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

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Proteasome-driven turnover of tryptophan hydroxylase is triggeredby phosphorylation in RBL2H3 cells, a serotonin producingmast cell lineYoshiko Iida1, Keiko Sawabe1, Masayo Kojima1, Kazuya Oguro1,2, Nobuo Nakanishi3andHiroyuki Hasegawa1,21Department of Bioscience, and2Biotechnology Research Center, Teikyo University of Science and Technology, Yamanashi, Japan;3Departments of Biochemistry, Meikai University School of Dentistry, Sakado, Saitama, JapanWe previously demonstrated in mast cell lines RBL2H3 andFMA3 that tryptophan hydroxylase (TPH) undergoes veryfast 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 examinedan 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 okadaicacid, a protein phosphatase inhibitor. Incorporation of32Pinto a 53-kDa protein, which was judged to be TPH based onautoradiography and Western blot analysis using anti-TPHserum and purified TPH as the size marker, was observed inFMA3 cells only in the presence of both okadaic acid andMG132, inhibitors of protein phosphatase and proteasome,respectively. In a cell-free proteasome system constitutedmainly of RBL2H3 cell extracts, degradation of exogenousTPH isolated from mastocytoma P-815 cells was inhibitedby protein kinase inhibitors KN-62 and K252a but not byH89. Consistent with the inhibitor specificity, the same TPHwas phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+andcalmodulin but not by protein kinase A (catalytic subunit).TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in thecell-free proteasome system than was the nonphosphoryl-ated enzyme. These results indicated that the phosphoryla-tion of TPH was a prerequisite for proteasome-driven TPHdegradation.Keywords: tetrahydrobiopterin; CaM kinase II; proteasometarget; ubiquitin ligase; enzyme turnover.Tryptophan hydroxylase (TPH, EC, a member ofa family of pterin-dependent aromatic amino acid hydroxy-lases [1], catalyzes the conversion ofL-tryptophan to5-hydroxy-L-tryptophan. This reaction is the initial andrate-limiting step in the biosynthesis of serotonin [2–5]. TPHhas been extensively purified from various sources such asbovine pineal gland [6], mouse mastocytoma [7,8], andmammalian brains [9–11]. Physicochemical, enzymic andimmunochemical properties differed between TPHs ofneural and non-neural tissue origin, and it is accepted thatneural TPH might be a different entity from the non-neuralenzyme [8,10,12,13]. Complimentary DNAs of TPH havebeen cloned from various sources but no differences or onlytrivial variation in amino acid sequences were found amongthem [14–19]. The molecular basis of differences between theneural and non-neural enzymes has not yet been explained.Both types of cytosolic environment should be studiedfurther to detect differences in the control of gene expres-sion, post-translational modification, and turnover of theenzyme protein in a tissue-specific way.We have demonstrated with RBL2H3, an established cellline that expresses TPH in culture while retaining many ofthe characteristics of mast cells, that: (a) cellular TPHactivity was seriously limited by insufficient supply with theenzyme’s essential cofactor, ferrous iron, and the substratestryptophan and 6R-tetrahydrobiopterin [20]; (b) immunestimulation lead to a marked increase in TPH level bymeans of enhanced expression of the TPH gene [21]; and(c) the steady state TPH level of this cell was maintainedat extremely low levels by rapid degradation of the enzyme(T1/2, 15–60 min) [22,23]. In the latter report, the turnover ofTPH protein was shown to be driven by ATP-dependentaction of 26S-proteasomes including, at least in part,ubiquitinylation of TPH. Furthermore, it was noted thatthis rapid turnover was suppressed by a protein kinaseinhibitor. Since proteasomes might, in general, be ubiquit-ous in the cell, recognition of the specific target is crucial interms of the specific protein to be digested. Poly-ubiquiti-nylation represents a major tag for proteasomes. Theubiquitinylation of a specific protein is determined bythe ubiquitin ligase complex E3. The molecular basis of thestructure–function relationship enabling E3 to specificallyrecognize a wide variety of substrates is one of the majorsubjects of investigation in this field. In the ubiquitinylationCorrespondence 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.jpAbbreviations: TPH, tryptophan hydroxylase; CaM kinase II,calcium/calmodulin-dependent protein kinase II; PKA, cyclicAMP-dependent protein kinase; 5HTP, 5-hydroxy-L-tryptophan.Enzyme: Tryptophan hydroxylase (EC 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.xof TPH, a specific tag might be required for targeting by theligase. In many cases, phosphorylation of the target proteinprovides the tag for the ubiquitinylation system, especiallyof such families as the SCF-complex, Skp1/Cullin-1/F-boxprotein (reviewed in [24,25]). Involvement of phosphoryla-tion in TPH degradation was expected, however, phos-phorylation of non-neural TPH has never beendemonstrated, although TPH of brain origin and recom-binant TPH have been known to be phosphorylated byPKA and by CaM kinase II [26–28]. On the other hand,proteasome-driven turnover has only been demonstratedwith mast cell lines. The aim of this work was to elucidatewhether the phosphorylation of non-neural TPH takesplace and, if it does, whether it provides the tag for targetingby the proteasomes involved in the rapid turnover of theenzyme.MATERIALS AND METHODSMaterialsMG132 (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), okadaicacid 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 werepurchased from Sigma. Sodium fluoride was obtained fromNacalai Tesque (Kyoto, Japan). The concentrations ofinhibitors 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]. Rabbitpolyclonal anti-TPH serum was raised against the purifiedTPH [13]. Bovine liver dihydropteridine reductase waspurified up to the second ammonium sulfate fractionationstep [29]. (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin wasdonated by Suntory (Tokyo, Japan). CaM kinase II andcalmodulin, both purified from rat brain, were donated byT. Yamauchi (Department of Biochemistry, Faculty ofPharmaceutical Science, The University of Tokushima,Japan). [32P]H3PO4(500 mCiÆmL)1)and[c-32P]ATP (tetra-triethylammonium salt; 4500 CiÆmmol)1) were purchasedfrom ICN Biochemicals.Cell cultureRBL2H3, a mast cell line derived from rat basophilicleukemia cells, was obtained from The Japanese CancerResearch Resources Bank (Tokyo). RBL2H3 cells andFMA3 (Furth’s mastocytoma) cells were maintained asdescribed [23]. One day before experiments, cells were platedto well of a 96-well culture plate (Falcon, Cat. No. 35072) at1 · 105cells per well. Two hours before the experimentaltreatment, cells were placed in serum-free medium bufferedwith 25 mMHepes/NaOH containing 100 UÆmL)1ofpenicillin and 100 lgÆmL)1of streptomycin, then kept at37 °C under 10% CO2/90% air throughout the experimentsexcept at the time of manipulation. Agents of lowsolubility in water were dissolved in dimethylsulfoxide ata concentration 100-fold greater than final one used, unlessotherwise stated, so that dimethylsulfoxide would be at anequivalent level in each experimental culture with novehicle effect.Tryptophan hydroxylase assayTPH activity was determined essentially as describedpreviously [13,23]. Cells in monolayer culture in wells ofthe96-wellplatewereplacedin20lLofNaCl/Pi(–), thensubjected twice to freezing in liquid nitrogen and thawing inwater. Reaction mixtures for the cell-free treatment ofpurified TPH (phosphorylation and proteolysis as describedbelow) were prepared just prior to measuring the enzymeactivity. The disrupted cells or TPH mixture were preincu-bated for 15 min at 30 °Cin0.1MTris/HCl ( pH 8.0)containing 30 mMdithiothreitol, 50 lMFe(NH4)2(SO4)2,and 4 mgÆmL)1catalase in a total volume of 100 lL.Subsequently, 50 lL of another cocktail were added toafford a final reaction mixture of 250 lMtryptophan,400 lM6R-tetrahydrobiopterin, 500 lMNADH, 1 mMNSD-1015, 2 mgÆmL)1catalase, and 50 lgÆmL)1dihydrop-teridine reductase in 0.1Mpotassium phosphate buffer( pH 6.9). The enzyme reaction was allowed to proceed for10 min at 30 °C, then was terminated by 1Mperchloricacid. The 5HTP formed was measured using an HPLCsystem equipped with a fluorescence monitor (JASCOmodel, FP920) set at 302 nm and 350 nm for excitation andemission, respectively. The solid phase was ODS(4.6 · 250 mm, JASCO, Finepak SIL-C18T5), the mobilephase was a 90 : 7 : 5 mixture of 40 mMsodium acetate(adjusted to pH 3.5 with formic acid), acetonitrile andmethanol and the flow rate was 1 mLÆmin)1[30].Cell-free proteolysis of TPHExtracts from RBL2H3 cells as the source of proteasomeswere prepared essentially as described [23]. The cells werehomogenized in 5 volumes of 50 mMTris/HCl (pH 7.5)containing 1 mMdithiothreitol, 2 mMATP, and 0.25Msucrose 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 wasperformed in a reaction mixture containing the RBL2H3cell extracts, 5 mMMgCl2,1mMCaCl2,2mMATP,10 lgÆmL)1creatine kinase, 10 mMphosphocreatine,0.2 mgÆmL)1catalase, and 1 mMdithiothreitol in 50 mMTris/HCl (pH 8.0). Purified TPH from P-815 cells with orwithout in vitro phosphorylation was used as the sub-strate. Inhibitors of proteasomes and protein kinases wereadded prior to addition of the substrate TPH. Aliquotswere taken after various intervals of incubation (30 °C) forthe TPH enzyme activity assay and for Western blotanalysis.Phosphorylation of TPHIn situ phosphorylation of TPH in FMA3 cells wasperformed as follows. Cells (2 · 106cells) were adapted tophosphate-free RPMI 1640 (Gibco, Cat. No. 11877–032)supplemented with 5 lMNaH2PO4for 90 min.Ó FEBS 2002 TPH phosphorylation as proteasome targeting (Eur. J. Biochem. 269) 4781Subsequently, cells were fed 0.4 mCiÆmL)1[32P]NaH2PO4for 30 min.32P-Loading was further continued for 120 minin the presence of protein-kinase inhibitors or protein-phosphatase inhibitors. Cells were then rinsed with NaCl/Piand disrupted with 1% NP-40 in 50 mMTris/HCl( pH 7.8) containing an inhibitor cocktail (1 mMphenyl-methanesulfonyl fluoride, 2 mMEDTA, 50 mMsodiumfluoride, and 1 mMsodium orthovanadate). The cell lysateswere mixed with anti-TPH serum (10 lL) and left overnightat 4 °C with agitation. Total IgG was collected by theaddition of staphylococcal ghosts (Pansorbin; Calbiochem,La Jolla, CA, USA) as a precipitant, solubilized in 1% SDS,and subjected to SDS/PAGE. Cell-free phosphorylation byPKA was carried out for 30 min at 37 °Cinareactionmixture containing 3 lg of purified TPH as substrate, or ratliver phenylalanine hydroxylase for comparison, 1 lgPKAcatalytic subunit and 2 lCi [c-32P]ATP in 50 mMTris/HCl( pH 7.4) containing 20 lMATP and 10 mMMgCl2in atotal volume of 210 lL. For SDS/PAGE, proteins wereprecipitated by the addition of trichloroacetic acid (5%) inthe cold and centrifuged. The pellets were then washed twicewith 400 lL of diethylether, dried and dissolved in 50 lLofthe lysis buffer for SDS/PAGE. Ca2+/calmodulin-depend-ent phosphorylation was carried out with 1 lgTPHassubstrate and 0.1 lg CaM kinase II for 30 min at 37 °Cinthe presence of 0.1 lMcalmodulin, in 210 lLof50mMTris/HCl ( pH 7.4) containing 10 lMATP (2 lCi[c-32P]ATP), 5 mMMgCl2,120lMCaCl2,and100lMEGTA. Aliquots were taken for the assay of TPH activityor for subjecting to the cell-free proteolysis described above.The remaining reaction mixture was mixed with affinity gelbeads DMPH4-Affigel-10 [8] for collecting TPH in thepresence of the inhibitor cocktail as above and 150 mMNaCl in 50 mMTris/acetate (pH 8.0), then left overnight at4 °C with agitation. The proteins obtained were subjected toSDS/PAGE followed by immunoblotting and autoradio-graphy.SDS/PAGE, Western blot analysis, and autoradiographyMonolayer cultures washed with NaCl/Pior proteinscollected as a pellet as described above were solubilized in1% SDS and subjected to SDS/PAGE according toLaemmli [31]. Western blot analysis was performed asdescribed previously [23]. The protein signal was visualizedusing an enhanced chemiluminescence detection system(ECL; Amersham, Buckinghamshire, England). Proteinbands were exposed to an X-ray film (Konica, Medical Film20287). For autoradiography with32P, gels following SDS/PAGE were dried on filter paper, then subjected to exposureeither to an X-ray film (Konica) at )80 °Cfor3dayswithan 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 Westernblot analysis or autoradiograms were analyzed usingNIHIMAGEver 1.62 software, Wayne Rasband, NationalInstitute of Health, USA.Other methodsProteins were determined by Bradford’s method [32] usingbovine serum albumin as the standard. Data were expressedas means ± SD (n ¼ 4) unless otherwise stated.RESULTSInvolvement of protein phosphorylation in TPHdegradation in living cellsIn previous works [22,23], we demonstrated in mast celllines RBL2H3 and FMA3 that de novo biosynthesis ofTPH enzyme protein was accompanied by rapid degra-dation with 26S-proteasomes and that ubiquitinylation ofTPH protein was involved in the process, presumably byproviding the targeting tag. In search for any connectionbetween protein phosphorylation and TPH turnover, weexamined the effect of okadaic acid, a protein phospha-tase inhibitor, on TPH degradation in the living cellsystem 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 activitywas observed in the absence of the inhibitors (T1/2:around 30 min, Fig. 1A). This decrease was much sloweror was virtually stopped by proteasome inhibitors MG132and lactacystin but was not affected by a cystein proteaseinhibitor, E-64-d; a representative finding showing thatthe steady state level of TPH was determined by aproteasome-driven degradation process. TPH degradationin the cells was accelerated by okadaic acid (0.25 lM): thehalf-life of TPH (T1/2) were estimated to be 29 min and38 min in the presence and absence of okadaic acid,respectively (Fig. 1A, OA), suggesting an involvement ofTPH phosphorylation in recognition of the enzyme by theubiquitinylation system. Based on this observation, weexamined whether TPH was phosphorylated in situ usingFMA3 cells in which cytosolic TPH was also rapidlydegradated by the proteasome-driven process while thesteady state TPH level was roughly 20-fold higher thanthat of RBL2H3 cells [22,23]. Cellular proteins werelabeled by incubating the cells with [32P]orthophosphate,and steady state phosphorylation levels of proteins wereperformed in the presence and absence of okadaic acidand/or MG132. By Western blot analysis of the wholecell extracts, TPH of molecular mass 53 kDa was locali-zed side-by-side with purified TPH and the anti-TPHserum (Fig. 1B, WB). Addition of both okadaic acid andMG132 caused the immunoreactive band to be twofoldthicker than the control band (see plot profiles of WB,right-most patterns), however, no discrete bands of32P-incorporation were recognized over the dense back-ground by autoradiography of this blot membrane. Inorder to concentrate the proteins of interest, immunopre-cipitation of the same cell extracts with the anti-TPHserum was performed before SDS/PAGE as described inMaterials and methods. Even after the immunoprecipita-tion,32P-labeled TPH-like protein was not detected (lane1inFig.1B,32P), indicating a very low steady state levelof phosphorylated TPH or none at all. Addition of eitherokadaic acid (lane 2 in Fig. 1B,32P)orMG132(notshown) made little difference. By simultaneous additionof okadaic acid (0.5 lM) and MG132 (3 lM), a proteinband of 53 kDa became detectable among several inten-sified proteins (lane 3). We conducted an Ôimage-mathÕoperation to obtain clearer difference by subtracting theimage of lane 2 (okadaic acid alone) from the image oflane 3 (okadaic acid plus MG132). A clear band of32P-incorporated protein with a molecular mass of 53 kDa4782 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002was obtained (Fig. 1B,32P, lane 4) and was coincidentwith the TPH visualized by Western blot analysis(Fig. 1B, WB, lane 3 and 4). This operation visualizes32P-incorporation into the specific proteins which wereprotected from proteasome-driven digestion by MG132among those32P-phosphorylated and protected fromdephosphorylation by okadaic acid, proteins which oth-erwise would have been digested by proteasomes. Thusthe phosphorylated form of TPH was detectable onlywhen the proteasome action and phosphatase wereeffectively blocked (lane 3 in both Fig. 1B,32P andWB). Together with the fact that the blocking of proteinphosphatase by okadaic acid resulted in the accelerationof TPH degradation (Fig. 1A, OA), the present result isevidence that phosphorylation takes place on this proteinwhere it functions as the tag for the targeting of TPH bythe proteasomes. It was noteworthy that TPH detectableunder steady state conditions was unphosphorylated,presumably because the phosphorylated TPH might havebeen digested away in the absence of proteasomeinhibitors (lane 1 in Fig. 1B,32P vs. WB).Inhibition of TPH degradation in the cell-freeproteasome system by protein kinase inhibitorsWe examined the involvement of TPH phosphorylation inproteasome-driven degradation of the enzyme in vitro.Oursystem contained extracts of RBL2H3 cells as the source ofproteasomes and ubiquitinylating enzymes [23], and purifiedTPH from mouse mastocytoma P-815 cells as the substratefor 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-likeimmunoreactive polypeptide bands of smaller molecularmass were not present; this was in contrast to TPH digestionin cell extracts without ATP, which presumably occurred bythe action of lysozomal enzymes [33]. Taken together, oursystem was representative of a TPH-proteasome systemwith regard to the sensitivity to specific proteasomeinhibitors (Fig. 2A). Degradation of TPH in this cell-freesystem was inhibited by KN-62 (100 lM), a potent inhibitorof 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 · 105cells per well) were cultured in the presence (closed circle) or absence (vehicle, open circle) ofrespective inhibitors for 60 min to allow the reagents to penetrate the cells. Then, 10 lgÆmL)1cycloheximide were added to each well and the culturecontinued. TPH activities at 0, 10, 30, and 60 min after addition of cycloheximide were measured. Inhibitors used were: 10 lMMG132 (A-MG132),30 lMlactacystin (A-Lact), 10 lME-64-d (A-E64d), and 0.25 lMokadaic acid (A-OA). Values are means ± SD (n ¼ 4). (B) FMA3 cells (2 · 106cellsÆmL)1) were placed in phosphate-free RPMI 1640 medium supplemented with 5 lMNaH2PO4for 1.5 h, and were exposed to 0.4 mCiÆmL)132P-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 disruptedwith 1% NP-40 in 50 mMTris/HCl (pH 7.8) containing the protease inhibitor cocktail. (32P): In order to examine32P-incorporation into proteins,the cell extracts were subjected to immunoprecipitation followed by SDS/PAGE for autoradiography as described in Materials and methods. Theautoradiogram (left panel,32P) represents treatment with vehicle (lane 1), 0.5 lMokadaic acid (lane 2), and okadaic acid plus 3 lMMG132 (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 expectedmigration of TPH with a molecular mass of 53 kDa. The adjacent pattern (PP) is the vertical Ôplot profileÕ of lane 4. (WB): The same extractsdescribed in (32P) incorporation were applied (15 lg protein per lane) to SDS/PAGE without the immunoprecipitation, then subjected to Westernblot analysis as described in Materials and methods. Lanes 1–3 corresponded to those in the (32P) panel, and lane 4 was the purifed TPH (2.7 ng) asa reference marker for immunostaining. The right-most patterns are the vertical Ôplot profileÕ of lanes 1–4. The Ôimage-mathÕ and Ôplot profileÕoperations were conducted usingNIH-IMAGEver 1.62 software.Ó FEBS 2002 TPH phosphorylation as proteasome targeting (Eur. J. Biochem. 269) 4783KN62 for protein analysis by Western blotting). KN-62 at50 lMwas also significantly effective, while at concentra-tions higher than 100 lM, the inhibitor showed no furthereffect (not shown). K252a and K252b (both 50 lM), proteinkinase inhibitors with relatively broad specificity, were alsoeffective as tested (Fig. 2B, K252a), but H-89 was notsignificantly effective at 50 lM(Fig. 2B, H89). Staurospo-rine and chelerythrine, potent inhibitors of protein kinase C,were not significantly effective (not shown). These resultsindicated that TPH digestion by proteasomes involvedphosphorylation of certain protein(s) as an essential step.Taken together with the outcome of the experiment inFig. 1, it is likely that the TPH molecule was the protein tobe phosphorylated in the selective degradation. Further-more, from the specificity of the protein kinase inhibitorstested above, CaM kinase II seemed to function in the cellextracts, at least in part.Stimulation of TPH degradation in the cell freeproteasome system by the phosphorylationof the enzyme by CaM kinase IIAs described above (Fig. 1), the phosphorylation of non-neuronal TPH was suggested for the first time usingRBL2H3 and FMA3 cells. We then examined TPHphosphorylation in vitro in order to determine the type ofprotein kinase responsible. TPH from brain is known to bephosphorylated by PKA [26,28] and CaM kinase II [27].Involvement of CaM kinase II was also suggested by resultsof the experiments shown in Fig. 2. These kinases weretested for the phosphorylation of TPH purified frommastocytoma P-815 cells. Since the available proteinspecimens contain unidentified proteins, electrophoreto-grams were carefully compared among images visualized byCoomassie Brilliant Blue staining (CBB), by Westernblotting using anti-TPH serum (WB), and by autoradio-graphy (32P). PKA was first examined using its catalyticsubunit for phosphorylation of TPH. In this experiment, theamount of the PKA-catalytic subunit and that of the otheragents added were optimized using rat phenylalaninehydroxylase (PAH, molecular mass of 55 kDa in Fig. 3A).Virtually no phosphorylation of TPH was detected underthe conditions used in which phenylalanine hydroxylase(and two other proteins, molecular masses of 95 kDa and60 kDa that contaminated the preparation) was clearlyphosphorylated (Fig. 3A). When an effort was made toenhance the autoradiograph image (32P panel in Fig. 3B), afaint and diffuse signal appeared at a slightly higher positionaround the TPH region but the signal was too small todetermine whether it corresponded to TPH protein (majorband in panel WB, and the major band in panel CBB withmolecular mass of 53 kDa). The only obviously labeledband (arrowhead, molecular mass of 41 kDa, also seen onthe32P panel of Fig. 3A) was judged to be from contami-nation of the PKA preparation because this band was notdetectable with Western blot analysis (WB in Fig. 3B) andwas seen with CBB staining only when PKA was added(CBB panel in Fig. 3B; note that PKA alone had poorrecovery through trichloroacetic acid precipitation anddiethylether washing to remove trichloroacetic acid forsampling). These results indicated that TPH protein incor-porated virtually no32P or far less than the stoichiometricamount of32P.On the other hand, TPH was clearly32P-labeled by CaMkinase II in vitro in the presence of both Ca2+andcalmodulin (Fig. 4). Besides TPH (molecular mass of53 kDa), two diffuse bands appeared on autoradiography(Fig. 4A, lanes 2 and 3). These were thought to be due toautophosphorylation of CaM kinase II proteins of 54 kDaand 63 kDa described by Yamauchi & Fujisawa [34]. ThisFig. 2. Inhibitory effect of protein kinase inhibitors on degradation of purified TPH in the cell-free proteasome system. TPH (1 lg) purified frommastocytoma P-815 cells was subjected to a cell-free proteasome system composed of freshly prepared extracts (400 lg protein). The reactionmixture (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 forWestern blot analysis (B). TPH activity was measured as described in Materials and methods after appropriate dilution (200-fold). Preparation ofRBL2H3 extracts, composition of the reaction mixtures, and analytical procedures are described in Materials and methods. (A) TPH activityremained after incubation for the indicated times in the absence (open circles) or presence of MG132 (50 lM, closed squares) or KN-62 (100 lM,closed circles). (B) TPH protein remained after the indicated period of incubation. Upper panels: immunoblot images visualized with anti-TPHserum. Lower panels: the digitized densities of corresponding spots were plotted relative to the 0-time density as 100%. The in vitro proteasomesystem included a vehicle control (open circles in all panels), 50 lMMG132 (closed squares for KN62), 100 lMKN-62 (closed circles for KN62),50 lMK252a (closed circles for K252a), and 50 lMH-89 (closed circles for H89).4784 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002phosphorylation was prevented by 50 lMKN-62 (notshown). In addition, PKA run for comparison again gaveno indication of phosphorylation of TPH (lane 4,32P-panel). These results indicated that TPH purified frommouse mastocytoma P-815 was readily phosphorylated byCaM kinase II. Although the TPH from P-815 cells waseasily phosphorylated by this kinase, its enzymic activitywas not altered by the phosphorylation reaction.We examined the effect of TPH phosphorylation by CaMkinase II on the susceptibility of the enzyme to degradationin our cell-free proteasome system. As shown in Fig. 4B,degradation of TPH phosphorylated by CaM kinase IIprior to the proteasome reaction was much more rapid thanthat of the nonphosphorylated TPH. This is presumablybecause the nonphosphorylated TPH had to undergo priorphosphorylation in situ to be targeted by the proteasomes inthe reconstituted cell-free system.DISCUSSIONIn the present study, using RBL2H3 and FMA3 cells asrepresentative non-neural cells, we examined whether TPHis actually phosphorylated and whether phosphorylation isthe prerequisite step in the proteasome-driven TPH degra-dation process. We presented evidence that: (a) TPH inFMA3 cells was phosphorylated in vivo; (b) TPH purifiedfrom mastocytoma P-815 cells was also phosphorylatedin vitro by CaM kinase II but not by PKA; (c) TPH thusphosphorylated was degraded in vitro at a higher rate thanwas the nonphosphorylated TPH; and (d) living RBL2H3cells are furnished with a whole proteasome system inclu-ding 26S-proteasomes and a specific ubiquitinylation systemthat recognizes phosphorylated TPH. As to non-neuralTPH, rat pineal enzyme was reported to increase in enzymicactivity by treatment with cAMP [35]. The authors,Fig. 3. Insignificant incorporation of32P into TPH by protein kinase A. TPH or rat liver phenylalanine hydroxylase (PAH), 3 lg each, were exposedto 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%) onice, centrifuged, washed with diethylether, and then subjected to SDS/PAGE followed by autoradiography for32P incorporation or Western blotanalysis using anti-TPH serum. Compositions of the reaction mixtures, and analytical procedures are described in Materials and methods. Sizemarkers are shown in kDa at left. Arrows indicate the estimated position of TPH and the arrowheads indicate contaminating protein in the PKApreparation for comparison between panels. (A) Autoradiogram of phosphorylation products of TPH and PAH (1 lg per lane assuming proteinrecovery to be consistent in washing procedure). (B) Comparison of protein staining (CBB), Western blot analysis (WB), and autoradiography (32P)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 phosphorylationconditions consisting of CaM kinase II, Ca2+andcalmodulinat37°Cfor30mininatotalvolumeof210lL. Exclusion of kinase or the PKAcatalytic subunit was also used for comparison. (A) Proteins in the reaction mixture were collected by preferential adsorption of TPH to DMPH4-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 ofTPH. The two right-most patterns (PP) are the Ôplot profileÕ of lanes 2 and 3 of (32P) panels, respectively. (B) TPH in the phosphorylation reactionmixture 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. TPHremaining undigested was visualized by Western blot immunostaining after SDS/PAGE (WB). The left panel represents the density of theremaining TPH relative to the 0-time density taken as 100.Ó FEBS 2002 TPH phosphorylation as proteasome targeting (Eur. J. Biochem. 269) 4785however, did not observe phosphorylation of pineal TPHprotein, though they described PKA-dependent phosphory-lation of brain TPH. The pineal gland is anatomicallyclassified as being outside the central nervous system and theenzymic properties of pineal TPH were obviously peripheralnature in every aspect we examined [6,36]. Careful investi-gation of TPH from mouse mastocytoma P-815 failed touncover any activation of enzyme activity by cAMP-dependent protein kinase action [37]. Thus far, no clearevidence has appeared for phosphorylation of non-neuralTPH, including that of pineal or neoplastic mastocytomacells.Evidence of TPH phosphorylation in living cellsWe first observed that okadaic acid, a protein phosphataseinhibitor, accelerated the degradation of the enzyme, whichwas already quite rapid (Fig. 1A, OA). This fact suggestedthat phosphorylation of certain proteins stimulates theirdegradation. TPH was the protein most likely to bephosphorylated, however, this meant that phosphorylatedTPH would be hardly detected unless the proteasome actionwas blocked. Indeed, in the absence of proteasomeinhibitor, phosphorylated TPH was not detectable in thesteady state labeling experiment where numerous cellularproteins were32P-labeled as shown in Fig. 1B,32P.Eveninthe presence of either okadaic acid (lane 2) or MG132 (notshown),32P-labeled TPH was undetectable, indicating thatthe 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 onlywhen both processes were blocked by simultaneous additionof okadaic acid, a protein phosphatase inhibitor, andMG132, a proteasome inhibitor (Fig. 1B,32P,lane3).The‘image math’ operation visualized the net increase in densityin lane 3 over lane 2 (Fig. 1B,32P, lane 4). This imagerepresented protein bands that fulfilled the following threeconditions simultaneously: (a) phosphorylation by cellularprotein kinases; (b) protection from dephosphorylation byokadaic 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 TPHvisualized by Western blot analysis of the same cell extractsrun with the authentic TPH protein as the staining referencemark (Fig. 1B, WB). This suggests that the proteasomeinhibitor MG132 might accumulate phosphorylated, sub-sequently ubiquitinylated TPH, the presumable substrate ofthe proteasomes. The experimental result was that MG132administered to living cells somehow raised TPH activityand increased the amount of TPH-like protein of molecularmass of 53 kDa (Fig. 1A, MG132 and 1B, WB lane 3),suggesting that de-ubiquinylating enzyme is considerablyactive [23]. Based on this outcome, phosphorylation of non-neural TPH and its role as an essential tag for proteindegradation were explored.Phosphorylation of non-neural TPHin vitroPurified TPH from mastocytoma P-815 cells, i.e. TPH ofnon-neural origin, was demonstrated to be phosphorylatedby CaM kinase II in vitro (Fig. 4). Although neural TPHand recombinant enzymes were reportedly phosphorylatedby PKA (reviewed in [38]), in the present study, we couldnot obtain positive evidence for PKA phosphorylation ofthe TPH from P-815 (Fig. 3). A possibility remains that ourTPH preparation was fully phosphorylated at the PKA-specific phosphorylation site before isolation and thereforeleft no room for further phosphorylation. Indeed, phenyl-alanine hydroxylase purified from rat liver containedendogenously phosphorylated subunits with 1.3 mol ofphosphate per tetramer and was fully phosphorylatedin vitro to give 1 mol per subunit by the catalytic subunitof PKA [39]. This possibility is difficult to rule out beforedirect measurement of endogenous phosphate [40]. Thepresent observation that TPH protein incorporated32P-phosphate by the action of CaM kinase II but not by PKAdoes not appear compatible with the idea that the site wasshared with PKA and CaM kinase II as is Ser58 of rat TPH[28]. For a solid conclusion, however, further investigation isrequired, such as site determination of kinase-specificphosphorylation.Phosphorylation of TPH as the tag for proteolysistargetingThe role of TPH phosphorylation is that it down regulatesthe TPH level in the cell by serving a tag for targeting fordigestion by proteasomes. This idea is based on thefollowing observations: (a) proteasome-driven TPH degra-dation in vivo (in RBL2H3 cells) was enhanced by okadaicacid, a protein phosphatase inhibitor (Fig. 1); (b) degrada-tion of exogenous TPH in a reconstituted proteasomesystem composed of RBL2H3 cell extracts was inhibited byboth KN-62, a CaM kinase II inhibitor, and K252a, apotent protein kinase inhibitor with broad specificity(Fig. 2); and (c) TPH previously phosphorylated by CaMkinase II was more rapidly degraded than nonphosphoryl-ated TPH in the same in vitro system. The cell-freeproteasome system employed in the present study wasconstructed with fresh extracts from RBL2H3 cells culturedunder ordinary conditions and prepared in the presence of2mMATP, 1 mMdithiothreitol and 0.25Msucrose tominimize the dissociation of proteasomes and possibledisruption of lysosomes [41]. Based on the sensitivity toprotein kinase inhibitors and to proteasome inhibitors, it isobvious that the cell-free proteasome system per se includeda relevant protein kinase system for TPH. Although theendogenous protein kinase shares properties with CaMkinase II in terms of sensitivity to inhibitors, the presence ofmultiple kinase species was also possible since KN-62 alonedid not completely prevent the proteolysis, even at concen-trations higher than 100 lM, while K252a of broadspecificity did. It was noteworthy that H-89, chelerythrineand staurospoline were not effective in preventing TPHdigestion, suggesting little contribution from PKA or PKCas far as the proteasome system in the RBL2H3 cells wasconcerned.Our observations supported the idea that phosphoryla-tion is a prerequisite for proteasome digestion of non-neural TPH. This phosphorylation enables the cell toseverely down-regulate the enzyme level by means ofstimulation of proteasome-driven degradation. This is anew role for TPH phosphorylation, which does notnecessarily alter its enzyme activity. It will be interesting tolearn whether the phosphorylation of neural TPH also has4786 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002an association with proteasome-dependent degradation,but there is as yet little information about TPH turnoverin the central nervous system. In addition, the questionremains as to how neural and non-neural TPH can appearso differentiated in view of their common amino acidsequence [19]. Studies on the cellular management of theircommon TPH gene, including RNA-processing, transla-tion of the message, post-translational processing,and cytosolic machinery such as phosphorylation andproteasome systems are just beginning to elucidate thedifferences between these two types of TPH.ACKNOWLEDGEMENTSWe are grateful to Dr T. Yamauchi for donating purified calmodulinand CaM kinase II and for instructions on its use. 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(1993) Purification and char-acterization of the 26S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins from rat liver.J. Biochem. (Tokyo) 113, 754–768.4788 Y. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line Yoshiko Iida1,. phosphorylated by this kinase, its enzymic activitywas not altered by the phosphorylation reaction.We examined the effect of TPH phosphorylation by CaMkinase
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