited by KN-62 (100 l
M
), a potent inhibitor
of CaM kinase II (Fig. 2A for TPH activity and Fig. 2B,
Fig. 1. Effect of protein phosphatase inhibitor, okadaic acid, and proteasome inhibitors on TPH degradation in RBL2H3 cells and on TPH phos-
phorylation in FMA3 cells. (A) RBL2H3 cells (1 · 10
5
cells per well) were cultured in the presence (closed circle) or absence (vehicle, open circle) of
respective inhibitors for 60 min to allow the reagents to penetrate the cells. Then, 10 lgÆmL

)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

32
P-incorporation into proteins,
the cell extracts were subjected to immunoprecipitation followed by SDS/PAGE for autoradiography as described in Materials and methods. The
autoradiogram (left panel,
32
P) represents treatment with vehicle (lane 1), 0.5 l
M
okadaic acid (lane 2), and okadaic acid plus 3 l
M
MG132 (lane 3).
Lane 4 is a graphically created image representing the net increase in the density of lane 3 over lane 2, which was obtained by image math operation
(image 3 minus image 2). To create this image, the contrast was pushed twice (pixel densities were doubled). The arrow indicates the expected
migration of TPH with a molecular mass of 53 kDa. The adjacent pattern (PP) is the vertical Ôplot profileÕ of lane 4. (WB): The same extracts
described in (
32
P) incorporation were applied (15 lg protein per lane) to SDS/PAGE without the immunoprecipitation, then subjected to Western
blot analysis as described in Materials and methods. Lanes 1–3 corresponded to those in the (
32
P) panel, and lane 4 was the purifed TPH (2.7 ng) as
a reference marker for immunostaining. The right-most patterns are the vertical Ôplot profileÕ of lanes 1–4. The Ôimage-mathÕ and Ôplot profileÕ
operations were conducted using
NIH
-
IMAGE
ver 1.62 software.
Ó FEBS 2002 TPH phosphorylation as proteasome targeting (Eur. J. Biochem. 269) 4783
KN62 for protein analysis by Western blotting). KN-62 at
50 l
M
was also significantly effective, while at concentra-

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].

Involvement of CaM kinase II was also suggested by results
of the experiments shown in Fig. 2. These kinases were
tested for the phosphorylation of TPH purified from
mastocytoma P-815 cells. Since the available protein
specimens contain unidentified proteins, electrophoreto-
grams were carefully compared among images visualized by
Coomassie Brilliant Blue staining (CBB), by Western
blotting using anti-TPH serum (WB), and by autoradio-
graphy (
32
P). PKA was first examined using its catalytic
subunit for phosphorylation of TPH. In this experiment, the
amount of the PKA-catalytic subunit and that of the other
agents added were optimized using rat phenylalanine
hydroxylase (PAH, molecular mass of 55 kDa in Fig. 3A).
Virtually no phosphorylation of TPH was detected under
the conditions used in which phenylalanine hydroxylase
(and two other proteins, molecular masses of 95 kDa and
60 kDa that contaminated the preparation) was clearly
phosphorylated (Fig. 3A). When an effort was made to
enhance the autoradiograph image (
32
P panel in Fig. 3B), a
faint and diffuse signal appeared at a slightly higher position
around the TPH region but the signal was too small to
determine whether it corresponded to TPH protein (major
band in panel WB, and the major band in panel CBB with
molecular mass of 53 kDa). The only obviously labeled
band (arrowhead, molecular mass of 41 kDa, also seen on
the

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

RBL2H3 extracts, composition of the reaction mixtures, and analytical procedures are described in Materials and methods. (A) TPH activity
remained after incubation for the indicated times in the absence (open circles) or presence of MG132 (50 l
M
, closed squares) or KN-62 (100 l
M
,
closed circles). (B) TPH protein remained after the indicated period of incubation. Upper panels: immunoblot images visualized with anti-TPH
serum. Lower panels: the digitized densities of corresponding spots were plotted relative to the 0-time density as 100%. The in vitro proteasome
system included a vehicle control (open circles in all panels), 50 l
M
MG132 (closed squares for KN62), 100 l
M
KN-62 (closed circles for KN62),
50 l
M
K252a (closed circles for K252a), and 50 l
M
H-89 (closed circles for H89).
4784 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphorylation was prevented by 50 l
M
KN-62 (not
shown). In addition, PKA run for comparison again gave
no indication of phosphorylation of TPH (lane 4,
32
P-panel). These results indicated that TPH purified from
mouse mastocytoma P-815 was readily phosphorylated by
CaM kinase II. Although the TPH from P-815 cells was
easily phosphorylated by this kinase, its enzymic activity
was not altered by the phosphorylation reaction.

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

P] ATP at 37 °C for 30 min. Proteins were precipitated by trichloroacetic acid (5%) on
ice, centrifuged, washed with diethylether, and then subjected to SDS/PAGE followed by autoradiography for
32
P incorporation or Western blot
analysis using anti-TPH serum. Compositions of the reaction mixtures, and analytical procedures are described in Materials and methods. Size
markers are shown in kDa at left. Arrows indicate the estimated position of TPH and the arrowheads indicate contaminating protein in the PKA
preparation for comparison between panels. (A) Autoradiogram of phosphorylation products of TPH and PAH (1 lg per lane assuming protein
recovery to be consistent in washing procedure). (B) Comparison of protein staining (CBB), Western blot analysis (WB), and autoradiography (
32
P)
with a common gel after SDS/PAGE.
Fig. 4. Phosphorylation of TPH and stimulation of its proteasome driven degradation by CaM kinase II. TPH (3 lg) was placed in phosphorylation
conditions consisting of CaM kinase II, Ca
2+
andcalmodulinat37°Cfor30mininatotalvolumeof210lL. Exclusion of kinase or the PKA
catalytic subunit was also used for comparison. (A) Proteins in the reaction mixture were collected by preferential adsorption of TPH to DMPH
4
-
conjugated Affigel-10 gel beads overnight with agitation. Proteins were then extracted from the gel with 1% SDS for SDS/PAGE, followed by (WB)
Western blot analysis using anti-TPH serum as the primary antibody and by (
32
P) autoradiography using a FLA-3000 fluorescence image analyzer
(details are described in Materials and methods). Size markers are shown in kDa at left. The arrow on the right indicates the estimated position of
TPH. The two right-most patterns (PP) are the Ôplot profileÕ of lanes 2 and 3 of (
32
P) panels, respectively. (B) TPH in the phosphorylation reaction
mixture including CaM kinase II (closed circles in left panel are marked (+) in right panel) or lacking kinase (open circles in left panel are marked
(–) in right panel) was subjected to cell-free proteolytic conditions for the indicated period of time, as described in the legend to Fig. 2. TPH
remaining undigested was visualized by Western blot immunostaining after SDS/PAGE (WB). The left panel represents the density of the
remaining TPH relative to the 0-time density taken as 100.

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

shown),
32
P-labeled TPH was undetectable, indicating that
the rate of either dephosphorylation or proteolytic degra-
dation of the putative
32
P-TPH was higher than phosphory-
lation of TPH. However, phosphorylated (
32
P-labeled)
TPH-like protein became detectable in FMA3 cells only
when both processes were blocked by simultaneous addition
of okadaic acid, a protein phosphatase inhibitor, and
MG132, a proteasome inhibitor (Fig. 1B,
32
P,lane3).The
‘image math’ operation visualized the net increase in density
in lane 3 over lane 2 (Fig. 1B,
32
P, lane 4). This image
represented protein bands that fulfilled the following three
conditions simultaneously: (a) phosphorylation by cellular
protein kinases; (b) protection from dephosphorylation by
okadaic acid; and (c) protection by MG132 from protea-
some action. A protein band (molecular mass of 53 kDa)
had the highest intensity and coincided with that of TPH
visualized by Western blot analysis of the same cell extracts
run with the authentic TPH protein as the staining reference
mark (Fig. 1B, WB). This suggests that the proteasome
inhibitor MG132 might accumulate phosphorylated, sub-

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-

phosphate by the action of CaM kinase II but not by PKA
does not appear compatible with the idea that the site was
shared with PKA and CaM kinase II as is Ser58 of rat TPH
[28]. For a solid conclusion, however, further investigation is
required, such as site determination of kinase-specific
phosphorylation.
Phosphorylation of TPH as the tag for proteolysis
targeting
The role of TPH phosphorylation is that it down regulates
the TPH level in the cell by serving a tag for targeting for
digestion by proteasomes. This idea is based on the
following observations: (a) proteasome-driven TPH degra-
dation in vivo (in RBL2H3 cells) was enhanced by okadaic
acid, a protein phosphatase inhibitor (Fig. 1); (b) degrada-
tion of exogenous TPH in a reconstituted proteasome
system composed of RBL2H3 cell extracts was inhibited by
both KN-62, a CaM kinase II inhibitor, and K252a, a
potent protein kinase inhibitor with broad specificity
(Fig. 2); and (c) TPH previously phosphorylated by CaM
kinase II was more rapidly degraded than nonphosphoryl-
ated TPH in the same in vitro system. The cell-free
proteasome system employed in the present study was
constructed with fresh extracts from RBL2H3 cells cultured
under ordinary conditions and prepared in the presence of
2m
M
ATP, 1 m
M
dithiothreitol and 0.25
M

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

remains as to how neural and non-neural TPH can appear
so differentiated in view of their common amino acid
sequence [19]. Studies on the cellular management of their
common TPH gene, including RNA-processing, transla-
tion of the message, post-translational processing,
and cytosolic machinery such as phosphorylation and
proteasome systems are just beginning to elucidate the
differences between these two types of TPH.
ACKNOWLEDGEMENTS
We are grateful to Dr T. Yamauchi for donating purified calmodulin
and CaM kinase II and for instructions on its use. This research was
supported by the Japan Private School Promotion Foundation and by
a Grant-in-Aid for Advanced Scientific Research on Bioscience/
Biotechnology Areas from the Ministry of Education, Science, Sports
and Culture of Japan.
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