O-glycosylation of Sp1 and transcriptional regulation of the calmodulin gene by insulin and glucagon
Tóm tắt
Both insulin and glucagon stimulate steady-state levels of Sp1 transcription factor, but only insulin stimulates transcription of the calmodulin (CaM) gene in liver. Because O-glycosylation of Sp1 by O-linked N-acetylglucosamine ( O-GlcNAc) is thought to regulate its ability to activate transcription, we assayed the levels of Sp1 with anti-Sp1 and anti- O-GlcNAc antibodies in Western blots by use of extracts of H-411E liver cells treated with insulin (10,000 μU/ml) or glucagon (1.5 × 10-5M). We also assessed subcellular localization of the native and glycosylated Sp1 in H411E cells treated with either hormone in the presence of deoxynorleucine (DON, an indirect inhibitor of O-glycosylation) or streptozotocin (STZ, an indirect stimulator of O-glycosylation). Insulin stimulated both total and O-GlcNAc-modified Sp1 primarily in the nucleus and induced CaM gene transcription ( P < 0.0001). In contrast, glucagon promoted accumulation of Sp1 in the cytoplasm but not the nucleus, without significantly stimulating ( P = not significant) either its O-glycosylation or transcription of the CaM gene. DON inhibited O-glycosylation of Sp1 and its ability to migrate to the nucleus and transactivate CaM gene transcription. In contrast, cotreatment of cells with STZ and glucagon enhanced O-glycosylation of Sp1, promoting its migration to the nucleus and resulting in increased CaM gene transcription. Thus O-glycosylation of Sp1 by insulin, but not glucagon, apparently enhances its (Sp1) nuclear recruitment and results in activation of CaM gene transcription.
Từ khóa
Tài liệu tham khảo
Holt GDand Hart GW.The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc.J Biol Chem261: 8049-8057, 1986.
Roos MD, Xie W, Su K, Clark JA, Yang X, Chin E, Paterson AJ, and Kudlow JE.Streptozotocin, an analog of N-acetylglucosamine, blocks the removal of O-GlcNAc from intracellular proteins.Proc Assoc Am Physicians110: 422-432, 1998.
Solomon SS, Palazzolo MR, Takahashi T, and Raghow R.Insulin stimulates rat calmodulin I gene transcription through activation of Sp1.Proc Assoc Am Physicians109: 470-477, 1997.
Solomon SSand Raghow R.Molecular mechanisms of insulin modulated signaling and regulation of gene transcription.Recent Res Devel Endocrinol3: 79-99, 2002.