Dynamic Interplay between O-Glycosylation and O-Phosphorylation of Nucleocytoplasmic Proteins

Previously, we reported that c-Myc is glycosylated by O-linked N-acetylglucosamine at Thr-58, a known phosphorylation site and a mutational hot spot in lymphomas. In this paper, we describe the production and characterization of two Thr-58 site-specific antibodies and use them to examine the modification of Thr-58 in living cells. One antibody specifically reacts with the Thr-58-glycosylated form of c-Myc, and the other reacts only with unmodified Thr-58 in c-Myc. Using these antibodies together with a commercial anti-Thr-58-phosphorylated c-Myc antibody, we simultaneously detected three forms of c-Myc (Thr-58-unmodified, -phosphorylated, and -glycosylated). It has been reported that Thr-58 phosphorylation is dependent on a prior phosphorylation of Ser-62. Mutagenesis of Ser-62 to Ala showed a marked decrease of Thr-58 phosphorylation and a marked increase of Thr-58 glycosylation. Growth inhibition of HL60 cells by serum starvation increases Thr-58 glycosylation and correspondingly decreases its phosphorylation. Serum stimulation has the opposite effect upon the modification status of Thr-58. A candidate kinase responsible for Thr-58 phosphorylation is the glycogen synthase kinase 3 (GSK3). Lithium, a competitive inhibitor of GSK3, decreased Thr-58 phosphorylation and increased its glycosylation. Finally, we show that the Thr-58-phosphorylated form of c-Myc predominantly accumulates in the cytoplasm rather than the nucleus upon inhibition of proteasome activity. These data suggest that hierarchical phosphorylation of Ser-62 and Thr-58 and alternative glycosylation/phosphorylation of Thr-58 together regulate the myriad functions of c-Myc in cells. Previously, we reported that c-Myc is glycosylated by O-linked N-acetylglucosamine at Thr-58, a known phosphorylation site and a mutational hot spot in lymphomas. In this paper, we describe the production and characterization of two Thr-58 site-specific antibodies and use them to examine the modification of Thr-58 in living cells. One antibody specifically reacts with the Thr-58-glycosylated form of c-Myc, and the other reacts only with unmodified Thr-58 in c-Myc. Using these antibodies together with a commercial anti-Thr-58-phosphorylated c-Myc antibody, we simultaneously detected three forms of c-Myc (Thr-58-unmodified, -phosphorylated, and -glycosylated). It has been reported that Thr-58 phosphorylation is dependent on a prior phosphorylation of Ser-62. Mutagenesis of Ser-62 to Ala showed a marked decrease of Thr-58 phosphorylation and a marked increase of Thr-58 glycosylation. Growth inhibition of HL60 cells by serum starvation increases Thr-58 glycosylation and correspondingly decreases its phosphorylation. Serum stimulation has the opposite effect upon the modification status of Thr-58. A candidate kinase responsible for Thr-58 phosphorylation is the glycogen synthase kinase 3 (GSK3). Lithium, a competitive inhibitor of GSK3, decreased Thr-58 phosphorylation and increased its glycosylation. Finally, we show that the Thr-58-phosphorylated form of c-Myc predominantly accumulates in the cytoplasm rather than the nucleus upon inhibition of proteasome activity. These data suggest that hierarchical phosphorylation of Ser-62 and Thr-58 and alternative glycosylation/phosphorylation of Thr-58 together regulate the myriad functions of c-Myc in cells. c-Myc, the product of the c-myc protooncogene, is a helix-loop-helix leucine zipper (HLHLZ) 1The abbreviations used are: HLHLZhelix-loop-helix leucine zipperTADtranscriptional activation domainα-T58Panti-phosphorylated Thr-58-specific antibodyα-T58Ganti-glycosylated Thr-58-specific antibodyα-T58Nanti-unmodified Thr-58-specific antibodyDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumDTTdithiothreitolPMSFphenylmethylsulfonyl fluorideALLNN-acetyl-Leu-Leu-Norleu-alERKextracellular signal-regulated kinase. 1The abbreviations used are: HLHLZhelix-loop-helix leucine zipperTADtranscriptional activation domainα-T58Panti-phosphorylated Thr-58-specific antibodyα-T58Ganti-glycosylated Thr-58-specific antibodyα-T58Nanti-unmodified Thr-58-specific antibodyDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumDTTdithiothreitolPMSFphenylmethylsulfonyl fluorideALLNN-acetyl-Leu-Leu-Norleu-alERKextracellular signal-regulated kinase. protein that regulates gene transcription in cell proliferation, apoptosis, and metabolism (1Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1370) Google Scholar). Two regions of c-Myc required for its biological activities are the N-terminal transcriptional activation domain (TAD) and the C-terminal basic-HLHLZ-specific DNA-binding domain (2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1023) Google Scholar). The HLHLZ domain mediates heterodimerization of c-Myc with its partner, Max, permitting binding to specific DNA sequences (2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1023) Google Scholar). c-Myc activity is precisely controlled at various levels, including transcription, translation, and posttranslation (3Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar). c-Myc can be phosphorylated at more than a dozen Ser and Thr residues (3Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar). Phosphorylation at Thr-58 and/or Ser-62 in the TAD has been shown to be particularly important for regulating transformation of cells by c-Myc (4Henriksson M. Bakardjiev A. Klein G. Luscher B. Oncogene. 1993; 8: 3199-3209PubMed Google Scholar, 5Pulverer B.J. Fisher C. Vousden K. Littlewood T. Evan G. Woodgett J.R. Oncogene. 1994; 9: 59-70PubMed Google Scholar). It has also been shown recently that c-Myc turnover appears to be regulated by the ubiquitin-proteasome pathway (6Flinn E.M. Busch C.M.C. Wright A.P.H. Mol. Cell. Biol. 1998; 18: 5961-5969Crossref PubMed Scopus (103) Google Scholar, 7Gross-Mesilaty S. Reinstein E. Bercovich B. Tobias K.E. Schwartz A.L. Kahana C. Ciechanover A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8058-8063Crossref PubMed Scopus (195) Google Scholar, 8Salghetti S.E. Kim S.Y. Tansey W.P. EMBO J. 1999; 18: 717-726Crossref PubMed Scopus (375) Google Scholar, 9Sears R. Leone G. DeGregori J. Nevins J.R. Mol. Cell. 1999; 3: 169-179Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Mutation of Thr-58 increases c-Myc stability (10Bahram F. von der Lehr N. Cetinkaya C. Larsson L.-G. Blood. 2000; 95: 2104-2110Crossref PubMed Google Scholar, 11Gregory M.A. Hann S.R. Mol. Cell. Biol. 2000; 20: 2423-2435Crossref PubMed Scopus (363) Google Scholar, 12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar), and phosphorylation of Thr-58 is associated with rapid degradation of c-Myc (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar). helix-loop-helix leucine zipper transcriptional activation domain anti-phosphorylated Thr-58-specific antibody anti-glycosylated Thr-58-specific antibody anti-unmodified Thr-58-specific antibody Dulbecco's modified Eagle's medium fetal calf serum dithiothreitol phenylmethylsulfonyl fluoride N-acetyl-Leu-Leu-Norleu-al extracellular signal-regulated kinase. helix-loop-helix leucine zipper transcriptional activation domain anti-phosphorylated Thr-58-specific antibody anti-glycosylated Thr-58-specific antibody anti-unmodified Thr-58-specific antibody Dulbecco's modified Eagle's medium fetal calf serum dithiothreitol phenylmethylsulfonyl fluoride N-acetyl-Leu-Leu-Norleu-al extracellular signal-regulated kinase. Our previous studies showed that the TAD of c-Myc is also glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) (13Chou T.-Y. Dang C.V. Hart G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4417-4421Crossref PubMed Scopus (182) Google Scholar) and that Thr-58 is a major glycosylation site of c-Myc (14Chou T.-Y. Hart G.W. Dang C.V. J. Biol. Chem. 1995; 270: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). O-GlcNAc is an abundant posttranslational modification of nuclear and cytoplasmic proteins in eukaryotes (15Hart G.W. Annu. Rev. Biochem. 1997; 66: 315-335Crossref PubMed Scopus (449) Google Scholar). Virtually all known O-GlcNAc-modified proteins are also phosphoproteins that form reversible multimeric protein complexes, suggesting that O-GlcNAc may regulate protein phosphorylation, protein-protein interaction, or both (16Comer F.I. Hart G.W. J. Biol. Chem. 2000; 275: 29179-29182Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 17Wells L. Vosseller K. Hart G.W. Science. 2001; 291: 2376-2378Crossref PubMed Scopus (800) Google Scholar). Thr-58 is also a known mutational hot spot in lymphomas, and this mutation is thought to be involved in tumor progression (1Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1370) Google Scholar). This evidence led us to the hypothesis that alternative modification of Thr-58 by O-phosphate or O-GlcNAc regulates the c-Myc function differentially. Here we present evidence for the occurrence of both glycosylation and phosphorylation at Thr-58 in a cell line using both site- and modification state-specific antibodies. We also demonstrate the interplay between hierarchical phosphorylation of Ser-62/Thr-58 and alternative glycosylation/phosphorylation of Thr-58 in living cells. A mouse anti-c-Myc antibody (C-33) is from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse anti-α-tubulin antibody (B-5–1-2) is from Sigma. A rabbit anti-phospho-c-Myc(Thr-58/Ser-62) antibody is from Cell Signaling Technology (Beverly, MA), and we designated this as a phosphorylated Thr-58-specific antibody (α-T58P). To generate anti-glycosylated Thr-58 antibody (α-T58G) and anti-unmodified Thr-58 antibody (α-T58N), a synthetic glycosylated peptide (KKFELLP(T-O-GlcNAc)PPLSPSRR) and a synthetic peptide (KKFELLPTPPLSPSRR) corresponding to amino acids 51–66 in the human c-Myc protein were used as antigens. After five immunizations in BALB/c mice, cells from the popliteal and inguinal lymph nodes were collected and fused with the P3X63Ag8.653 myeloma line according to the standard procedures. After the HAT selection, supernatants were screened for the reactivity with respective antigen. Confirmation of specificity was obtained by dot-blot analysis. Human embryonic kidney cell line 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) (heat inactivated at 56 °C for 30 min) at 37 °C in humidified air with 5% CO2. Human promyelocytic leukemia cell line HL60 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) FCS. pRSV-c-myc, an expression vector containing the cDNA of human c-Myc2 (a kind gift of Dr. C. V. Dang, Johns Hopkins University School of Medicine), was used for transfection. To prepare c-mycT58A and c-mycS62Amutant cDNAs, mutagenesis of pRSV-c-myc was performed with PCR-based QuikChange site-directed mutagenesis kits (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, synthetic DNA primers (5′-GAAATTCGAGCTGCTGCCCGCCCCGCCCCTGTC-3′ and 5′-GACAGGGGCGGGGCGGGCAGCAGCTCGAATTTC-3′ for c-mycT58A and 5′-CTGCTGCCCACCCCGCCCCTGGCCCCTAGC-3′ and 5′-GCTAGGGGCCAGGGGCGGGGTGGGCAGCAG-3′ for c-mycS62A) were used in the PCR with the template plasmid. For transfection of 293 cells, cells were in a and for in (v/v) prior to transfection. was performed using according to the manufacturer's instructions. were in and for and with of dithiothreitol phenylmethylsulfonyl fluoride and The were for at and the supernatants were with the antibody for at The were with protein and with and For the of the proteins in the were in and for were in MA), and to using the were by using antibody or antibody and according to the manufacturer's To of the c-Myc, with was performed as T.-Y. Hart G.W. 1994; PubMed Scopus Google Scholar, F.I. Vosseller K. L. M.A. Hart G.W. Biochem. 2001; PubMed Scopus Google Scholar). were by and to The was in serum in The was at with of serum of of and of calf The was with containing and and to using according to the manufacturer's 293 cells were cultured in the of for the and collected by and cytoplasmic and nuclear were as E. PubMed Scopus Google Scholar) with Briefly, collected cells were on in of and and and for The was to of 10% and for The was for 30 The was to as cytoplasmic The was in of and and at °C for The was for at and the was to as nuclear Thr-58 the TAD of c-Myc has been as both a glycosylation (14Chou T.-Y. Hart G.W. Dang C.V. J. Biol. Chem. 1995; 270: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar) and a phosphorylation site S. A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). These suggest that are three of at Thr-58 on and the phosphorylation status of Thr-58 has been using an antibody specific for Thr-58-phosphorylated c-Myc (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar, K. C. A. T. Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. B. 2001; PubMed Scopus Google Scholar, K. A. C. Y. Biochem. 2001; PubMed Scopus Google Scholar), has been to the other To all three c-Myc forms we two is specific for Thr-58-glycosylated c-Myc and the other is specific for c-Myc The specificity of antibody is shown in For the characterization of the we used forms of synthetic TAD modification status including phosphorylation at Ser-62. has been reported that Ser-62 on c-Myc is also a phosphorylation site S. A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar) and phosphorylation of Ser-62 may by the we also the both the unmodified and the peptide the other two forms that is specific for c-Myc and that the Ser-62 phosphorylation status its the of is than that of the other specifically reacts with the Thr-58-glycosylated peptide the other three forms The for to be a peptide in the TAD including O-GlcNAc-modified Thr-58 at all with other synthetic O-GlcNAc-modified we F.I. Vosseller K. L. M.A. Hart G.W. Biochem. 2001; PubMed Scopus Google Scholar) and the between and the Thr-58-glycosylated TAD peptide is by c-Myc in 293 cells with It has been reported that c-Myc can be phosphorylated at more than a dozen Ser and Thr including Ser-62 and Thr-58 (3Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar). This evidence the occurrence of or a of c-Myc by O-GlcNAc in 293 cells was by we that is a Thr-58-glycosylated reacts only with the Thr-58-phosphorylated peptide has also been reported that c-Myc that is phosphorylated at Thr-58 and phosphorylated at Thr-58 and Ser-62 c-Myc that is phosphorylated at Ser-62 or that is unmodified (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar), we as a Thr-58-phosphorylated Using these we detected Thr-58 modification status of c-Myc in HL60 cells, is of of the c-myc in HL60 cells F. A. S.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). c-Myc in were by with a anti-c-Myc antibody (C-33) and by analysis. shown in were with In to phosphorylation as has been reported that the c-myc gene to two major of human c-Myc with an of and a (3Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar). This evidence the occurrence of The with were in a and were to by by The of antibody upon serum stimulation were and to the of as shown in The of increased upon serum is to the of the major and the of were in cells and decreased upon serum stimulation and The of also to decrease upon serum stimulation to a the of be of the of antibody for the of c-Myc forms these that alternative glycosylation/phosphorylation at Thr-58 on c-Myc in cells. It has been reported that phosphorylation of Thr-58 is dependent on a prior phosphorylation of Ser-62 B. Hann S.R. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). To examine the effect of Ser-62 phosphorylation on the modification status of Thr-58, we the modification status of c-Myc and c-Myc in the cell was with and by analysis. shown in a protein of both c-Myc were detected by c-Myc with and with both and both and of were than that of c-Myc, and with at These that both the glycosylation and phosphorylation of Thr-58 are by Ser-62 phosphorylation A candidate kinase responsible for Thr-58 phosphorylation is (4Henriksson M. Bakardjiev A. Klein G. Luscher B. Oncogene. 1993; 8: 3199-3209PubMed Google Scholar, 5Pulverer B.J. Fisher C. Vousden K. Littlewood T. Evan G. Woodgett J.R. Oncogene. 1994; 9: 59-70PubMed Google Scholar, 12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar, B. Hann S.R. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). It has been reported that activity Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. Klein J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) by for Biochem. 2001; PubMed Scopus Google Scholar). To examine the of on the modification status of Thr-58, c-myc 293 cells were with the of After for c-Myc was with and by The of a are shown in The of antibodies were and to the of as shown in A decrease of was as a of the (a decrease of increased with the decrease of to also increased in these to To the between the two HL60 cells were with or three that the of the cells. c-Myc was with or from the cells cultured in the of the for and by shown in of c-Myc decreased in the of all three and the was cells were with of c-Myc increased in to cells were with a of Thr-58-phosphorylated c-Myc and a increase of the Thr-58-glycosylated In the of or serum stimulation a increase in Thr-58 phosphorylation and a corresponding decrease in Thr-58 glycosylation and data has been reported that activity A. G. A. G. F. J. 2001; PubMed Scopus Google Scholar), also an effect on the Thr-58 modification status to a It has recently been reported that phosphorylation of Thr-58 is associated with degradation of c-Myc by the ubiquitin-proteasome pathway (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar). To examine the modification status at Thr-58 is to or we the effect of proteasome inhibition on the Thr-58 modification status in 293 cells. to the of the c-Myc forms the proteasome inhibitor was c-Myc was with and by shown in c-Myc in the nuclear and a of c-Myc was also in the cytoplasmic at and the of the proteasome was detected only in the cytoplasmic we that was c-Myc in the cytoplasmic rather than the nuclear upon of of the cells with and were obtained cells were with proteasome data Finally, we that the c-Myc in the cytoplasmic is predominantly with all antibodies with both the cytoplasmic and nuclear forms of c-Myc It is known that phosphorylation (15Hart G.W. Annu. Rev. Biochem. 1997; 66: 315-335Crossref PubMed Scopus (449) Google Scholar), and in the of proteins including c-Myc (14Chou T.-Y. Hart G.W. Dang C.V. J. Biol. Chem. 1995; 270: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), R.N. J. Hart G.W. 2000; PubMed Scopus Google Scholar), L. K. 1998; 8: PubMed Scopus Google Scholar), and C. M. J. 2001; PubMed Scopus Google Scholar), O-GlcNAc and O-phosphate for the In this using both site- and modification we the occurrence of both O-GlcNAc and O-phosphate at Thr-58 on c-Myc in cell We also the Thr-58 modification status of c-Myc in living cells. suggest that Thr-58 phosphorylation is dependent on the prior phosphorylation of Ser-62 B. Hann S.R. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). Our data show that mutation of Ser-62 increases Thr-58 glycosylation and decreases Thr-58 phosphorylation, that Thr-58 glycosylation prior to its phosphorylation studies suggest that is responsible for the phosphorylation of Thr-58 (4Henriksson M. Bakardjiev A. Klein G. Luscher B. Oncogene. 1993; 8: 3199-3209PubMed Google Scholar, 5Pulverer B.J. Fisher C. Vousden K. Littlewood T. Evan G. Woodgett J.R. Oncogene. 1994; 9: 59-70PubMed Google Scholar, 12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar, B. Hann S.R. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar) and that as an inhibitor of Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. Klein J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with we a decrease of Thr-58 phosphorylation and a corresponding increase of Thr-58 glycosylation. The increase of Thr-58-glycosylated c-Myc by appears to be a with to inhibition of phosphorylation. This is by studies that shown that of the phosphorylation of cells in the of O-GlcNAc in a K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google B. J. Biochem. 1999; PubMed Scopus Google Scholar). these were cells are with O-GlcNAc modification at Thr-58 on c-Myc may an important in the of stimulation to Ser-62 phosphorylation reported Ser-62 phosphorylation is by (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar) and/or kinase B. T. T.-Y. M. Hann S.R. Dang C.V. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). Thr-58 is by O-GlcNAc M.A. Hart G.W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L. F.I. Hart G.W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the interplay between alternative glycosylation/phosphorylation of Thr-58 and hierarchical phosphorylation of Ser-62/Thr-58 be regulated by these c-Myc is a protein S.R. Eisenman R.N. Mol. Cell. Biol. PubMed Scopus Google Scholar), and of c-Myc is by the ubiquitin-proteasome pathway (6Flinn E.M. Busch C.M.C. Wright A.P.H. Mol. Cell. Biol. 1998; 18: 5961-5969Crossref PubMed Scopus (103) Google Scholar, 7Gross-Mesilaty S. Reinstein E. Bercovich B. Tobias K.E. Schwartz A.L. Kahana C. Ciechanover A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8058-8063Crossref PubMed Scopus (195) Google Scholar, 8Salghetti S.E. Kim S.Y. Tansey W.P. EMBO J. 1999; 18: 717-726Crossref PubMed Scopus (375) Google Scholar, 9Sears R. Leone G. DeGregori J. Nevins J.R. Mol. Cell. 1999; 3: 169-179Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). has been shown that c-Myc is phosphorylated at Thr-58 (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar). we the Thr-58 modification status of the c-Myc, data show that all three forms of predominantly Thr-58-phosphorylated are by proteasome inhibitor These suggest that the major of c-Myc for is the Thr-58-phosphorylated form Thr-58 phosphorylation is required for the This is in with the that degradation of c-Myc appears to Thr-58 phosphorylation in the of Ser-62 phosphorylation (12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar) and that are between and c-Myc the K. A. C. Y. Biochem. 2001; PubMed Scopus Google Scholar). the other the turnover of c-Myc may be by its modification status has been that mutation of Thr-58 increases c-Myc stability S.E. Kim S.Y. Tansey W.P. EMBO J. 1999; 18: 717-726Crossref PubMed Scopus (375) Google Scholar, F. von der Lehr N. Cetinkaya C. Larsson L.-G. Blood. 2000; 95: 2104-2110Crossref PubMed Google Scholar, 11Gregory M.A. Hann S.R. Mol. Cell. Biol. 2000; 20: 2423-2435Crossref PubMed Scopus (363) Google Scholar, 12Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (944) Google Scholar). has been that Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, Hart G.W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We also a of c-Myc in the cytoplasm by the proteasome inhibitor in cells the proteasome is both in the cytoplasm and the nucleus Annu. Rev. Biochem. 1999; PubMed Scopus Google Scholar), and c-Myc is a nuclear Our data suggest that the modification status of Thr-58 the of c-Myc for the degradation in the turnover of c-Myc. are the of the Thr-58 is in the TAD and is the amino in (1Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1370) Google Scholar). Mutation of Thr-58 can in a with (4Henriksson M. Bakardjiev A. Klein G. Luscher B. Oncogene. 1993; 8: 3199-3209PubMed Google Scholar, 5Pulverer B.J. Fisher C. Vousden K. Littlewood T. Evan G. Woodgett J.R. Oncogene. 1994; 9: 59-70PubMed Google Scholar), the effect of Thr-58 mutation in c-Myc activity S. A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, B. Hann S.R. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). It has been that the biological activities of c-Myc are by its with other in to heterodimerization with (1Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1370) Google Scholar, C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1023) Google Scholar, Oncogene. 1999; 18: PubMed Scopus Google Scholar, B. S.R. S. 2001; PubMed Scopus Google Scholar, R.N. Genes Dev. 2001; PubMed Scopus Google Scholar). are of proteins to with the c-Myc including the as S. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar), E.M. L. R. EMBO J. 1994; PubMed Scopus Google Scholar), N. J. V. M. M. Mol. Cell. Biol. 1995; PubMed Google Scholar), R. 14: PubMed Scopus Google Scholar), protein associated with J. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and C. Penn L.Z. A. K. K. J. Cell Sci. 2001; PubMed Google Scholar). The alternative glycosylation/phosphorylation of Thr-58 and its interplay with Ser-62 phosphorylation may in regulating the of with these are of c-Myc to various functions including metabolism (1Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1370) Google Scholar). of cells was to apoptosis, and that is thought to be to increased A expression Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google on proteins is by O-GlcNAc and the is the of the pathway S. V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This pathway is by S. V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of cells by may be a of an of the status of c-Myc including Thr-58. This is in with a that modification by of transcription as may function as a for regulating gene transcription M. 2001; PubMed Scopus Google Scholar). alternative glycosylation/phosphorylation are thought to be and of nuclear and cytoplasmic proteins in It be important to the of O-phosphate and O-GlcNAc at Thr-58 for the of the myriad functions of c-Myc in cells. We are to Dr. C. V. Dang for the kind gift of We all the of the Hart for and of the