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c-Myc Is Glycosylated at Threonine 58, a Known Phosphorylation Site and a Mutational Hot Spot in Lymphomas

c-Myc Is Glycosylated at Threonine 58, a Known Phosphorylation Site and a Mutational Hot Spot in... Val. 270, No. 32, Issue of August 11, pp. 18961-18965, 1995 THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. © 1995 by The American Society for Biochemistry and Molecular Biology. Inc. c-Myc Is Glycosylated at Threonine 58, a Known Phosphorylation Site and a Mutational Hot Spot in Lymphomas* (Received for publication, June 13, 1995) Teh-Ying Chout, Gerald W. Harl§ll, and Chi V. Dan~I** From the :J;Biochemistry, Cellular, and Molecular Biology Training Program and l!Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the §Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294 cosamine (O-GlcNAc). The TAD is required for neoplastic c-Myc is a helix-loop-helix leucine zipper phosphopro­ transformation (5), inhibition of cellular differentiation (6), and tein that heterodimerizes with Max and regulates gene transcription in cell proliferation, cell differentiation, induction of apoptosis (7) mediated by c-Myc, and programmed cell death. Previously, we demon­ c-Myc can be phosphorylated by casein kinase II (8), MAP strated that c-Myc is modified by O-linked N-acetylglu­ kinase (9), or glycogen synthase kinase 3 (10). Phosphorylation cosamine (O-GlcNAc) within or nearby the N-terminal at Thr-58 and/or Ser-62 in the TAD of c-Myc has been sug­ transcriptional activation domain (Chou, T.-Y., Dang, C. gested to modulate the transactivation (11) and cellular trans­ V., and Hart, G. W. (1995) Proc, Natl. Acad. Sci. U.S.A. 92, formation (12) by c-Myc. Our previous study (13) showed that 4417-4421). In this paper, we identified the O-GlcNAc the TAD of c-Myc is also modified by O-GlcNAc, a form of attachment sitets) on c-Myc. c-Myc purified from sf9 in­ protein glycosylation composed of a single monosaccharide, sect cells was trypsinized, and its GlcNAc moieties were GlcNAc, linked to the side chain hydroxyl of serine or threonine enzymically labeled with [3H]galactose. The [3H]galac­ (14). O-GlcNAc has been found almost exclusively in the nu­ tose-labeled glycopeptides were isolated by reverse cleus and cytoplasm of eukaryotic cells. The known O-GlcNAc­ phase high performance liquid chromatography and bearing proteins share two common features; all of them are then subjected to gas-phase sequencing, manual Edman phosphoproteins and all form reversible multimeric complexes. degradation, and laser desorption/ionization mass spec­ Although the role of O-GIcNAc in altering protein function trometry. These analyses show that threonine 58, an in remains unknown, experiments to date suggest that the vivo phosphorylation site in the transactivation domain, O-GIcNAc modification is dynamic and appears to have a is the major O-GlcNAc glycosylation site of c-Myc. Muta­ reciprocal relationship with protein phosphorylation (15). tion of threonine 58, frequently found in retroviral v­ Myc proteins and in human Burkitt and AIDS-related In this report, using a variety of analytical techniques on lymphomas, is associated with enhanced transforming c-Myc overexpressed in insect cells, we provide evidence that activity and tumorigenicity. The reciprocal glycosyla­ O-GlcNAc occurs at c-Myc threonine 58, a known phos­ tion and phosphorylation at this biologically significant phorylation site and a frequently mutated hot spot in human amino acid residue may play an important role in the lymphomas. regulation of the functions of c-Myc, EXPERIMENTAL PROCEDURES Materials-sill insect cells were from Paragon. Monoclonal mouse c-Myc, the product of the c-myc protooncogene, is a nuclear anti-Myc antibody 9ElO was from American Type Culture Collection (ATCC). Sequencing grade trypsin (tosylphenylalanyl chloromethyl ke­ phosphoprotein of 439 amino acids that plays a critical role in 3H]galactose tone-treated) was from Worthington. UDP-[6- (38 Ci/mmo!) the regulation of gene transcription in normal and neoplastic was from Amersham Corp. Bovine milk galactosyltransferase from cells. Mutations of c-myc are associated with different types of Sigma (37.2 units/ml) was pregalactosylated as described (16). All other tumors in human and other species (1). c-Myc has several chemicals were of the highest quality commercially available. structural features conserved among many transcription fac­ Expression and Purification of c-Myc in Insect Cells-sill insect cells tors. A basic helix-loop-helix leucine zipper motif in the C­ grown in suspension culture were infected with recombinant baculovi­ terminal region mediates heterodimerization with Max (2) and rus Ac373lhc-myc (Ref. 17; a gift of G. Ju, Hoffmann-La Roche) accord­ ing to the method of Summers and Smith (18). The cells were harvested DNA binding to a specific E-box sequence, CACGTG or EMS 40 h postinfection, and the c-Myc protein was purified as described by (E-box myc site) (3). The N-terminal transcriptional activation Papoulas, Williams, and Kingston (19) with some changes. Briefly, ~1 domain (TAD)l (amino acids 1-143) (4) has a proline-rich ele­ X 10'0 cells were washed twice in phosphate-buffered saline and resus­ ment spanning from amino acid 41 to amino acid 103 (1), which pended at 2.5 X 10 cells/ml in low salt lysis buffer (20 mx Hepes, pH contains several potential sites for O-linked N-acetylglu- 6.8,5 mM KCl, 5 mM MgC12' 0.5% Nonidet P-40, 0.1% sodium deoxy­ cholate, 1 mg/ml aprotinin, 0.1 mMPMSF, 50 mM GlcNAc). After 10 min cells were subjected to 40 strokes in a Dounce homogenizer with a type * This work was supported by National Institutes of Health Grants A pestle. Nuclei were pelleted at 1,000 x g for 5 min at 4°C, washed CA42486 (to G. W. H.) and CA57341 (to C. V. D.). The costs of publica­ once in low salt lysis buffer, resuspended at 2 X 10 nuclei/ml in low salt tion of this article were defrayed in part by the payment of page lysis buffer containing 600 units/ml DNase I, and incubated at 4 °C for charges. This article must therefore be hereby marked "advertisement" 2 h. An equal volume of 2 X high salt lysis buffer (20 mM Tris, pH 7.4, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 MNaCl, 1 mMMgCI , 0.1% Nonidet P-40, 50 mM GIcNAc) was added, 11 To whom correspondence should be addressed. Tel.: 205-934-4786; mixed, and incubated for 10 min. The residual nuclear material was Fax: 205-975-6685; Internet: [email protected]. pelleted at 2,000 X g for 10 min at 4°C, resuspended for solubilization ** Scholar of the Leukemia Society of America. at 5 X 10 nuclear eq/ml in buffer A (50 mMTris, pH 8.0, 2 mM EDTA, 1 The abbreviations used are: TAD, transcriptional activation do­ main; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel 5% glycerol, 0.1 mM dithiothreitol, 0.1 mM PMSF, 5 Murea), vigorously electrophoresis; RP-HPLC, reverse phase high performance liquid chro­ stirred on ice for 30 min, and then centrifuged at 5,000 X g for 10 min matography; MALDI-MS, matrix-assisted laser desorption/ionization at 4°C. The supernatant was loaded at 0.1 mllmin on a 80-ml DEAE­ mass spectrometry. Sepharose CL-6B (Sigma) column pre-equilibrated with 5 column vol- This is an Open Access article under the CC BY license. 18962 Glyco sy lation of c-Myc Threonine 58 u rnes of bu ffer A. After loadin g, t he colu m n wa s wa sh ed at 0.4 m llmin 2 3 4 5 6 7 with 3 volu mes of buffe r A a n d th en 4 volu mes of bu ffer A contain ing 0. 15 ~I NaC!. c-Myc was elu te d by buffer A con ta in ing 0 .35 MN a C!. Th e c-Myc-con ta in ing frac t ions wer e pooled a nd diluted wit h buffer A to 0 .1 M N aC l a n d load ed a t 0 .5 ml/m in onto a 1-ml fast protein liq u id ch ro ­ ma t ogr aphy Mon o Q colu m n (P h a r ma cia Biot ech I nc.) pr e-equi librated wit h buffe r A contain in g 0.1 MNaC!. Th e colu m n wa s elute d in buffer A with a pr ogr amm ed gra die nt of 0.1- 2 M Na C!. Th e c-Myc wa s elu te d a t 0.2-0.4 M NaC l, and th e c-Myc con t a inin g fra ct ions we re pooled a n d di a ly zed aga ins t buffer con ta in ing 20 mxt T ri s , pH 7.8 , 50 mM KCl , 10% glycerol, 0. 1% dithi othreit ol, a nd 0 .1 mM PM SF in ba gs of Sp ectroP or 2 membran e for four ch an ges of 1 lit e r eac h and for 6 h eac h . All purifi ­ ca t ion ste ps we r e carr ied ou t on ice or with ice-cold buffer s. 2 3 4 5 6 7 SD S ·PA GE , Sil ver S ta in i ng , and West ern Bl ot A nal y s is-Pu rifi ed c-Myc wa s a na lyze d by 10% SDS -PA GE , s tai ne d wit h silver s tai ni ng, or tran s ferred t o nitr ocellu lose pap er for Western blot a na lys is u sin g mon oclon al mous e a n t i-Myc a n t ibody 9 E 10 (AT CC). 70­ T rypsi n Dig est ion of Purified c-My c and Gala ctosyltransfera se Lab el­ i ng of T ryp t ic Pep tides-Purifi ed c-Myc wa s di gested with tryp sin a t a 43- r a ti o of 1:10 t ryp sin t o pr ot ein (d is solved in 1 mM H Cn in 100 mM Tri s-H Cl, pH 8 .5, a t 37 °C for 18 h . Th e r eac t ion mi xt ure wa s ac idified by a dding 10% (v/v) triflu or oa ceti c ac id t o mak e a fin al con cen trat ion of 1% (v/ v) triflu or oa ce ti c ac id a n d loa ded onto a Sep-Pak C 18 ca rtrid ge (Wa te rs) . Th e t r yp tic peptid es we re elu te d with 60 % (v/v ) ace to n it r ile, d ri ed , r esu sp end ed in 80 J-ll of H 0 , a n d lab eled by ga la ctosy lt ra ns ­ fe r a s e (20) in lab elin g buffe r (10 mst Hep es , pH 7.4, 10 mM D( + )­ FIG. 1. Puri fi cation o f reco m b ina n t c-Myc from s ffi i n s e c t c e ll s. ga la ct ose, 5 mxt Mn ClJ with 50 J-lCi of U DP -["H ]ga lac tose a n d 0.1 unit Protein sa m ples from seq ue nt ia l s te ps of th e purification pro cedures of ga la ctosyltra nsfera se a t 37 °C for 4 h . An oth er 0.1 uni t of ga la ctosyl ­ were r esolved on t wo ide nt ica l 10% S DS-PA GE ge ls for s ilver s tai n ing t ra ns fera se a n d 5 J-lg of UDP -ga la ctose we re a dde d a fter 1 h of in cub a ­ (top p an el ) a n d Western blot a na lysis (bott om panel) a s described und er t io n . Th e lab el ed tryptic peptid es wer e pu r ified by a S ep-Pak C 18 "E xpe r ime n t a l Pr ocedures. " lan e 1, low sa lt lysis , tota l lys a te ; lane 2, cartrid ge . Th e 60% (v/ v) ace to n it r ile el ue n t wa s dri ed and a pplied to low sa lt lysi s , su pe rna ta n t ; lan e 3 , low sa lt lys is , pellet; lan e 4, hi gh sa lt lysi s , s u pe rnata n t; lan e 5, h igh sa lt lysis , pellet , 5 M u r ea ex t ra ct ; lan e RP -HPLC. 6 , a fte r DEAE CL-6 B column ; lan e 7, a fte r Mono Q column . Molecular I solation of {' H ]Ga lact ose-labeled Gly copep ti d es by RP·HPLC­ ma ss marker s of 70 kDa ( 70) a nd 43 kD a (43 ) a re sho wn on th e left . ["H] Ga lactose -la be le d glycope pt ide s wer e isolated by three rounds of RP -HPLC on a Rainin HPLC syst e m eq u ippe d wit h a Vyd a c 5-J-l m C 18 colu m n (0 .46 x 25 em) . Glycopeptid es we re load ed on to th e colu m n wi t h sm insect cells to map the O-GlcNAc a ddition sites of c-Myc , It 0 .5 rnxt sodiu m ph osp hate buffer , pH 7.0 (fir st dim en sion ), 0 .1% (v/v) is es t ima t ed t h at s m in sect cells infect ed wit h r ecom bin a n t t rifluo r oac etic a cid , pH 2 .0 (second dim en si on ), or 0.1% (w/v) a m m o­ baculovirus Ac373lhc -myc (17 ) expr ess mor e than 1 millio n nium acetate, pH 4 .0 (t h ir d dim en si on ) and elu te d with a gra die nt of molecu les of c-Myc per cell (da t a not s h own), an a mou nt at a ce t on it r ile fro m 0 t o 60 % (v/v). T h e flow rate wa s 1 mllmin , t h e elue n t lea st 10-fold mor e ab un dant than t h e c-Myc molecu les in H eLa wa s mon it or ed by a bsor ba nce a t A = 2 14 nm , frac t ions were collected cell s . Studies h a ve sh own t hat sm in sect cells , lik e vertebrate e ve ry minute , a n d t he ["H]ga lac to se -la be led glycope pt ides we r e de­ tected by liq uid sc in t illa tio n counting. cells, are ab le to pr oces s post-translationa l pr ot ein modifica ­ Ga s -p ha se S equ en cing - Isolated ["H]ga lactose -la be le d glycope pt ides tions , inclu din g O-GlcNAc (26, 27 ). A recent stu dy on hu ma n from th e third dim en si on RP -HPLC were se qu e nced by a u t oma te d cytomegalovirus tegument ba sic phosphoprotei n in dicated that Edm a n degradati on in a model 470A gas- phase se q ue nce r (Appl ied t he O-GlcNAc sit es of t h e recombinant bac u loviral protein Biosys tem s , In c .), faithfu lly corres pon d to tho se of the nativ e virion protein (28). M anu al Se q ue nt ial Edm an Degrada t ion-Aliquots of isolated ["H]ga lactose -la be led glycope pt ides fr om th e third dim en sion RP- HPLC c-Myc was pu r ified from 5 lit ers of in fecte d sm cells, h a r ­ wer e su bj ect ed to th e m anual Edm an degr ad ation method of Sulliv an vested at - 2 X 10 cell s/m!. Du r in g t he initial lysi s procedure, a n d Won g (2 1) wit h modification s as des crib ed by Kelly et al . (22 ). 50 mM GlcNAc wa s a dded to the buffers to partia lly inhibit th e Ma ss S pectro me try - Mat rix -ass is te d la se r des orpti on/i on ization en doge n ous h exosa min id a se activity. Most of the over ex­ ma ss s pect ro me t ry (MALDI-MS) wa s perform ed on a lin ear time-of­ pressed c-Myc pr ot ein app eare d to be bound to DNA a n d could flight ma s s s pect r ome te r built in-h ou se a n d describ ed pr evio u sl y by on ly be ext r a ct ed by 5 Mu r ea or SDS (19 ). Protein s a m ples from Che vr ier a nd Cot ter (23). seque n t ia l steps of purification wer e su bj ect ed to SDS -PAGE RESULTS AND DI SCUSSION for silver st a in in g and Western blot a na lysi s (F ig. 1). Proteins c-Myc protein level s h a ve been s h own to be very low in co-electrophoresing with c-Myc comprised mor e than 95% of norma l a n d transformed cells t hroug h t h e u se of imm u no logi­ th e tota l after purification by Mono Q chromatograp hy. When ca l ass ays (24). There are approximate ly 750 molec u le s of c­ O-GlcNAc in th is puri fied c-Myc pr eparation wa s la beled wit h Myc per cell in se r u m -st a rv ed fibrob la sts . After serum stim u­ [3H]ga lactose by ga lactosyltransferase, c-Myc wa s th e major la t ion , the numb er increa ses to 6,300 per eel!. HeLa cells , ra dioactive sign a l (13 ). whic h are tran sfor me d , contain - 9 7,000 molecule s of c-Myc per P urifie d c-Myc wa s fir st try psinized to ga in better accessibil­ eel!. Th e level s of cellu lar c-Myc polypepti de a ppe a r to be con­ ity to O-GlcNAc re sidues for subs eq uent ga lacto syltransferase st a n t t hroug hout t he cell cycle (25). Th e low ab un dance of lab eling (20). The O-GlcNAc-modified glycop epti des , labe le d c-Myc an d t h e in h erent lim itation of t he sens it ivit y of t r it iu m wit h [3H ]ga la ct os e by galactosyltransferase , were sepa ra t ed by la belin g render the det ecti on of carbohy drate moieties on c-Myc RP-H PLC at pH 7.0 on a C18 column in t he first dim ension . As an d th e su bse que n t m appi n g of carbohydrate sit es on c-Myc illustrate d in Fig. 2a , on ly on e major radioactive pea k wa s ext re me ly difficu lt . In our previou s wor k (13 ), we devel oped det ect ed , whic h contained - 48 pmol of la beled glycope pti de . se ns it ive methods to detect O-GlcNAc on in vitro translated T he fractio n corresponding to t h e ra dioactive pea k (fr a ct ion 65 ) c-Myc a n d id en tifi ed a N-t e rmina l r egion of c-Myc modifi ed by wa s ap pli ed to a s econd dimen sio n RP-HPLC at pH 2.0 and t he O-GlcNAc. We al so overex pre s se d t he c-Myc protein in either radioactivity elu te d as a si ngle peak (F ig. 2b ). Wh en the frac ­ s m in s ect cells or Ch inese ham ster ovary cells an d demon­ tion containin g thi s r adi oa ct ive peak wa s appli ed to a third st r a te d that c-Myc ex presse d in the se cell s is modifi ed by 0 ­ dim en sion RP -HPLC a t pH 4 .0, a sing le r a dioactive peak wa s GlcNAc. In t h e pr esent s t u dy, we used c-Myc overexpres se d in elu t ed (F ig. 2c ). Th e yie ld of radio labeled glycope pt ide a fte r 18963 Glycosylation of c-Myc Threonine 58 Phenylthiohydantoin-Amino Acids: cme AmIno Acids 1. D,G>S>A,F 2. T> F, E 3. H. F, L> V 4. K>A > E, L 5. S>L>P 6. E 7. I>D>P.V 8. A>Q>K,P 9. H > I, L, F, K 10. R> P 11. F>E>K 12. K > L Amino Acid Sequence of Peptides: 1. DTHKSEIAHRFK(DLGEEHFK) 2. SFFAL(R) I SFFAL(R)DQIPEL(ENNEK) 3. FELLP(T)PPL(SPSR) FIG. 3. Amino acids and peptide sequences derived from gas­ )( phase sequencing. [3HJGalactose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to gas-phase sequencing. u Phenylthiohydantoin-amino acids released in each cycle in order of abundance (high> low) are listed in the top panel. Peptide sequences deduced from the phenylthiohydantoin-amino acids released and tryp­ tic maps of c-Myc and bovine serum albumin (as described under 40 60 80 100 120 140 0 20 "Results and Discussion") are listed in the bottom panel. Fraction Number ... f 100 :E c. '0 "' 0.0 140.0 ~ 2 3 4 5 6 7 8 9 1 0 1 1 1 2 DISC ... 3 Cycle Number )( 2: 2 FIG. 4. Determination of O-GlcNAc site on a c-Myc tryptic gly. copeptide by sequential manual Edman degradation. [3HJGalac­ tose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to manual sequential Edman degradation. Counts released in 0 each cycle (numbers) and counts from the disc after all 12 cycles (DISC) 0 20 40 60 II 1110 120 140 were plotted. Fraction Number these multiple HPLC analyses was -12.5% due to the "sticky" C nature of this glycopeptide. After the third round of HPLC, the UV absorbance profile suggested that this peptide was fairly ... homogeneous. Therefore, further fractionation (e.g. ion-ex­ /'00 change chromatography) was not performed (however, see • 0 below) . • 0 An aliquot containing 5.5 pmol of [3H]galactose-Iabeled gly­ copeptide from the third dimension RP-HPLC was subjected to gas-phase sequencing. The relative abundance of amino acids recovered in each sequencing cycle surprisingly suggested the 0.0 presence of three major co-purified peptides (Fig. 3): DTHK· SEIAHRFK(DLGEEHFK) (-15 pmol) , SFFAL(R) or SF­ FAL(R)DQIPEL(ENNEK) (-10 pmol), and FELLP(T)PPL­ (SPSR) (-5 pmol). The two less abundant peptides were from c-Myc tryptic fragments of amino acids 373-378, SFFALR (or )( 373-389, SFFALRDQIPELENNEK), in the first helix (and loop) region and amino acids 53-65, FELLPTPPLSPSR, in the syltransferase, and separated by RP-HPLC as described under "Exper­ .. .. imental Procedures." a, first dimension; b, second dimension; c, third • • • •• dimension. Top panels, absorbance profile of eluted peptides; bottom Fraction Number panels, tritium profile of eluted peptides. The straight line in the top FIG. 2. Isolation of [3Hlgalactose-Iabeled tryptic glycopep­ panel represents acetonitrile gradient. %B is percentage of 60% (v/v) tides. Purified c-Myc was digested with trypsin, labeled with galacto- acetonitrile. 18964 Glycosylation of c-Myc Threonine 58 FELLPTPPISPSR + GlcNAc-[3H]GaI + 2 x Na FIG. 5. Identification of [3Hlgalac­ tose-Iabeled FELLPTPPLSPSR by mass spectrometry. [3HlGalactose-la­ .0 ~ 60 271.7.7 beled tryptic glycopeptides from third di­ mension RP-HPLC were subjected to ~ so MALDI-MS. The m l z of 1867.3 repre­ sents the molecular mass ofFELLPTPPL­ 865.6 SPSR (molecular mass, 1453.71) plus Q) 40­ [3H]Galj31-4GIcNAc (367.33) plus two so- ~ dium (2 X 22.99) (total = 1867.02). For ~ 30 the assignment of the other peaks, see Q) "Results and Discussion." ~ 20 10 -- sao 'BOO Maas/Charge transactivation domain. A protein data bank search (BLAST, sponding to FELLPTPPLSPSR (1453.71) plus GlcNAc (203.18) National Institutes of Health) revealed the more abundant plus sodium (22.99) plus H 0 (18.02) (total = 1697.9). Since peptide, DTHKSEIAHRFKDLGEEHFK, was from tryptic frag­ both glycosylated and non-glycosylated forms of c-Myc are pres­ ments of bovine serum albumin. Fetal bovine serum (10%) ent in cells and the galactosyltransferase labeling is not com­ added in the insect cell culture medium is likely the source of pletely efficient (20), the co-existence of FELLPTPPLSPSR, this contaminating peptide. Apparently, this serum-derived FELLPTPPLSPSR with GlcNAc, and FELLPTPPLSPSR with peptide either exactly co-migrates with the c-myc peptides or [3Hlgalactosylated GlcNAc is expected. In addition, RP-HPLC binds to them with high affinity during HPLC purification. The generally does not resolve unmodified, O-GlcNAc-modified, or presence of this contaminant is surprising since we have typi­ galactosylated O-GlcNAc-modified peptides (20). Furthermore, cally found this iterative RP-HPLC method to provide more we have recently found that O-GlcNAc saccharides are rapidly than adequate purification of glycopeptides for sequencing and selectively lost during ionization in electrospray mass spec­ (20,28). trometry." Also found in the mass spectrometry were a peak Since the three major peptides detected by the gas-phase with m/z 865.6 corresponding to SFFALR (739.88) plus phos­ sequencing contain serine or threonine at different positions, phate (79.98) plus two sodium (2 X 22.99) (total = 865.84) and sequential manual Edman degradation, followed by scintilla­ a peak with mlz 2717.7 corresponding to DTHKSEIAHRFKDL­ tion counting to detect the released radiolabeled saccharide, GEEHFK (2405.13) plus copper (63.55) plus hexose (162.14) provides an unambiguous assignment of the site of O-GlcNAc plus three sodium (3 x 22.99) plus H 0 (18.02) (total = 2717.54). modification. The result of sequential manual Edman degrada­ The peptide DTHKSEIAHRFKDLGEEHFK contains a copper tion (Fig. 4) indicates that the threonine residue in FELLPT­ chelating site at its histidine residues. A non-enzymatic glycation PPLSPSR (threonine 58) is glycosylated. Only the peptide at lysine 12 of the peptide DTHKSEIAHRFKDLGEEHFK has FELLPTPPLSPSR has a threonine or serine at the sixth amino been reported (29). From the data of gas-phase sequencing, se­ acid, the cycle in which the radiolabel is released. The assign­ quential Edman degradation, and mass spectrometry we con­ ment of O-GlcNAc glycosylation to threonine 58 is also sup­ clude that threonine 58 is the major O-GlcNAc site of c-Myc. ported by two other observations. First, the major radioactive Threonine 58 is in the TAD of c-Myc within the region where peak from the first, second, and third round ofRP-HPLC eluted we previously localized O-GlcNAc by more indirect methods at 22.5, 26.8, and 26.0% acetonitrile, respectively, consistent (13). It has been shown that threonine 58 is a phosphorylation with the predicted retention times for the peptide FELLPTP­ site in vivo (30) and can be phosphorylated in vitro by glycogen PLSPSR (41). Second, the gas-phase sequencing data showed synthase kinase 3 (10). Threonine 58 is altered to a methionine the amount of the released phenylthiohydantoin-threonine in MC29 and HB1 v-Myc and to an alanine in OKlO and MH2 was smaller than other internal residues of the peptide v-Myc (31). These mutations of threonine 58 in v-Myc enhance F(5.4)E(1.5)L( 4.1)L(2.4)P(2. 7)T( <0.5)P(1.1)P(0.5)L(4.3)S­ the transforming activity of Myc protein (32, 33). On the other «0.5)P( <0.5)S( <0.5)R (repetitive yields are in parentheses hand, a v-Myc protein with a threonine at amino acid 58 has a following each amino acid), suggesting that threonine 58 has reduced capability to induce growth in soft agar by non-trans­ been modified. This low recovery of threonine 58 also ruled out formed embryo fibroblasts (34, 35). Comparison of c-Myc and v-Myc by a variety of transformation assays also revealed that the possibility that the released radioactivity in the sixth se­ quencing cycle came from a small amount of contaminating c-Myc has a reduced ability to induce tumor formation (33). peptide. These results suggest that threonine 58 has a key role in The conclusion that threonine 58 is the site of O-GlcNAc transducing a negative growth signal of c-Myc through its was further confirmed by analyzing the peptides by post-translational modifications. This working hypothesis is MALDI-MS (Fig. 5). A peak with mlz 1867.3 was assigned to supported by the observations that mutations of c-Myc at or near threonine 58 are frequently found in Burkitt or AIDS­ the mass of FELLPTPPLSPSR (1453.71) plus [3H1Galf31­ 4GlcNAc (367.33) plus two sodium (2 x 22.99) (total = related lymphomas, and threonine 58 is the most frequently 1867.02). We also noted a peak with mlz 1493.9 corresponding to FELLPTPPLSPSR (1453.71) plus sodium (22.99) plus H 0 (18.02) (total = 1494.72) and a peak with m/z 1696.5 corre- 2 K. Greis, B. Hayes, and G. W. Hart, unpublished observations. Glycosylation of c-Myc Threonine 58 18965 Biochem. 58,841-874 mutated amino acid of c-Myc in these tumors (36-40). 15. Hart, G. W., Kelly, W. G., Blomberg, M. A., Roquemore, E. P., Dong, L.-Y. D., Since mutations altering threonine 58 augment c-Myc trans­ Kreppel, L., Chou, T.-Y., Snow, D., and Greis, K. (1993) in 44. Colloquium forming ability, we speculate that reciprocal phosphorylation! Mosbach 1993 Glyco- and Cell Biology, pp. 91-103, Springer-Verlag, Berlin 16. Holt, G. D., and Hart, G. W. (1986) J. Bioi. Chem. 261, 8049-8057 O-GlcNAc glycosylation modulate the activity of c-Myc. With 17. Miyamoto, C., Smith, G. E., Farrell-Towt, J., Chizzonite, R., Summers, M. D., the observation that c-Myc polypeptide levels remain relatively and Ju, G. (1985) Mol. Cell. BioI. 5,2860-2865 constant throughout the cell cycle (25), we also propose that 18. Summers, M. D., and Smith, G. E. (1987)A Manual of Me thuds for Baculovirus Vectors and Insect Cell Culture Procedures, Department of Entomology, these reciprocal post-translational modifications of threonine Texas Agricultural Experiment Station and Texas A&M University, College 58 differentially regulate c-Myc functions in different stages of Station, TX the cell cycle. 19. Papoulas, 0., Williams, N. G., and Kingston, R. E. (1992) J. Bioi. Chem. 267, 10470-10480 20. Roquemore, E. P., Chou, T.-Y., and Hart, G. W. (1994) Methods Enzymol. 230, Acknowledgments-We thank Dr. Grace Ju for providing Ac373/hc­ 443-460 myc, Dr. Dennis L-Y. Dong for helpful suggestions, Dr. Wu-Schyong Liu 21. Sullivan, S., and Wong, T. W. (1991) Anal. Biochem. 197,65-68 for help in gas-phase sequencing, and Drs. Amina S. Woods and Mar­ 22. Kelly, W. G., Dahmus, M. E., and Hart, G. W. (1993) J. BioI. Chem. 268, cela M. Cordero for help in mass spectrometry. We also thank Dr. 10416-10424 Joseph Eiden for help with the recombinant baculoviral protein expres­ 23. Chevrier, M. R., and Cotter, R. J. (1991) Rapid Commun. Mass Spectrom. 5, sion system. 611-617 24. Moore, J. P., Hancock, D. C., Littlewood, T. D., and Evan, G. 1. (1987) Oncogene REFERENCES Res. 2, 65- 80 1. Kato, G. J., and Dang, C. V. (1992) FASEB J. 6,3065-3072 25. Hann, S. R., Thompson, C. B., and Eisenman, R. N. (1985) Nature 314, 2. Blackwood, E. M., and Eisenman, R. N. (1991) Science 251, 1211-1217 366-369 3. Blackwell, T. K., Kretzner, I.., Blackwood, E. M., Eisenman, R. N., and 26. Faulkner, P., and Whitford, M. (1992) J. Virol. 3324-3329 Weintraub, H. (1990) Science 251, 1149-1151 27. Ku, N.-O., and Omary, M. B. (1994) Exp. Cell Res. 211,24-35 4. Kato, G. J., Barrett, J., Villa-Garcia, M. C., and Dang, C. V. (1989) Mol. Cell. 28. Greis, K. D., Gibson, W., and Hart, G. W. (1994) J. Virol. 68,8339-8349 BioI. 10, 5914-5920 29. Iberg, N., and Fluckiger, R. (1986) J. Bioi. Chem. 261, 13542-13545 5. Stone, J., De Lange, T., Ramsay, G., Jakobovits, E., Bishop, J. M., Varmus, H., 30. Lutterbach, B., and Hann, S. R. (1994) Mol. Cell. BioI. 14,5510-5522 and Lee, W. (1987) Mol. Cell. BioI. 7, 1697-1709 31. Papas, T. S., and Lautenberger, J. A. (1985) Nature 318, 237 6. Freytag, S. 0., Dang, C. V., and Lee, W. M.-F. (1990) Cell Growth & Differ. 1, 32. Raffeld, M., Yano, T., Hoang, A. T., Lewis, B. C., Clark, H. M., Otsuki, T., and 339-343 Dang, C. V. (1995) Curro Top. Microbial. Immunol. 194,265-272 7. Evan, G. 1., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., 33. Frykberg, L., Graf, T., and Vennstrom, B. (1987) Oncogene 1,415-421 Waters, C. M., Penn, L. Z., and Hancock, D. C. (1992) Cell 69, 119-128 34. Palmieri, S., Kahn, P., and Graf, T. (1983) EMBO J. 2,2385-2389 8. Luscher, B., Kuenzel, E. A., Krebs, E. G., and Eisenman, R. N. (1989) EMBO 35. Symonds, G., Hartshorn, A., Kennewell, A., O'Mara, M.-A., Bruskin, A., and J. 8, 1111-1119 Bishop, J. M. (1989) Oncogene 4, 285-294 9. Seth, A., Alvarez, E., Gupta, S., and Davis, R. J. (1991) J. Bioi. Chem. 226, 36. Rabbitts, T:, Hamlyn, P., and Baer, R. (1983) Nature 306, 760-765 23521-23524 37. Yano, T., Sander, C. A., Clark, H. M., Dolezal, M. V., Jaffe, E. S., and Raffeld, 10. Henriksson, M., Bakardjiev, A., Klein, G., and Luscher, B. (1993) Oncogene 8, M. (1993) Oncogene 8, 2741-2748 3199-3209 38. Clark, H. M., Yano, T., Otsuki, T., Jaffe, E. S., Shibata, D., and Raffeld, M. 11. Albert, T., Urlbauer, B., Kohlhuber, F., Hammersen, B., and Eick, D. 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c-Myc Is Glycosylated at Threonine 58, a Known Phosphorylation Site and a Mutational Hot Spot in Lymphomas

Chou, Teh-Ying; Hart, Gerald W.; Dang, Chi V.
Journal of Biological Chemistry – Aug 1, 1995
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10.1074/jbc.270.32.18961
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Val. 270, No. 32, Issue of August 11, pp. 18961-18965, 1995 THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. © 1995 by The American Society for Biochemistry and Molecular Biology. Inc. c-Myc Is Glycosylated at Threonine 58, a Known Phosphorylation Site and a Mutational Hot Spot in Lymphomas* (Received for publication, June 13, 1995) Teh-Ying Chout, Gerald W. Harl§ll, and Chi V. Dan~I** From the :J;Biochemistry, Cellular, and Molecular Biology Training Program and l!Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the §Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294 cosamine (O-GlcNAc). The TAD is required for neoplastic c-Myc is a helix-loop-helix leucine zipper phosphopro­ transformation (5), inhibition of cellular differentiation (6), and tein that heterodimerizes with Max and regulates gene transcription in cell proliferation, cell differentiation, induction of apoptosis (7) mediated by c-Myc, and programmed cell death. Previously, we demon­ c-Myc can be phosphorylated by casein kinase II (8), MAP strated that c-Myc is modified by O-linked N-acetylglu­ kinase (9), or glycogen synthase kinase 3 (10). Phosphorylation cosamine (O-GlcNAc) within or nearby the N-terminal at Thr-58 and/or Ser-62 in the TAD of c-Myc has been sug­ transcriptional activation domain (Chou, T.-Y., Dang, C. gested to modulate the transactivation (11) and cellular trans­ V., and Hart, G. W. (1995) Proc, Natl. Acad. Sci. U.S.A. 92, formation (12) by c-Myc. Our previous study (13) showed that 4417-4421). In this paper, we identified the O-GlcNAc the TAD of c-Myc is also modified by O-GlcNAc, a form of attachment sitets) on c-Myc. c-Myc purified from sf9 in­ protein glycosylation composed of a single monosaccharide, sect cells was trypsinized, and its GlcNAc moieties were GlcNAc, linked to the side chain hydroxyl of serine or threonine enzymically labeled with [3H]galactose. The [3H]galac­ (14). O-GlcNAc has been found almost exclusively in the nu­ tose-labeled glycopeptides were isolated by reverse cleus and cytoplasm of eukaryotic cells. The known O-GlcNAc­ phase high performance liquid chromatography and bearing proteins share two common features; all of them are then subjected to gas-phase sequencing, manual Edman phosphoproteins and all form reversible multimeric complexes. degradation, and laser desorption/ionization mass spec­ Although the role of O-GIcNAc in altering protein function trometry. These analyses show that threonine 58, an in remains unknown, experiments to date suggest that the vivo phosphorylation site in the transactivation domain, O-GIcNAc modification is dynamic and appears to have a is the major O-GlcNAc glycosylation site of c-Myc. Muta­ reciprocal relationship with protein phosphorylation (15). tion of threonine 58, frequently found in retroviral v­ Myc proteins and in human Burkitt and AIDS-related In this report, using a variety of analytical techniques on lymphomas, is associated with enhanced transforming c-Myc overexpressed in insect cells, we provide evidence that activity and tumorigenicity. The reciprocal glycosyla­ O-GlcNAc occurs at c-Myc threonine 58, a known phos­ tion and phosphorylation at this biologically significant phorylation site and a frequently mutated hot spot in human amino acid residue may play an important role in the lymphomas. regulation of the functions of c-Myc, EXPERIMENTAL PROCEDURES Materials-sill insect cells were from Paragon. Monoclonal mouse c-Myc, the product of the c-myc protooncogene, is a nuclear anti-Myc antibody 9ElO was from American Type Culture Collection (ATCC). Sequencing grade trypsin (tosylphenylalanyl chloromethyl ke­ phosphoprotein of 439 amino acids that plays a critical role in 3H]galactose tone-treated) was from Worthington. UDP-[6- (38 Ci/mmo!) the regulation of gene transcription in normal and neoplastic was from Amersham Corp. Bovine milk galactosyltransferase from cells. Mutations of c-myc are associated with different types of Sigma (37.2 units/ml) was pregalactosylated as described (16). All other tumors in human and other species (1). c-Myc has several chemicals were of the highest quality commercially available. structural features conserved among many transcription fac­ Expression and Purification of c-Myc in Insect Cells-sill insect cells tors. A basic helix-loop-helix leucine zipper motif in the C­ grown in suspension culture were infected with recombinant baculovi­ terminal region mediates heterodimerization with Max (2) and rus Ac373lhc-myc (Ref. 17; a gift of G. Ju, Hoffmann-La Roche) accord­ ing to the method of Summers and Smith (18). The cells were harvested DNA binding to a specific E-box sequence, CACGTG or EMS 40 h postinfection, and the c-Myc protein was purified as described by (E-box myc site) (3). The N-terminal transcriptional activation Papoulas, Williams, and Kingston (19) with some changes. Briefly, ~1 domain (TAD)l (amino acids 1-143) (4) has a proline-rich ele­ X 10'0 cells were washed twice in phosphate-buffered saline and resus­ ment spanning from amino acid 41 to amino acid 103 (1), which pended at 2.5 X 10 cells/ml in low salt lysis buffer (20 mx Hepes, pH contains several potential sites for O-linked N-acetylglu- 6.8,5 mM KCl, 5 mM MgC12' 0.5% Nonidet P-40, 0.1% sodium deoxy­ cholate, 1 mg/ml aprotinin, 0.1 mMPMSF, 50 mM GlcNAc). After 10 min cells were subjected to 40 strokes in a Dounce homogenizer with a type * This work was supported by National Institutes of Health Grants A pestle. Nuclei were pelleted at 1,000 x g for 5 min at 4°C, washed CA42486 (to G. W. H.) and CA57341 (to C. V. D.). The costs of publica­ once in low salt lysis buffer, resuspended at 2 X 10 nuclei/ml in low salt tion of this article were defrayed in part by the payment of page lysis buffer containing 600 units/ml DNase I, and incubated at 4 °C for charges. This article must therefore be hereby marked "advertisement" 2 h. An equal volume of 2 X high salt lysis buffer (20 mM Tris, pH 7.4, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 MNaCl, 1 mMMgCI , 0.1% Nonidet P-40, 50 mM GIcNAc) was added, 11 To whom correspondence should be addressed. Tel.: 205-934-4786; mixed, and incubated for 10 min. The residual nuclear material was Fax: 205-975-6685; Internet: [email protected]. pelleted at 2,000 X g for 10 min at 4°C, resuspended for solubilization ** Scholar of the Leukemia Society of America. at 5 X 10 nuclear eq/ml in buffer A (50 mMTris, pH 8.0, 2 mM EDTA, 1 The abbreviations used are: TAD, transcriptional activation do­ main; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel 5% glycerol, 0.1 mM dithiothreitol, 0.1 mM PMSF, 5 Murea), vigorously electrophoresis; RP-HPLC, reverse phase high performance liquid chro­ stirred on ice for 30 min, and then centrifuged at 5,000 X g for 10 min matography; MALDI-MS, matrix-assisted laser desorption/ionization at 4°C. The supernatant was loaded at 0.1 mllmin on a 80-ml DEAE­ mass spectrometry. Sepharose CL-6B (Sigma) column pre-equilibrated with 5 column vol- This is an Open Access article under the CC BY license. 18962 Glyco sy lation of c-Myc Threonine 58 u rnes of bu ffer A. After loadin g, t he colu m n wa s wa sh ed at 0.4 m llmin 2 3 4 5 6 7 with 3 volu mes of buffe r A a n d th en 4 volu mes of bu ffer A contain ing 0. 15 ~I NaC!. c-Myc was elu te d by buffer A con ta in ing 0 .35 MN a C!. Th e c-Myc-con ta in ing frac t ions wer e pooled a nd diluted wit h buffer A to 0 .1 M N aC l a n d load ed a t 0 .5 ml/m in onto a 1-ml fast protein liq u id ch ro ­ ma t ogr aphy Mon o Q colu m n (P h a r ma cia Biot ech I nc.) pr e-equi librated wit h buffe r A contain in g 0.1 MNaC!. Th e colu m n wa s elute d in buffer A with a pr ogr amm ed gra die nt of 0.1- 2 M Na C!. Th e c-Myc wa s elu te d a t 0.2-0.4 M NaC l, and th e c-Myc con t a inin g fra ct ions we re pooled a n d di a ly zed aga ins t buffer con ta in ing 20 mxt T ri s , pH 7.8 , 50 mM KCl , 10% glycerol, 0. 1% dithi othreit ol, a nd 0 .1 mM PM SF in ba gs of Sp ectroP or 2 membran e for four ch an ges of 1 lit e r eac h and for 6 h eac h . All purifi ­ ca t ion ste ps we r e carr ied ou t on ice or with ice-cold buffer s. 2 3 4 5 6 7 SD S ·PA GE , Sil ver S ta in i ng , and West ern Bl ot A nal y s is-Pu rifi ed c-Myc wa s a na lyze d by 10% SDS -PA GE , s tai ne d wit h silver s tai ni ng, or tran s ferred t o nitr ocellu lose pap er for Western blot a na lys is u sin g mon oclon al mous e a n t i-Myc a n t ibody 9 E 10 (AT CC). 70­ T rypsi n Dig est ion of Purified c-My c and Gala ctosyltransfera se Lab el­ i ng of T ryp t ic Pep tides-Purifi ed c-Myc wa s di gested with tryp sin a t a 43- r a ti o of 1:10 t ryp sin t o pr ot ein (d is solved in 1 mM H Cn in 100 mM Tri s-H Cl, pH 8 .5, a t 37 °C for 18 h . Th e r eac t ion mi xt ure wa s ac idified by a dding 10% (v/v) triflu or oa ceti c ac id t o mak e a fin al con cen trat ion of 1% (v/ v) triflu or oa ce ti c ac id a n d loa ded onto a Sep-Pak C 18 ca rtrid ge (Wa te rs) . Th e t r yp tic peptid es we re elu te d with 60 % (v/v ) ace to n it r ile, d ri ed , r esu sp end ed in 80 J-ll of H 0 , a n d lab eled by ga la ctosy lt ra ns ­ fe r a s e (20) in lab elin g buffe r (10 mst Hep es , pH 7.4, 10 mM D( + )­ FIG. 1. Puri fi cation o f reco m b ina n t c-Myc from s ffi i n s e c t c e ll s. ga la ct ose, 5 mxt Mn ClJ with 50 J-lCi of U DP -["H ]ga lac tose a n d 0.1 unit Protein sa m ples from seq ue nt ia l s te ps of th e purification pro cedures of ga la ctosyltra nsfera se a t 37 °C for 4 h . An oth er 0.1 uni t of ga la ctosyl ­ were r esolved on t wo ide nt ica l 10% S DS-PA GE ge ls for s ilver s tai n ing t ra ns fera se a n d 5 J-lg of UDP -ga la ctose we re a dde d a fter 1 h of in cub a ­ (top p an el ) a n d Western blot a na lysis (bott om panel) a s described und er t io n . Th e lab el ed tryptic peptid es wer e pu r ified by a S ep-Pak C 18 "E xpe r ime n t a l Pr ocedures. " lan e 1, low sa lt lysis , tota l lys a te ; lane 2, cartrid ge . Th e 60% (v/ v) ace to n it r ile el ue n t wa s dri ed and a pplied to low sa lt lysi s , su pe rna ta n t ; lan e 3 , low sa lt lys is , pellet; lan e 4, hi gh sa lt lysi s , s u pe rnata n t; lan e 5, h igh sa lt lysis , pellet , 5 M u r ea ex t ra ct ; lan e RP -HPLC. 6 , a fte r DEAE CL-6 B column ; lan e 7, a fte r Mono Q column . Molecular I solation of {' H ]Ga lact ose-labeled Gly copep ti d es by RP·HPLC­ ma ss marker s of 70 kDa ( 70) a nd 43 kD a (43 ) a re sho wn on th e left . ["H] Ga lactose -la be le d glycope pt ide s wer e isolated by three rounds of RP -HPLC on a Rainin HPLC syst e m eq u ippe d wit h a Vyd a c 5-J-l m C 18 colu m n (0 .46 x 25 em) . Glycopeptid es we re load ed on to th e colu m n wi t h sm insect cells to map the O-GlcNAc a ddition sites of c-Myc , It 0 .5 rnxt sodiu m ph osp hate buffer , pH 7.0 (fir st dim en sion ), 0 .1% (v/v) is es t ima t ed t h at s m in sect cells infect ed wit h r ecom bin a n t t rifluo r oac etic a cid , pH 2 .0 (second dim en si on ), or 0.1% (w/v) a m m o­ baculovirus Ac373lhc -myc (17 ) expr ess mor e than 1 millio n nium acetate, pH 4 .0 (t h ir d dim en si on ) and elu te d with a gra die nt of molecu les of c-Myc per cell (da t a not s h own), an a mou nt at a ce t on it r ile fro m 0 t o 60 % (v/v). T h e flow rate wa s 1 mllmin , t h e elue n t lea st 10-fold mor e ab un dant than t h e c-Myc molecu les in H eLa wa s mon it or ed by a bsor ba nce a t A = 2 14 nm , frac t ions were collected cell s . Studies h a ve sh own t hat sm in sect cells , lik e vertebrate e ve ry minute , a n d t he ["H]ga lac to se -la be led glycope pt ides we r e de­ tected by liq uid sc in t illa tio n counting. cells, are ab le to pr oces s post-translationa l pr ot ein modifica ­ Ga s -p ha se S equ en cing - Isolated ["H]ga lactose -la be le d glycope pt ides tions , inclu din g O-GlcNAc (26, 27 ). A recent stu dy on hu ma n from th e third dim en si on RP -HPLC were se qu e nced by a u t oma te d cytomegalovirus tegument ba sic phosphoprotei n in dicated that Edm a n degradati on in a model 470A gas- phase se q ue nce r (Appl ied t he O-GlcNAc sit es of t h e recombinant bac u loviral protein Biosys tem s , In c .), faithfu lly corres pon d to tho se of the nativ e virion protein (28). M anu al Se q ue nt ial Edm an Degrada t ion-Aliquots of isolated ["H]ga lactose -la be led glycope pt ides fr om th e third dim en sion RP- HPLC c-Myc was pu r ified from 5 lit ers of in fecte d sm cells, h a r ­ wer e su bj ect ed to th e m anual Edm an degr ad ation method of Sulliv an vested at - 2 X 10 cell s/m!. Du r in g t he initial lysi s procedure, a n d Won g (2 1) wit h modification s as des crib ed by Kelly et al . (22 ). 50 mM GlcNAc wa s a dded to the buffers to partia lly inhibit th e Ma ss S pectro me try - Mat rix -ass is te d la se r des orpti on/i on ization en doge n ous h exosa min id a se activity. Most of the over ex­ ma ss s pect ro me t ry (MALDI-MS) wa s perform ed on a lin ear time-of­ pressed c-Myc pr ot ein app eare d to be bound to DNA a n d could flight ma s s s pect r ome te r built in-h ou se a n d describ ed pr evio u sl y by on ly be ext r a ct ed by 5 Mu r ea or SDS (19 ). Protein s a m ples from Che vr ier a nd Cot ter (23). seque n t ia l steps of purification wer e su bj ect ed to SDS -PAGE RESULTS AND DI SCUSSION for silver st a in in g and Western blot a na lysi s (F ig. 1). Proteins c-Myc protein level s h a ve been s h own to be very low in co-electrophoresing with c-Myc comprised mor e than 95% of norma l a n d transformed cells t hroug h t h e u se of imm u no logi­ th e tota l after purification by Mono Q chromatograp hy. When ca l ass ays (24). There are approximate ly 750 molec u le s of c­ O-GlcNAc in th is puri fied c-Myc pr eparation wa s la beled wit h Myc per cell in se r u m -st a rv ed fibrob la sts . After serum stim u­ [3H]ga lactose by ga lactosyltransferase, c-Myc wa s th e major la t ion , the numb er increa ses to 6,300 per eel!. HeLa cells , ra dioactive sign a l (13 ). whic h are tran sfor me d , contain - 9 7,000 molecule s of c-Myc per P urifie d c-Myc wa s fir st try psinized to ga in better accessibil­ eel!. Th e level s of cellu lar c-Myc polypepti de a ppe a r to be con­ ity to O-GlcNAc re sidues for subs eq uent ga lacto syltransferase st a n t t hroug hout t he cell cycle (25). Th e low ab un dance of lab eling (20). The O-GlcNAc-modified glycop epti des , labe le d c-Myc an d t h e in h erent lim itation of t he sens it ivit y of t r it iu m wit h [3H ]ga la ct os e by galactosyltransferase , were sepa ra t ed by la belin g render the det ecti on of carbohy drate moieties on c-Myc RP-H PLC at pH 7.0 on a C18 column in t he first dim ension . As an d th e su bse que n t m appi n g of carbohydrate sit es on c-Myc illustrate d in Fig. 2a , on ly on e major radioactive pea k wa s ext re me ly difficu lt . In our previou s wor k (13 ), we devel oped det ect ed , whic h contained - 48 pmol of la beled glycope pti de . se ns it ive methods to detect O-GlcNAc on in vitro translated T he fractio n corresponding to t h e ra dioactive pea k (fr a ct ion 65 ) c-Myc a n d id en tifi ed a N-t e rmina l r egion of c-Myc modifi ed by wa s ap pli ed to a s econd dimen sio n RP-HPLC at pH 2.0 and t he O-GlcNAc. We al so overex pre s se d t he c-Myc protein in either radioactivity elu te d as a si ngle peak (F ig. 2b ). Wh en the frac ­ s m in s ect cells or Ch inese ham ster ovary cells an d demon­ tion containin g thi s r adi oa ct ive peak wa s appli ed to a third st r a te d that c-Myc ex presse d in the se cell s is modifi ed by 0 ­ dim en sion RP -HPLC a t pH 4 .0, a sing le r a dioactive peak wa s GlcNAc. In t h e pr esent s t u dy, we used c-Myc overexpres se d in elu t ed (F ig. 2c ). Th e yie ld of radio labeled glycope pt ide a fte r 18963 Glycosylation of c-Myc Threonine 58 Phenylthiohydantoin-Amino Acids: cme AmIno Acids 1. D,G>S>A,F 2. T> F, E 3. H. F, L> V 4. K>A > E, L 5. S>L>P 6. E 7. I>D>P.V 8. A>Q>K,P 9. H > I, L, F, K 10. R> P 11. F>E>K 12. K > L Amino Acid Sequence of Peptides: 1. DTHKSEIAHRFK(DLGEEHFK) 2. SFFAL(R) I SFFAL(R)DQIPEL(ENNEK) 3. FELLP(T)PPL(SPSR) FIG. 3. Amino acids and peptide sequences derived from gas­ )( phase sequencing. [3HJGalactose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to gas-phase sequencing. u Phenylthiohydantoin-amino acids released in each cycle in order of abundance (high> low) are listed in the top panel. Peptide sequences deduced from the phenylthiohydantoin-amino acids released and tryp­ tic maps of c-Myc and bovine serum albumin (as described under 40 60 80 100 120 140 0 20 "Results and Discussion") are listed in the bottom panel. Fraction Number ... f 100 :E c. '0 "' 0.0 140.0 ~ 2 3 4 5 6 7 8 9 1 0 1 1 1 2 DISC ... 3 Cycle Number )( 2: 2 FIG. 4. Determination of O-GlcNAc site on a c-Myc tryptic gly. copeptide by sequential manual Edman degradation. [3HJGalac­ tose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to manual sequential Edman degradation. Counts released in 0 each cycle (numbers) and counts from the disc after all 12 cycles (DISC) 0 20 40 60 II 1110 120 140 were plotted. Fraction Number these multiple HPLC analyses was -12.5% due to the "sticky" C nature of this glycopeptide. After the third round of HPLC, the UV absorbance profile suggested that this peptide was fairly ... homogeneous. Therefore, further fractionation (e.g. ion-ex­ /'00 change chromatography) was not performed (however, see • 0 below) . • 0 An aliquot containing 5.5 pmol of [3H]galactose-Iabeled gly­ copeptide from the third dimension RP-HPLC was subjected to gas-phase sequencing. The relative abundance of amino acids recovered in each sequencing cycle surprisingly suggested the 0.0 presence of three major co-purified peptides (Fig. 3): DTHK· SEIAHRFK(DLGEEHFK) (-15 pmol) , SFFAL(R) or SF­ FAL(R)DQIPEL(ENNEK) (-10 pmol), and FELLP(T)PPL­ (SPSR) (-5 pmol). The two less abundant peptides were from c-Myc tryptic fragments of amino acids 373-378, SFFALR (or )( 373-389, SFFALRDQIPELENNEK), in the first helix (and loop) region and amino acids 53-65, FELLPTPPLSPSR, in the syltransferase, and separated by RP-HPLC as described under "Exper­ .. .. imental Procedures." a, first dimension; b, second dimension; c, third • • • •• dimension. Top panels, absorbance profile of eluted peptides; bottom Fraction Number panels, tritium profile of eluted peptides. The straight line in the top FIG. 2. Isolation of [3Hlgalactose-Iabeled tryptic glycopep­ panel represents acetonitrile gradient. %B is percentage of 60% (v/v) tides. Purified c-Myc was digested with trypsin, labeled with galacto- acetonitrile. 18964 Glycosylation of c-Myc Threonine 58 FELLPTPPISPSR + GlcNAc-[3H]GaI + 2 x Na FIG. 5. Identification of [3Hlgalac­ tose-Iabeled FELLPTPPLSPSR by mass spectrometry. [3HlGalactose-la­ .0 ~ 60 271.7.7 beled tryptic glycopeptides from third di­ mension RP-HPLC were subjected to ~ so MALDI-MS. The m l z of 1867.3 repre­ sents the molecular mass ofFELLPTPPL­ 865.6 SPSR (molecular mass, 1453.71) plus Q) 40­ [3H]Galj31-4GIcNAc (367.33) plus two so- ~ dium (2 X 22.99) (total = 1867.02). For ~ 30 the assignment of the other peaks, see Q) "Results and Discussion." ~ 20 10 -- sao 'BOO Maas/Charge transactivation domain. A protein data bank search (BLAST, sponding to FELLPTPPLSPSR (1453.71) plus GlcNAc (203.18) National Institutes of Health) revealed the more abundant plus sodium (22.99) plus H 0 (18.02) (total = 1697.9). Since peptide, DTHKSEIAHRFKDLGEEHFK, was from tryptic frag­ both glycosylated and non-glycosylated forms of c-Myc are pres­ ments of bovine serum albumin. Fetal bovine serum (10%) ent in cells and the galactosyltransferase labeling is not com­ added in the insect cell culture medium is likely the source of pletely efficient (20), the co-existence of FELLPTPPLSPSR, this contaminating peptide. Apparently, this serum-derived FELLPTPPLSPSR with GlcNAc, and FELLPTPPLSPSR with peptide either exactly co-migrates with the c-myc peptides or [3Hlgalactosylated GlcNAc is expected. In addition, RP-HPLC binds to them with high affinity during HPLC purification. The generally does not resolve unmodified, O-GlcNAc-modified, or presence of this contaminant is surprising since we have typi­ galactosylated O-GlcNAc-modified peptides (20). Furthermore, cally found this iterative RP-HPLC method to provide more we have recently found that O-GlcNAc saccharides are rapidly than adequate purification of glycopeptides for sequencing and selectively lost during ionization in electrospray mass spec­ (20,28). trometry." Also found in the mass spectrometry were a peak Since the three major peptides detected by the gas-phase with m/z 865.6 corresponding to SFFALR (739.88) plus phos­ sequencing contain serine or threonine at different positions, phate (79.98) plus two sodium (2 X 22.99) (total = 865.84) and sequential manual Edman degradation, followed by scintilla­ a peak with mlz 2717.7 corresponding to DTHKSEIAHRFKDL­ tion counting to detect the released radiolabeled saccharide, GEEHFK (2405.13) plus copper (63.55) plus hexose (162.14) provides an unambiguous assignment of the site of O-GlcNAc plus three sodium (3 x 22.99) plus H 0 (18.02) (total = 2717.54). modification. The result of sequential manual Edman degrada­ The peptide DTHKSEIAHRFKDLGEEHFK contains a copper tion (Fig. 4) indicates that the threonine residue in FELLPT­ chelating site at its histidine residues. A non-enzymatic glycation PPLSPSR (threonine 58) is glycosylated. Only the peptide at lysine 12 of the peptide DTHKSEIAHRFKDLGEEHFK has FELLPTPPLSPSR has a threonine or serine at the sixth amino been reported (29). From the data of gas-phase sequencing, se­ acid, the cycle in which the radiolabel is released. The assign­ quential Edman degradation, and mass spectrometry we con­ ment of O-GlcNAc glycosylation to threonine 58 is also sup­ clude that threonine 58 is the major O-GlcNAc site of c-Myc. ported by two other observations. First, the major radioactive Threonine 58 is in the TAD of c-Myc within the region where peak from the first, second, and third round ofRP-HPLC eluted we previously localized O-GlcNAc by more indirect methods at 22.5, 26.8, and 26.0% acetonitrile, respectively, consistent (13). It has been shown that threonine 58 is a phosphorylation with the predicted retention times for the peptide FELLPTP­ site in vivo (30) and can be phosphorylated in vitro by glycogen PLSPSR (41). Second, the gas-phase sequencing data showed synthase kinase 3 (10). Threonine 58 is altered to a methionine the amount of the released phenylthiohydantoin-threonine in MC29 and HB1 v-Myc and to an alanine in OKlO and MH2 was smaller than other internal residues of the peptide v-Myc (31). These mutations of threonine 58 in v-Myc enhance F(5.4)E(1.5)L( 4.1)L(2.4)P(2. 7)T( <0.5)P(1.1)P(0.5)L(4.3)S­ the transforming activity of Myc protein (32, 33). On the other «0.5)P( <0.5)S( <0.5)R (repetitive yields are in parentheses hand, a v-Myc protein with a threonine at amino acid 58 has a following each amino acid), suggesting that threonine 58 has reduced capability to induce growth in soft agar by non-trans­ been modified. This low recovery of threonine 58 also ruled out formed embryo fibroblasts (34, 35). Comparison of c-Myc and v-Myc by a variety of transformation assays also revealed that the possibility that the released radioactivity in the sixth se­ quencing cycle came from a small amount of contaminating c-Myc has a reduced ability to induce tumor formation (33). peptide. These results suggest that threonine 58 has a key role in The conclusion that threonine 58 is the site of O-GlcNAc transducing a negative growth signal of c-Myc through its was further confirmed by analyzing the peptides by post-translational modifications. This working hypothesis is MALDI-MS (Fig. 5). A peak with mlz 1867.3 was assigned to supported by the observations that mutations of c-Myc at or near threonine 58 are frequently found in Burkitt or AIDS­ the mass of FELLPTPPLSPSR (1453.71) plus [3H1Galf31­ 4GlcNAc (367.33) plus two sodium (2 x 22.99) (total = related lymphomas, and threonine 58 is the most frequently 1867.02). We also noted a peak with mlz 1493.9 corresponding to FELLPTPPLSPSR (1453.71) plus sodium (22.99) plus H 0 (18.02) (total = 1494.72) and a peak with m/z 1696.5 corre- 2 K. Greis, B. Hayes, and G. W. Hart, unpublished observations. Glycosylation of c-Myc Threonine 58 18965 Biochem. 58,841-874 mutated amino acid of c-Myc in these tumors (36-40). 15. Hart, G. W., Kelly, W. G., Blomberg, M. A., Roquemore, E. P., Dong, L.-Y. D., Since mutations altering threonine 58 augment c-Myc trans­ Kreppel, L., Chou, T.-Y., Snow, D., and Greis, K. (1993) in 44. Colloquium forming ability, we speculate that reciprocal phosphorylation! Mosbach 1993 Glyco- and Cell Biology, pp. 91-103, Springer-Verlag, Berlin 16. Holt, G. D., and Hart, G. W. (1986) J. Bioi. Chem. 261, 8049-8057 O-GlcNAc glycosylation modulate the activity of c-Myc. With 17. Miyamoto, C., Smith, G. E., Farrell-Towt, J., Chizzonite, R., Summers, M. D., the observation that c-Myc polypeptide levels remain relatively and Ju, G. (1985) Mol. Cell. BioI. 5,2860-2865 constant throughout the cell cycle (25), we also propose that 18. Summers, M. D., and Smith, G. E. (1987)A Manual of Me thuds for Baculovirus Vectors and Insect Cell Culture Procedures, Department of Entomology, these reciprocal post-translational modifications of threonine Texas Agricultural Experiment Station and Texas A&M University, College 58 differentially regulate c-Myc functions in different stages of Station, TX the cell cycle. 19. Papoulas, 0., Williams, N. G., and Kingston, R. E. (1992) J. Bioi. Chem. 267, 10470-10480 20. Roquemore, E. P., Chou, T.-Y., and Hart, G. W. (1994) Methods Enzymol. 230, Acknowledgments-We thank Dr. Grace Ju for providing Ac373/hc­ 443-460 myc, Dr. Dennis L-Y. Dong for helpful suggestions, Dr. Wu-Schyong Liu 21. Sullivan, S., and Wong, T. W. (1991) Anal. Biochem. 197,65-68 for help in gas-phase sequencing, and Drs. Amina S. Woods and Mar­ 22. Kelly, W. G., Dahmus, M. E., and Hart, G. W. (1993) J. BioI. Chem. 268, cela M. Cordero for help in mass spectrometry. We also thank Dr. 10416-10424 Joseph Eiden for help with the recombinant baculoviral protein expres­ 23. Chevrier, M. R., and Cotter, R. J. (1991) Rapid Commun. Mass Spectrom. 5, sion system. 611-617 24. Moore, J. P., Hancock, D. C., Littlewood, T. D., and Evan, G. 1. (1987) Oncogene REFERENCES Res. 2, 65- 80 1. Kato, G. J., and Dang, C. V. (1992) FASEB J. 6,3065-3072 25. Hann, S. R., Thompson, C. B., and Eisenman, R. N. (1985) Nature 314, 2. Blackwood, E. M., and Eisenman, R. N. (1991) Science 251, 1211-1217 366-369 3. Blackwell, T. K., Kretzner, I.., Blackwood, E. 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Published: Aug 1, 1995

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