©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 Chou (1) Gerald W. Hart (3)(§),   Chi V. Dang (2)(¶)

From the (1)Biochemistry, Cellular, and Molecular Biology Training Program and (2)Division of Hematology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the (3)Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

c-Myc is a helix-loop-helix leucine zipper phosphoprotein that heterodimerizes with Max and regulates gene transcription in cell proliferation, cell differentiation, and programmed cell death. Previously, we demonstrated that c-Myc is modified by O-linked N-acetylglucosamine (O-GlcNAc) within or nearby the N-terminal transcriptional activation domain (Chou, T.-Y., Dang, C. V., and Hart, G. W.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4417-4421). In this paper, we identified the O-GlcNAc attachment site(s) on c-Myc. c-Myc purified from sf9 insect cells was trypsinized, and its GlcNAc moieties were enzymically labeled with [^3H]galactose. The [^3H]galactose-labeled glycopeptides were isolated by reverse phase high performance liquid chromatography and then subjected to gas-phase sequencing, manual Edman degradation, and laser desorption/ionization mass spectrometry. These analyses show that threonine 58, an in vivo phosphorylation site in the transactivation domain, is the major O-GlcNAc glycosylation site of c-Myc. Mutation of threonine 58, frequently found in retroviral v-Myc proteins and in human Burkitt and AIDS-related lymphomas, is associated with enhanced transforming activity and tumorigenicity. The reciprocal glycosylation and phosphorylation at this biologically significant amino acid residue may play an important role in the regulation of the functions of c-Myc.


INTRODUCTION

c-Myc, the product of the c-myc protooncogene, is a nuclear phosphoprotein of 439 amino acids that plays a critical role in the regulation of gene transcription in normal and neoplastic cells. Mutations of c-myc are associated with different types of tumors in human and other species(1) . c-Myc has several structural features conserved among many transcription factors. A basic helix-loop-helix leucine zipper motif in the C-terminal region mediates heterodimerization with Max (2) and DNA binding to a specific E-box sequence, CACGTG or EMS (E-box myc site)(3) . The N-terminal transcriptional activation domain (TAD) (^1)(amino acids 1-143) (4) has a proline-rich element spanning from amino acid 41 to amino acid 103(1) , which contains several potential sites for O-linked N-acetylglucosamine (O-GlcNAc). The TAD is required for neoplastic transformation(5) , inhibition of cellular differentiation(6) , and induction of apoptosis (7) mediated by c-Myc.

c-Myc can be phosphorylated by casein kinase II(8) , MAP kinase(9) , or glycogen synthase kinase 3(10) . Phosphorylation at Thr-58 and/or Ser-62 in the TAD of c-Myc has been suggested to modulate the transactivation (11) and cellular transformation (12) by c-Myc. Our previous study (13) showed that the TAD of c-Myc is also modified by O-GlcNAc, a form of protein glycosylation composed of a single monosaccharide, GlcNAc, linked to the side chain hydroxyl of serine or threonine(14) . O-GlcNAc has been found almost exclusively in the nucleus and cytoplasm of eukaryotic cells. The known O-GlcNAc-bearing proteins share two common features; all of them are phosphoproteins and all form reversible multimeric complexes. Although the role of O-GlcNAc in altering protein function remains unknown, experiments to date suggest that the O-GlcNAc modification is dynamic and appears to have a reciprocal relationship with protein phosphorylation(15) .

In this report, using a variety of analytical techniques on c-Myc overexpressed in insect cells, we provide evidence that O-GlcNAc occurs at c-Myc threonine 58, a known phosphorylation site and a frequently mutated hot spot in human lymphomas.


EXPERIMENTAL PROCEDURES

Materials

sf9 insect cells were from Paragon. Monoclonal mouse anti-Myc antibody 9E10 was from American Type Culture Collection (ATCC). Sequencing grade trypsin (tosylphenylalanyl chloromethyl ketone-treated) was from Worthington. UDP-[6-^3H]galactose (38 Ci/mmol) was from Amersham Corp. Bovine milk galactosyltransferase from Sigma (37.2 units/ml) was pregalactosylated as described(16) . All other chemicals were of the highest quality commercially available.

Expression and Purification of c-Myc in Insect Cells

sf9 insect cells grown in suspension culture were infected with recombinant baculovirus Ac373/hc-myc ((17) ; a gift of G. Ju, Hoffmann-La Roche) according to the method of Summers and Smith (18) . The cells were harvested 40 h postinfection, and the c-Myc protein was purified as described by Papoulas, Williams, and Kingston (19) with some changes. Briefly, 1 10 cells were washed twice in phosphate-buffered saline and resuspended at 2.5 10^7 cells/ml in low salt lysis buffer (20 mM Hepes, pH 6.8, 5 mM KCl, 5 mM MgCl(2), 0.5% Nonidet P-40, 0.1% sodium deoxycholate, 1 mg/ml aprotinin, 0.1 mM PMSF, 50 mM GlcNAc). After 10 min cells were subjected to 40 strokes in a Dounce homogenizer with a type A pestle. Nuclei were pelleted at 1,000 g for 5 min at 4 °C, washed once in low salt lysis buffer, resuspended at 2 10^8 nuclei/ml in low salt lysis buffer containing 600 units/ml DNase I, and incubated at 4 °C for 2 h. An equal volume of 2 high salt lysis buffer (20 mM Tris, pH 7.4, 4 M NaCl, 1 mM MgCl(2), 0.1% Nonidet P-40, 50 mM GlcNAc) was added, mixed, and incubated for 10 min. The residual nuclear material was pelleted at 2,000 g for 10 min at 4 °C, resuspended for solubilization at 5 10^7 nuclear eq/ml in buffer A (50 mM Tris, pH 8.0, 2 mM EDTA, 5% glycerol, 0.1 mM dithiothreitol, 0.1 mM PMSF, 5 M urea), vigorously stirred on ice for 30 min, and then centrifuged at 5,000 g for 10 min at 4 °C. The supernatant was loaded at 0.1 ml/min on a 80-ml DEAE-Sepharose CL-6B (Sigma) column pre-equilibrated with 5 column volumes of buffer A. After loading, the column was washed at 0.4 ml/min with 3 volumes of buffer A and then 4 volumes of buffer A containing 0.15 M NaCl. c-Myc was eluted by buffer A containing 0.35 M NaCl. The c-Myc-containing fractions were pooled and diluted with buffer A to 0.1 M NaCl and loaded at 0.5 ml/min onto a 1-ml fast protein liquid chromatography Mono Q column (Pharmacia Biotech Inc.) pre-equilibrated with buffer A containing 0.1 M NaCl. The column was eluted in buffer A with a programmed gradient of 0.1-2 M NaCl. The c-Myc was eluted at 0.2-0.4 M NaCl, and the c-Myc containing fractions were pooled and dialyzed against buffer containing 20 mM Tris, pH 7.8, 50 mM KCl, 10% glycerol, 0.1% dithiothreitol, and 0.1 mM PMSF in bags of SpectroPor 2 membrane for four changes of 1 liter each and for 6 h each. All purification steps were carried out on ice or with ice-cold buffers.

SDS-PAGE, Silver Staining, and Western Blot Analysis

Purified c-Myc was analyzed by 10% SDS-PAGE, stained with silver staining, or transferred to nitrocellulose paper for Western blot analysis using monoclonal mouse anti-Myc antibody 9E10 (ATCC).

Trypsin Digestion of Purified c-Myc and Galactosyltransferase Labeling of Tryptic Peptides

Purified c-Myc was digested with trypsin at a ratio of 1:10 trypsin to protein (dissolved in 1 mM HCl) in 100 mM Tris-HCl, pH 8.5, at 37 °C for 18 h. The reaction mixture was acidified by adding 10% (v/v) trifluoroacetic acid to make a final concentration of 1% (v/v) trifluoroacetic acid and loaded onto a Sep-Pak C18 cartridge (Waters). The tryptic peptides were eluted with 60% (v/v) acetonitrile, dried, resuspended in 80 µl of H(2)O, and labeled by galactosyltransferase (20) in labeling buffer (10 mM Hepes, pH 7.4, 10 mMD(+)-galactose, 5 mM MnCl(2)) with 50 µCi of UDP-[^3H]galactose and 0.1 unit of galactosyltransferase at 37 °C for 4 h. Another 0.1 unit of galactosyltransferase and 5 µg of UDP-galactose were added after 1 h of incubation. The labeled tryptic peptides were purified by a Sep-Pak C18 cartridge. The 60% (v/v) acetonitrile eluent was dried and applied to RP-HPLC.

Isolation of [^3H]Galactose-labeled Glycopeptides by RP-HPLC

[^3H]Galactose-labeled glycopeptides were isolated by three rounds of RP-HPLC on a Rainin HPLC system equipped with a Vydac 5-µm C18 column (0.46 25 cm). Glycopeptides were loaded onto the column with 0.5 mM sodium phosphate buffer, pH 7.0 (first dimension), 0.1% (v/v) trifluoroacetic acid, pH 2.0 (second dimension), or 0.1% (w/v) ammonium acetate, pH 4.0 (third dimension) and eluted with a gradient of acetonitrile from 0 to 60% (v/v). The flow rate was 1 ml/min, the eluent was monitored by absorbance at = 214 nm, fractions were collected every minute, and the [^3H]galactose-labeled glycopeptides were detected by liquid scintillation counting.

Gas-phase Sequencing

Isolated [^3H]galactose-labeled glycopeptides from the third dimension RP-HPLC were sequenced by automated Edman degradation in a model 470A gas-phase sequencer (Applied Biosystems, Inc.).

Manual Sequential Edman Degradation

Aliquots of isolated [^3H]galactose-labeled glycopeptides from the third dimension RP-HPLC were subjected to the manual Edman degradation method of Sullivan and Wong (21) with modifications as described by Kelly et al.(22) .

Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was performed on a linear time-of-flight mass spectrometer built in-house and described previously by Chevrier and Cotter(23) .


RESULTS AND DISCUSSION

c-Myc protein levels have been shown to be very low in normal and transformed cells through the use of immunological assays(24) . There are approximately 750 molecules of c-Myc per cell in serum-starved fibroblasts. After serum stimulation, the number increases to 6,300 per cell. HeLa cells, which are transformed, contain 97,000 molecules of c-Myc per cell. The levels of cellular c-Myc polypeptide appear to be constant throughout the cell cycle(25) . The low abundance of c-Myc and the inherent limitation of the sensitivity of tritium labeling render the detection of carbohydrate moieties on c-Myc and the subsequent mapping of carbohydrate sites on c-Myc extremely difficult. In our previous work(13) , we developed sensitive methods to detect O-GlcNAc on in vitro translated c-Myc and identified a N-terminal region of c-Myc modified by O-GlcNAc. We also overexpressed the c-Myc protein in either sf9 insect cells or Chinese hamster ovary cells and demonstrated that c-Myc expressed in these cells is modified by O-GlcNAc. In the present study, we used c-Myc overexpressed in sf9 insect cells to map the O-GlcNAc addition sites of c-Myc. It is estimated that sf9 insect cells infected with recombinant baculovirus Ac373/hc-myc (17) express more than 1 million molecules of c-Myc per cell (data not shown), an amount at least 10-fold more abundant than the c-Myc molecules in HeLa cells. Studies have shown that sf9 insect cells, like vertebrate cells, are able to process post-translational protein modifications, including O-GlcNAc(26, 27) . A recent study on human cytomegalovirus tegument basic phosphoprotein indicated that the O-GlcNAc sites of the recombinant baculoviral protein faithfully correspond to those of the native virion protein(28) .

c-Myc was purified from 5 liters of infected sf9 cells, harvested at 2 10^6 cells/ml. During the initial lysis procedure, 50 mM GlcNAc was added to the buffers to partially inhibit the endogenous hexosaminidase activity. Most of the overexpressed c-Myc protein appeared to be bound to DNA and could only be extracted by 5 M urea or SDS(19) . Protein samples from sequential steps of purification were subjected to SDS-PAGE for silver staining and Western blot analysis (Fig.1). Proteins co-electrophoresing with c-Myc comprised more than 95% of the total after purification by Mono Q chromatography. When O-GlcNAc in this purified c-Myc preparation was labeled with [^3H]galactose by galactosyltransferase, c-Myc was the major radioactive signal(13) .


Figure 1: Purification of recombinant c-Myc from sf9 insect cells. Protein samples from sequential steps of the purification procedures were resolved on two identical 10% SDS-PAGE gels for silver staining (top panel) and Western blot analysis (bottom panel) as described under ``Experimental Procedures.'' lane 1, low salt lysis, total lysate; lane 2, low salt lysis, supernatant; lane 3, low salt lysis, pellet; lane 4, high salt lysis, supernatant; lane 5, high salt lysis, pellet, 5 M urea extract; lane 6, after DEAE CL-6B column; lane 7, after Mono Q column. Molecular mass markers of 70 kDa (70) and 43 kDa (43) are shown on the left.



Purified c-Myc was first trypsinized to gain better accessibility to O-GlcNAc residues for subsequent galactosyltransferase labeling(20) . The O-GlcNAc-modified glycopeptides, labeled with [^3H]galactose by galactosyltransferase, were separated by RP-HPLC at pH 7.0 on a C18 column in the first dimension. As illustrated in Fig.2a, only one major radioactive peak was detected, which contained 48 pmol of labeled glycopeptide. The fraction corresponding to the radioactive peak (fraction 65) was applied to a second dimension RP-HPLC at pH 2.0 and the radioactivity eluted as a single peak (Fig.2b). When the fraction containing this radioactive peak was applied to a third dimension RP-HPLC at pH 4.0, a single radioactive peak was eluted (Fig.2c). The yield of radiolabeled glycopeptide after these multiple HPLC analyses was 12.5% due to the ``sticky'' 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-exchange chromatography) was not performed (however, see below).


Figure 2: Isolation of [^3H]galactose-labeled tryptic glycopeptides. Purified c-Myc was digested with trypsin, labeled with galactosyltransferase, and separated by RP-HPLC as described under ``Experimental Procedures.'' a, first dimension; b, second dimension; c, third dimension. Top panels, absorbance profile of eluted peptides; bottom panels, tritium profile of eluted peptides. The straight line in the top panel represents acetonitrile gradient. %B is percentage of 60% (v/v) acetonitrile.



An aliquot containing 5.5 pmol of [^3H]galactose-labeled glycopeptide 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 presence of three major co-purified peptides (Fig.3): DTHKSEIAHRFK(DLGEEHFK) (15 pmol), SFFAL(R) or SFFAL(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 transactivation domain. A protein data bank search (BLAST, National Institutes of Health) revealed the more abundant peptide, DTHKSEIAHRFKDLGEEHFK, was from tryptic fragments of bovine serum albumin. Fetal bovine serum (10%) added in the insect cell culture medium is likely the source of this contaminating peptide. Apparently, this serum-derived peptide either exactly co-migrates with the c-myc peptides or binds to them with high affinity during HPLC purification. The presence of this contaminant is surprising since we have typically found this iterative RP-HPLC method to provide more than adequate purification of glycopeptides for sequencing(20, 28) .


Figure 3: Amino acids and peptide sequences derived from gas-phase sequencing. [^3H]Galactose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to gas-phase sequencing. 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 tryptic maps of c-Myc and bovine serum albumin (as described under ``Results and Discussion'') are listed in the bottom panel.



Since the three major peptides detected by the gas-phase sequencing contain serine or threonine at different positions, sequential manual Edman degradation, followed by scintillation counting to detect the released radiolabeled saccharide, provides an unambiguous assignment of the site of O-GlcNAc modification. The result of sequential manual Edman degradation (Fig.4) indicates that the threonine residue in FELLPTPPLSPSR (threonine 58) is glycosylated. Only the peptide FELLPTPPLSPSR has a threonine or serine at the sixth amino acid, the cycle in which the radiolabel is released. The assignment of O-GlcNAc glycosylation to threonine 58 is also supported by two other observations. First, the major radioactive peak from the first, second, and third round of RP-HPLC eluted at 22.5, 26.8, and 26.0% acetonitrile, respectively, consistent with the predicted retention times for the peptide FELLPTPPLSPSR(41) . Second, the gas-phase sequencing data showed the amount of the released phenylthiohydantoin-threonine was smaller than other internal residues of the peptide 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(<0.5)P(<0.5)S(<0.5)R (repetitive yields are in parentheses following each amino acid), suggesting that threonine 58 has been modified. This low recovery of threonine 58 also ruled out the possibility that the released radioactivity in the sixth sequencing cycle came from a small amount of contaminating peptide.


Figure 4: Determination of O-GlcNAc site on a c-Myc tryptic glycopeptide by sequential manual Edman degradation. [^3H]Galactose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to manual sequential Edman degradation. Counts released in each cycle (numbers) and counts from the disc after all 12 cycles (DISC) were plotted.



The conclusion that threonine 58 is the site of O-GlcNAc was further confirmed by analyzing the peptides by MALDI-MS (Fig.5). A peak with m/z 1867.3 was assigned to the mass of FELLPTPPLSPSR (1453.71) plus [^3H]Galbeta1-4GlcNAc (367.33) plus two sodium (2 22.99) (total = 1867.02). We also noted a peak with m/z 1493.9 corresponding to FELLPTPPLSPSR (1453.71) plus sodium (22.99) plus H(2)O (18.02) (total = 1494.72) and a peak with m/z 1696.5 corresponding to FELLPTPPLSPSR (1453.71) plus GlcNAc (203.18) plus sodium (22.99) plus H(2)O (18.02) (total = 1697.9). Since both glycosylated and non-glycosylated forms of c-Myc are present in cells and the galactosyltransferase labeling is not completely efficient(20) , the co-existence of FELLPTPPLSPSR, FELLPTPPLSPSR with GlcNAc, and FELLPTPPLSPSR with [^3H]galactosylated GlcNAc is expected. In addition, RP-HPLC generally does not resolve unmodified, O-GlcNAc-modified, or galactosylated O-GlcNAc-modified peptides(20) . Furthermore, we have recently found that O-GlcNAc saccharides are rapidly and selectively lost during ionization in electrospray mass spectrometry. (^2)Also found in the mass spectrometry were a peak with m/z 865.6 corresponding to SFFALR (739.88) plus phosphate (79.98) plus two sodium (2 22.99) (total = 865.84) and a peak with m/z 2717.7 corresponding to DTHKSEIAHRFKDLGEEHFK (2405.13) plus copper (63.55) plus hexose (162.14) plus three sodium (3 22.99) plus H(2)O (18.02) (total = 2717.54). The peptide DTHKSEIAHRFKDLGEEHFK contains a copper chelating site at its histidine residues. A non-enzymatic glycation at lysine 12 of the peptide DTHKSEIAHRFKDLGEEHFK has been reported(29) . From the data of gas-phase sequencing, sequential Edman degradation, and mass spectrometry we conclude that threonine 58 is the major O-GlcNAc site of c-Myc.


Figure 5: Identification of [^3H]galactose-labeled FELLPTPPLSPSR by mass spectrometry. [^3H]Galactose-labeled tryptic glycopeptides from third dimension RP-HPLC were subjected to MALDI-MS. The m/z of 1867.3 represents the molecular mass of FELLPTPPLSPSR (molecular mass, 1453.71) plus [^3H]Galbeta1-4GlcNAc (367.33) plus two sodium (2 22.99) (total = 1867.02). For the assignment of the other peaks, see ``Results and Discussion.''



Threonine 58 is in the TAD of c-Myc within the region where we previously localized O-GlcNAc by more indirect methods(13) . It has been shown that threonine 58 is a phosphorylation site in vivo(30) and can be phosphorylated in vitro by glycogen synthase kinase 3(10) . Threonine 58 is altered to a methionine in MC29 and HB1 v-Myc and to an alanine in OK10 and MH2 v-Myc(31) . These mutations of threonine 58 in v-Myc enhance the transforming activity of Myc protein(32, 33) . On the other hand, a v-Myc protein with a threonine at amino acid 58 has a reduced capability to induce growth in soft agar by non-transformed embryo fibroblasts(34, 35) . Comparison of c-Myc and v-Myc by a variety of transformation assays also revealed that c-Myc has a reduced ability to induce tumor formation(33) . These results suggest that threonine 58 has a key role in transducing a negative growth signal of c-Myc through its post-translational modifications. This working hypothesis is supported by the observations that mutations of c-Myc at or near threonine 58 are frequently found in Burkitt or AIDS-related lymphomas, and threonine 58 is the most frequently mutated amino acid of c-Myc in these tumors(36, 37, 38, 39, 40) .

Since mutations altering threonine 58 augment c-Myc transforming ability, we speculate that reciprocal phosphorylation/O-GlcNAc glycosylation modulate the activity of c-Myc. With the observation that c-Myc polypeptide levels remain relatively constant throughout the cell cycle(25) , we also propose that these reciprocal post-translational modifications of threonine 58 differentially regulate c-Myc functions in different stages of the cell cycle.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA42486 (to G. W. H.) and CA57341 (to C. V. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 205-934-4786; Fax: 205-975-6685; GWHART{at}BMG.BHS.UAB.EDU.

Scholar of the Leukemia Society of America.

^1
The abbreviations used are: TAD, transcriptional activation domain; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; RP-HPLC, reverse phase high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry.

^2
K. Greis, B. Hayes, and G. W. Hart, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Grace Ju for providing Ac373/hc-myc, Dr. Dennis L-Y. Dong for helpful suggestions, Dr. Wu-Schyong Liu for help in gas-phase sequencing, and Drs. Amina S. Woods and Marcela M. Cordero for help in mass spectrometry. We also thank Dr. Joseph Eiden for help with the recombinant baculoviral protein expression system.


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