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.
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ABSTRACT: The discovery of nitric oxide (NO) as the endothelial-derived relaxing factor has led to significant research on NO and the proteins involved in its function, generation, location and regulation. Synthesis of NO by blood vessel endothelial cells results from the enzymatic oxidation of arginine by endothelial nitric oxide synthase (eNOS) resulting in the formation of equimolar amounts of NO and citrulline. Citrulline is sequentially recycled to arginine by successive reactions involving the enzymes argininosuccinate synthase (AS) and argininosuccinate lyase (AL), respectively. eNOS activity has been shown to be regulated by post-translational modifications including dynamic phosphorylation on multiple serine/threonine and tyrosine residues and dynamic O-linked beta-N-acetylglucosamine (O-GlcNAc) modifications on serine/threonine residues.Previous studies showed that even though intracellular endothelial arginine levels range from 0.1 to 0.8 mM and the Km of eNOS for arginine is 3 uM, the addition of exogenous arginine caused an increase in NO production. To explain this "arginine paradox" we hypothesize that there is a separate and distinct cellular source of arginine substrate directed to NO production and that this source is maintained through the regeneration of arginine via a citrulline-NO cycle. The presented research has provided the following evidence in support of this hypothesis: Citrulline stimulates NO production in an arginine-rich medium, without an increase in intracellular arginine. The enzymes of the citrulline-NO cycle, eNOS, AS and AL, co-fractionate with caveolin-1 in an endothelial cell caveolar membrane fraction. In vitro interaction assays demonstrate protein-protein interactions between fusion tagged AS or AL with eNOS or caveolin-1. Simultaneous monitoring of apparent citrulline and NO production demonstrates an efficient and essential coupling of the reactions of the citrulline-NO cycle. Glucosamine treatment of endothelial cells results in increased NO production in the basal state and decreased NO production in the stimulated state.Our findings demonstrate the enzymes of the citrulline-NO cycle, eNOS, AS and AL, are functionally associated, the reactions are efficiently coupled and enzyme activities are changed by post-translational modifications based on nutrient levels. These alterations ensure a constant and distinct source of arginine which is available for NO production to ensure vascular health.
Article: Sp1 and the "Hallmarks of Cancer"[Show abstract] [Hide abstract]
ABSTRACT: Transcription factor Sp1 was, for many years, viewed as a basal transcription factor and relegated to the regulation of so-called housekeeping genes. Sp1's role in recruiting the general transcription machinery in the absence of a TATA box increased its importance in gene regulation, particularly in light of recent estimates that the majority of mammalian genes lack a TATA box. In this review, we briefly address the history of Sp1, the founding member of the Sp family of transcription factors. We review the evidence suggesting Sp1 is highly regulated by post-translational modifications that both positively and negatively affect Sp1's activity on a wide array of genes. Sp1 is overexpressed in many cancers and is associated with poor prognosis. Targeting Sp1 in cancer treatment has been suggested; however, our review of the literature on Sp1-dependent regulation of genes that contribute to the "hallmarks of cancer", described by Hanahan and Weinberg, illustrates the extreme complexity of Sp1 functions. Sp1 both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors, as well as genes involved in essential cellular functions, including proliferation, differentiation, DNA damage response, apoptosis, senescence, and angiogenesis. Sp1 is also implicated in inflammation and genomic instability, as well as epigenetic silencing. Based on the seemingly opposing effects of Sp1, a more complete understanding of the function of Sp1 in cancer is needed to validate its potential as a therapeutic target. This article is protected by copyright. All rights reserved.FEBS Journal 11/2014; 282(2). DOI:10.1111/febs.13148 · 3.99 Impact Factor
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ABSTRACT: Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-linked β-N-acetylglucosamine (O-GlcNAc) is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify multiple rodent adipocytokines and secreted proteins whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. We have validated the relative levels of several of these factors using immunoblotting. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating secreted protein expression. Here, we show that upon the elevation of O-GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple secreted proteins reflect the modulation observed at the protein level. We validate the transcript levels in male mouse models of diabetes. Using inguinal fat pads from the severely IR db/db mouse model and the mildly IR diet-induced mouse model, we have confirmed that the secreted proteins regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. By comparing the promoters of similarly regulated genes, we determine that Sp1 is a common cis-acting element. Furthermore, we show that the LPL and SPARC promoters are enriched for Sp1 and O-GlcNAc modified proteins during insulin resistance in adipocytes. Thus, the O-GlcNAc modification of proteins bound to promoters, including Sp1, is linked to adipocytokine transcription during insulin resistance.Frontiers in Endocrinology 01/2014; 5:223. DOI:10.3389/fendo.2014.00223