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. 1997 May;17(5):2550–2558. doi: 10.1128/mcb.17.5.2550

Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility.

I Han 1, J E Kudlow 1
PMCID: PMC232104  PMID: 9111324

Abstract

Sp1 is a ubiquitously expressed transcription factor that is particularly important for the regulation of TATA-less genes that encode housekeeping proteins. Most growth factors and receptors are also encoded by such genes. Sp1 is multiply O glycosylated by covalent linkage of the monosaccharide N-acetylglucosamine (O-GlcNAc) to serine and threonine residues. Based on an earlier observation that growth factor gene transcription can be regulated by glucose and glucosamine in vascular smooth muscle cells, we determined whether Sp1 glycosylation could be regulated and if this modification altered Sp1 function. We found that Sp1 becomes hyperglycosylated when cells are exposed to 5 mM glucosamine, whereas under glucose starvation, stimulation with cyclic AMP (cAMP) results in nearly complete deglycosylation of this protein. Correlating with this hypoglycosylated state, Sp1 is rapidly proteolytically degraded by an enzyme(s) that can be inhibited by specific proteasome inhibitors, lactacystin and LLnL. Treatment of cells with glucose or glucosamine protects Sp1 from cAMP-mediated degradation, whereas blockade of glucosamine synthesis abrogates glucose but not glucosamine protection. This effect on Sp1 is specific, in that the Stat-3 and E2F transcription factors did not undergo degradation under these conditions. The O-GlcNAc modification of Sp1 may play a role as a nutritional checkpoint. In the absence of adequate nutrition, Sp1 becomes hypoglycosylated and thereby subject to proteasome degradation. This process could potentially result in reduced general transcription, thereby conserving nutrients.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aristarkhov A., Eytan E., Moghe A., Admon A., Hershko A., Ruderman J. V. E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4294–4299. doi: 10.1073/pnas.93.9.4294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Azizkhan J. C., Jensen D. E., Pierce A. J., Wade M. Transcription from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev Eukaryot Gene Expr. 1993;3(4):229–254. [PubMed] [Google Scholar]
  3. Barral Y., Jentsch S., Mann C. G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast. Genes Dev. 1995 Feb 15;9(4):399–409. doi: 10.1101/gad.9.4.399. [DOI] [PubMed] [Google Scholar]
  4. Biggs J. R., Kudlow J. E., Kraft A. S. The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells. J Biol Chem. 1996 Jan 12;271(2):901–906. doi: 10.1074/jbc.271.2.901. [DOI] [PubMed] [Google Scholar]
  5. Briggs M. R., Kadonaga J. T., Bell S. P., Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986 Oct 3;234(4772):47–52. doi: 10.1126/science.3529394. [DOI] [PubMed] [Google Scholar]
  6. Chakraborty A., Saha D., Bose A., Chatterjee M., Gupta N. K. Regulation of eIF-2 alpha-subunit phosphorylation in reticulocyte lysate. Biochemistry. 1994 May 31;33(21):6700–6706. doi: 10.1021/bi00187a041. [DOI] [PubMed] [Google Scholar]
  7. Chen L. I., Nishinaka T., Kwan K., Kitabayashi I., Yokoyama K., Fu Y. H., Grünwald S., Chiu R. The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator. Mol Cell Biol. 1994 Jul;14(7):4380–4389. doi: 10.1128/mcb.14.7.4380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chou T. Y., Dang C. V., Hart G. W. Glycosylation of the c-Myc transactivation domain. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4417–4421. doi: 10.1073/pnas.92.10.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou T. Y., Hart G. W., Dang C. V. c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas. J Biol Chem. 1995 Aug 11;270(32):18961–18965. doi: 10.1074/jbc.270.32.18961. [DOI] [PubMed] [Google Scholar]
  10. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. doi: 10.1016/0092-8674(94)90396-4. [DOI] [PubMed] [Google Scholar]
  11. Cook S. J., McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science. 1993 Nov 12;262(5136):1069–1072. doi: 10.1126/science.7694367. [DOI] [PubMed] [Google Scholar]
  12. Daniels M. C., Kansal P., Smith T. M., Paterson A. J., Kudlow J. E., McClain D. A. Glucose regulation of transforming growth factor-alpha expression is mediated by products of the hexosamine biosynthesis pathway. Mol Endocrinol. 1993 Aug;7(8):1041–1048. doi: 10.1210/mend.7.8.8232303. [DOI] [PubMed] [Google Scholar]
  13. Datta B., Chakrabarti D., Roy A. L., Gupta N. K. Roles of a 67-kDa polypeptide in reversal of protein synthesis inhibition in heme-deficient reticulocyte lysate. Proc Natl Acad Sci U S A. 1988 May;85(10):3324–3328. doi: 10.1073/pnas.85.10.3324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Datta B., Ray M. K., Chakrabarti D., Wylie D. E., Gupta N. K. Glycosylation of eukaryotic peptide chain initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) and its possible role in the inhibition of eIF-2 kinase-catalyzed phosphorylation of the eIF-2 alpha-subunit. J Biol Chem. 1989 Dec 5;264(34):20620–20624. [PubMed] [Google Scholar]
  15. Deshaies R. J., Chau V., Kirschner M. Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway. EMBO J. 1995 Jan 16;14(2):303–312. doi: 10.1002/j.1460-2075.1995.tb07004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dynan W. S., Saffer J. D., Lee W. S., Tjian R. Transcription factor Sp1 recognizes promoter sequences from the monkey genome that are simian virus 40 promoter. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4915–4919. doi: 10.1073/pnas.82.15.4915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Elias S., Bercovich B., Kahana C., Coffino P., Fischer M., Hilt W., Wolf D. H., Ciechanover A. Degradation of ornithine decarboxylase by the mammalian and yeast 26S proteasome complexes requires all the components of the protease. Eur J Biochem. 1995 Apr 1;229(1):276–283. [PubMed] [Google Scholar]
  19. Fenteany G., Standaert R. F., Lane W. S., Choi S., Corey E. J., Schreiber S. L. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science. 1995 May 5;268(5211):726–731. doi: 10.1126/science.7732382. [DOI] [PubMed] [Google Scholar]
  20. Hochstrasser M. Protein degradation or regulation: Ub the judge. Cell. 1996 Mar 22;84(6):813–815. doi: 10.1016/s0092-8674(00)81058-2. [DOI] [PubMed] [Google Scholar]
  21. Horowitz J. M., Yandell D. W., Park S. H., Canning S., Whyte P., Buchkovich K., Harlow E., Weinberg R. A., Dryja T. P. Point mutational inactivation of the retinoblastoma antioncogene. Science. 1989 Feb 17;243(4893):937–940. doi: 10.1126/science.2521957. [DOI] [PubMed] [Google Scholar]
  22. Ishii H., Jirousek M. R., Koya D., Takagi C., Xia P., Clermont A., Bursell S. E., Kern T. S., Ballas L. M., Heath W. F. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science. 1996 May 3;272(5262):728–731. doi: 10.1126/science.272.5262.728. [DOI] [PubMed] [Google Scholar]
  23. Jackson S. P., MacDonald J. J., Lees-Miller S., Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell. 1990 Oct 5;63(1):155–165. doi: 10.1016/0092-8674(90)90296-q. [DOI] [PubMed] [Google Scholar]
  24. Jackson S. P., Tjian R. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell. 1988 Oct 7;55(1):125–133. doi: 10.1016/0092-8674(88)90015-3. [DOI] [PubMed] [Google Scholar]
  25. Jariel-Encontre I., Pariat M., Martin F., Carillo S., Salvat C., Piechaczyk M. Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome. J Biol Chem. 1995 May 12;270(19):11623–11627. doi: 10.1074/jbc.270.19.11623. [DOI] [PubMed] [Google Scholar]
  26. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  27. Kato J. Y., Matsuoka M., Polyak K., Massagué J., Sherr C. J. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell. 1994 Nov 4;79(3):487–496. doi: 10.1016/0092-8674(94)90257-7. [DOI] [PubMed] [Google Scholar]
  28. Kelly W. G., Dahmus M. E., Hart G. W. RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc. J Biol Chem. 1993 May 15;268(14):10416–10424. [PubMed] [Google Scholar]
  29. Lubas W. A., Smith M., Starr C. M., Hanover J. A. Analysis of nuclear pore protein p62 glycosylation. Biochemistry. 1995 Feb 7;34(5):1686–1694. doi: 10.1021/bi00005a025. [DOI] [PubMed] [Google Scholar]
  30. McClain D. A., Paterson A. J., Roos M. D., Wei X., Kudlow J. E. Glucose and glucosamine regulate growth factor gene expression in vascular smooth muscle cells. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8150–8154. doi: 10.1073/pnas.89.17.8150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Measday V., Moore L., Ogas J., Tyers M., Andrews B. The PCL2 (ORFD)-PHO85 cyclin-dependent kinase complex: a cell cycle regulator in yeast. Science. 1994 Nov 25;266(5189):1391–1395. doi: 10.1126/science.7973731. [DOI] [PubMed] [Google Scholar]
  32. Moss B. Vaccinia virus: a tool for research and vaccine development. Science. 1991 Jun 21;252(5013):1662–1667. doi: 10.1126/science.2047875. [DOI] [PubMed] [Google Scholar]
  33. Pugh B. F., Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev. 1991 Nov;5(11):1935–1945. doi: 10.1101/gad.5.11.1935. [DOI] [PubMed] [Google Scholar]
  34. Reason A. J., Morris H. R., Panico M., Marais R., Treisman R. H., Haltiwanger R. S., Hart G. W., Kelly W. G., Dell A. Localization of O-GlcNAc modification on the serum response transcription factor. J Biol Chem. 1992 Aug 25;267(24):16911–16921. [PubMed] [Google Scholar]
  35. Richter-Ruoff B., Wolf D. H. Proteasome and cell cycle. Evidence for a regulatory role of the protease on mitotic cyclins in yeast. FEBS Lett. 1993 Dec 20;336(1):34–36. doi: 10.1016/0014-5793(93)81603-w. [DOI] [PubMed] [Google Scholar]
  36. Roberts A. B., Anzano M. A., Wakefield L. M., Roche N. S., Stern D. F., Sporn M. B. Type beta transforming growth factor: a bifunctional regulator of cellular growth. Proc Natl Acad Sci U S A. 1985 Jan;82(1):119–123. doi: 10.1073/pnas.82.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roos M. D., Han I. O., Paterson A. J., Kudlow J. E. Role of glucosamine synthesis in the stimulation of TGF-alpha gene transcription by glucose and EGF. Am J Physiol. 1996 Mar;270(3 Pt 1):C803–C811. doi: 10.1152/ajpcell.1996.270.3.C803. [DOI] [PubMed] [Google Scholar]
  38. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993 Apr 29;362(6423):801–809. doi: 10.1038/362801a0. [DOI] [PubMed] [Google Scholar]
  39. Saffer J. D., Jackson S. P., Annarella M. B. Developmental expression of Sp1 in the mouse. Mol Cell Biol. 1991 Apr;11(4):2189–2199. doi: 10.1128/mcb.11.4.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saffer J. D., Jackson S. P., Thurston S. J. SV40 stimulates expression of the transacting factor Sp1 at the mRNA level. Genes Dev. 1990 Apr;4(4):659–666. doi: 10.1101/gad.4.4.659. [DOI] [PubMed] [Google Scholar]
  41. Santos R. C., Waters N. C., Creasy C. L., Bergman L. W. Structure-function relationships of the yeast cyclin-dependent kinase Pho85. Mol Cell Biol. 1995 Oct;15(10):5482–5491. doi: 10.1128/mcb.15.10.5482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sayeski P. P., Kudlow J. E. Glucose metabolism to glucosamine is necessary for glucose stimulation of transforming growth factor-alpha gene transcription. J Biol Chem. 1996 Jun 21;271(25):15237–15243. doi: 10.1074/jbc.271.25.15237. [DOI] [PubMed] [Google Scholar]
  43. Seamon K. B., Daly J. W. Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J Cyclic Nucleotide Res. 1981;7(4):201–224. [PubMed] [Google Scholar]
  44. Shin T. H., Paterson A. J., Grant J. H., 3rd, Meluch A. A., Kudlow J. E. 5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression. Mol Cell Biol. 1992 Sep;12(9):3998–4006. doi: 10.1128/mcb.12.9.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Snow C. M., Senior A., Gerace L. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol. 1987 May;104(5):1143–1156. doi: 10.1083/jcb.104.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weinberg R. A. The retinoblastoma protein and cell cycle control. Cell. 1995 May 5;81(3):323–330. doi: 10.1016/0092-8674(95)90385-2. [DOI] [PubMed] [Google Scholar]
  47. Wu J., Dent P., Jelinek T., Wolfman A., Weber M. J., Sturgill T. W. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science. 1993 Nov 12;262(5136):1065–1069. doi: 10.1126/science.7694366. [DOI] [PubMed] [Google Scholar]
  48. Yaglom J., Linskens M. H., Sadis S., Rubin D. M., Futcher B., Finley D. p34Cdc28-mediated control of Cln3 cyclin degradation. Mol Cell Biol. 1995 Feb;15(2):731–741. doi: 10.1128/mcb.15.2.731. [DOI] [PMC free article] [PubMed] [Google Scholar]

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