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 triggeredby phosphorylation in RBL2H3 cells, a serotonin producing mast cell line Yoshiko Iida1, Keiko Sawabe1, Masayo Kojima1, Ka

Proteasome-driven turnover of tryptophan hydroxylase is triggered

by phosphorylation in RBL2H3 cells, a serotonin producing

mast cell line

Yoshiko Iida1, Keiko Sawabe1, Masayo Kojima1, Kazuya Oguro1,2, Nobuo Nakanishi3and

Hiroyuki Hasegawa1,2

1

Department of Bioscience, and2Biotechnology 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 of32P

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 Ca2+ /calmodulin-dependent protein kinase II in the presence of Ca2+and calmodulin but not by protein kinase A (catalytic subunit) TPH protein thus phosphorylated by Ca2+ /calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphoryl-ated enzyme These results indicnonphosphoryl-ated 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 (T1/2, 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: hasegawa@ntu.ac.jp

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)

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 degradaphosphoryla-tion 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

M A T E R I A L S A N D M E T H O D S

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) [32P]H3PO4(500 mCiÆmL)1) and [c-32P]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· 105cells per well Two hours before the experimental

treatment, cells were placed in serum-free medium buffered

with 25 mM Hepes/NaOH containing 100 UÆmL)1 of

penicillin and 100 lgÆmL)1 of streptomycin, then kept at

37C under 10% CO/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 the 96-well plate were placed in 20 lL of NaCl/Pi(–), 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 30C in 0.1 M Tris/HCl ( pH 8.0) containing 30 mM dithiothreitol, 50 lM Fe(NH4)2(SO4)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 lM tryptophan,

400 lM 6R-tetrahydrobiopterin, 500 lM NADH, 1 mM NSD-1015, 2 mgÆmL)1catalase, and 50 lgÆmL)1 dihydrop-teridine reductase in 0.1M potassium phosphate buffer ( pH 6.9) The enzyme reaction was allowed to proceed for

10 min at 30C, then was terminated by 1M perchloric acid The 5HTPformed 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 mM 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 mM Tris/HCl (pH 7.5) containing 1 mM dithiothreitol, 2 mM 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 mM MgCl2, 1 mM CaCl2, 2 mM ATP,

10 lgÆmL)1 creatine kinase, 10 mM phosphocreatine, 0.2 mgÆmL)1 catalase, and 1 mM dithiothreitol in 50 mM 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 (30C) 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· 106cells) were adapted to phosphate-free RPMI 1640 (Gibco, Cat No 11877–032)

Subsequently, cells were fed 0.4 mCiÆmL)1[32P]NaH2PO4

for 30 min.32P-Loading was further continued for 120 min

in the presence of kinase inhibitors or

protein-phosphatase inhibitors Cells were then rinsed with NaCl/Pi

and disrupted with 1% NP-40 in 50 mM Tris/HCl

( pH 7.8) containing an inhibitor cocktail (1 mM

phenyl-methanesulfonyl fluoride, 2 mM EDTA, 50 mM sodium

fluoride, and 1 mMsodium orthovanadate) The cell lysates

were mixed with anti-TPH serum (10 lL) and left overnight

at 4C 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 37C in a reaction

mixture containing 3 lg of purified TPH as substrate, or rat

liver phenylalanine hydroxylase for comparison, 1 lg P KA

catalytic subunit and 2 lCi [c-32P]ATP in 50 mMTris/HCl

( pH 7.4) containing 20 lMATPand 10 mM MgCl2 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 lL of

the lysis buffer for SDS/PAGE Ca2+

/calmodulin-depend-ent phosphorylation was carried out with 1 lg TP H as

substrate and 0.1 lg CaM kinase II for 30 min at 37C in

the presence of 0.1 lM calmodulin, in 210 lL of 50 mM

Tris/HCl ( pH 7.4) containing 10 lM ATP(2 lCi

[c-32P]ATP), 5 mM MgCl2, 120 lM CaCl2, and 100 lM

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 DMPH4-Affigel-10 [8] for collecting TPH in the

presence of the inhibitor cocktail as above and 150 mM

NaCl in 50 mMTris/acetate (pH 8.0), then left overnight at

4C 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/Pi 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 with32P, gels following SDS/

PAGE were dried on filter paper, then subjected to exposure

either to an X-ray film (Konica) at)80 C for 3 days with

an intensifying screen (Kodak, Bio Max MS) or to a

fluoro-image analyser (Fujifilm, FLA-3000) using an imaging plate

(Fujifilm, BSA-IPMS2040) Graphic images of Western

blot analysis or autoradiograms were analyzed usingNIH

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 (n¼ 4) unless otherwise stated

R E S U L T S

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 (T1/2: 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 lM): the half-life of TPH (T1/2) 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 [32P]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

32P-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,32P-labeled TPH-like protein was not detected (lane

1 in Fig 1B,32P), indicating a very low steady state level

of phosphorylated TPH or none at all Addition of either okadaic acid (lane 2 in Fig 1B, 32P ) or MG132 (not shown) made little difference By simultaneous addition

of okadaic acid (0.5 lM) and MG132 (3 lM), a protein band of 53 kDa became detectable among several inten-sified proteins (lane 3) We conducted an image-math 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 of32 P-incorporated protein with a molecular mass of 53 kDa

was obtained (Fig 1B, 32P, lane 4) and was coincident

with the TPH visualized by Western blot analysis

(Fig 1B, WB, lane 3 and 4) This operation visualizes

32P-incorporation into the specific proteins which were

protected from proteasome-driven digestion by MG132

among those 32P-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, 32Pand

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,32Pvs 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 lM), 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)1cycloheximide 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 plot profile 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 plot profile of lanes 1–4 The image-math and plot profile operations were conducted using NIH - IMAGE ver 1.62 software.

KN62 for protein analysis by Western blotting) KN-62 at

50 lMwas also significantly effective, while at

concentra-tions higher than 100 lM, the inhibitor showed no further

effect (not shown) K252a and K252b (both 50 lM), protein

kinase inhibitors with relatively broad specificity, were also

effective as tested (Fig 2B, K252a), but H-89 was not

significantly effective at 50 lM(Fig 2B, H89)

Staurospo-rine and chelerythStaurospo-rine, 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 (32P) 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 (32Ppanel 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 the32Ppanel 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 no32Por far less than the stoichiometric amount of32P

On the other hand, TPH was clearly32P-labeled by CaM kinase II in vitro in the presence of both Ca2+ 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).

phosphorylation was prevented by 50 lM KN-62 (not

shown) In addition, PKA run for comparison again gave

no indication of phosphorylation of TPH (lane 4,

32P-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

D I S C U S S I O N

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 vitroby 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-32P] 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 Pincorporation 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, Ca2+and calmodulin at 37 C for 30 min in a total volume of 210 lL 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 (32P) 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 plot profile 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.

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 were32P-labeled as shown in Fig 1B,32P Even in

the presence of either okadaic acid (lane 2) or MG132 (not

shown),32P-labeled TPH was undetectable, indicating that

the rate of either dephosphorylation or proteolytic

degra-dation of the putative32P-TPH was higher than

phosphory-lation of TPH However, phosphorylated (32P-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,32P , lane 3) The

‘image math’ operation visualized the net increase in density

in lane 3 over lane 2 (Fig 1B, 32P, 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 TPHin 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 vitroto 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

2 mM ATP, 1 mM dithiothreitol and 0.25M 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 lM, 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 digesphosphoryla-tion 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

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

A C K N O W L E D G E M E N T S

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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