Insulin Dynamically Regulates Calmodulin Gene Expression by Sequential O-Glycosylation and Phosphorylation of Sp1 and Its Subcellular Compartmentalization in Liver Cells

Abstract

O-glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (approximately 30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Cellular Content of UDP-N-acetylhexosamines Controls Hyaluronan Synthase 2 Expression and Correlates with O-Linked N-Acetylglucosamine Modification of Transcription Factors YY1 and SP1

“…The protein is known to be O-GlcNAcylated, and this modification regulates its effects on the expression of other genes, especially those important in insulin signaling and metabolic syndrome (32). Insulininduced O-GlcNAcylation of SP1 causes its transition to the nucleus (34), and its glucosamine-induced glycosylation is known to protect SP1 from proteasomal degradation (35). We found that the level of O-GlcNAc on SP1 was modified according to the content of cellular UDP-HexNAc and that SP1 binding to the HAS2 promoter and expression of the HAS2 gene were inversely related to the level of O-GlcNAc in SP1.…”

Sp1 and Sp3 regulate glucokinase gene transcription in the liver of gilthead sea bream (Sparus aurata)

“…Insulin stimulates synthesis, O-glycosylation and phosphorylation of Sp1 (Bouwman & Philipsen 2002, Samson & Wong 2002, Chu & Ferro 2005, Majumdar et al 2006. The fact that Sp1 binding sites are found in the insulin-responsive regions of numerous genes suggests that Sp1 is involved in mediating insulin action.…”

Glutamine enhances heat shock protein 70 expression via increased hexosamine biosynthetic pathway activity

“…This pathway appears to be part of an early cellular protective response to stress because this protein modification can occur rapidly (29). Known transcription factors modified by the HBP include Sp1, a key transcription factor required for optimal HSP expression following stress (4,7). The O-glycosylation of Sp1 causes its translocation to the nucleus, at which point it can then become active and induce gene promoter activity (7).…”

“…Known transcription factors modified by the HBP include Sp1, a key transcription factor required for optimal HSP expression following stress (4,7). The O-glycosylation of Sp1 causes its translocation to the nucleus, at which point it can then become active and induce gene promoter activity (7). As previously mentioned, this finding is vital to HSP expression because expression of heat HSF-1, HSP70, and HSP27 all rely on Sp1 binding in their promoter sequences for optimal transcriptional activity (1,10,14).…”