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

University of Tennessee, Knoxville, Tennessee, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2006; 281(6):3642-50. DOI: 10.1074/jbc.M511223200
Source: PubMed


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.

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    • "In addition, high glucose (HG) concentrations can potentially stimulate the expression of genes associated with the development of diabetic nephropathy [58]. A recent report also indicated that insulin dynamically regulates calmodulin gene expression by sequential O-glycosylation and phosphorylation of Sp1 in liver cells [59]. In addition, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates the expression of genes that contribute to the pathogenesis of diabetic complications [60]. "
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    • "SP1 is a ubiquitously expressed, prototypic C2H2-type zinc fingercontaining DNA binding protein that can activate or repress transcription through GC-rich elements in response to physiological and pathological stimuli. SP1 can be phosphorylated by various kinases at different sites, and the effects of these modifications can lead to its translocation from the cytoplasm to the nucleus, followed by the regulation of gene expression (Majumdar et al. 2006, Solomon et al. 2008, Tan & Khachigian 2009). A report from Chu & Ferro (2005) shows that AMPK downstream signaling molecules, such as ERK, can mediate the phosphorylation of transcription factor SP1. "
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