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.
- SourceAvailable from: Kathleen Dickson[Show abstract] [Hide abstract]
ABSTRACT: Injury-induced expression of p75 neurotrophin receptor (p75NTR) in the CNS induces neuronal apoptosis and prevents neuronal regrowth. The mechanisms regulating injury-induced p75NTR expression are poorly characterized but previous studies have established that reductions in extracellular osmolarity which mimic cytotoxic edema induce p75NTR gene expression through pathways that activate the Sp1 transcription factor. In this report, we examined how extracellular osmolarity converges on Sp1 to regulate p75NTR expression. We report that levels of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme that mediates O-linked attachment of GlcNAc, are reduced by extracellular hypo-osmolarity and that global levels of protein O-GlcNAcylation and of Sp1 show a corresponding decline. We demonstrate that chemical and RNAi-based treatments that reduce cellular O-GlcNAcylation facilitate p75NTR induction by hypo-osmolarity, directly linking protein O-GlcNAcylation to p75NTR induction. To determine if Sp1 O-GlcNAc content regulates p75NTR expression, we replaced endogenous Sp1 with a Sp1 mutated at O-GlcNAc target residues. This O-GlcNAc-deficient form of Sp1-enhanced p75NTR expression, demonstrating that O-GlcNAcylation of Sp1 negatively regulates p75NTR expression. We conclude that a stress-induced decline in the O-GlcNAc content of Sp1 drives expression of p75NTR.Journal of Neurochemistry 02/2011; 116(3):396-405. DOI:10.1111/j.1471-4159.2010.07120.x · 4.24 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In an attempt to search for novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (EPS) on the differential change in pancreatic proteome and transcriptome in streptozotocin (STZ)-induced diabetic rats using 2-DE-based protein mapping and oligonucleotide microarray analysis. The 2-DE system separated more than 2000 individual spots, demonstrating that 34 proteins out of about 500 matched spots were differentially expressed. A total of 22 overexpressed and 12 underexpressed proteins in 2-DE map were observed (p<0.05) between the healthy and diabetic rats, of which 26 spots were identified by PMF analysis. Of these, significant down regulation of carbonyl reductase (Cbr), hydroxymethylglutaryl-CoA synthase (HMGCS), and putative human mitogen-activated protein kinase activator with WD repeats-binding protein (MAWDBP) in diabetic pancreas were reported for the first time in this study. When treated with EPS, all these four proteins were reverted to normal levels. The microarray analysis revealed that 96 out of 1272 genes were down- or up-regulated in the diabetic rats and the altered transcript levels of many of these genes were reversed after EPS treatment. In particular, ROS generation in rat islets was significantly increased after STZ treatment, thereafter EPS treatment was likely to play a preventive role in beta-cell destruction mediated by STZ. Taken together, EPS may act as a potent regulator of gene expression for a wide variety of genes in diabetic rats, particularly in antioxidative stress, insulin biosynthesis, and cell proliferation.Proteomics 06/2008; 8(11):2344-61. DOI:10.1002/pmic.200700779 · 3.97 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Phosphorylation and O-GlcNAcylation of keratin 18 (K18) are highly dynamic and involve primarily independent K18 populations. We used in vitro phosphorylation and O-GlcNAcylation of wild-type, phospho-Ser52, glyco-Ser48, and Ser-to-Ala mutant 17mer peptides (K18 amino acids 40-56), which include the major K18 glycosylation (Ser48) and phosphorylation (Ser52) sites, to address whether each modification blocks the other. The glyco-K18 peptide blocks Ser52 phosphorylation by protein kinase C, an in vivo K18 kinase, while the phospho-K18 peptide blocks its O-GlcNAcylation. Our findings support the reciprocity of these two post-translational modifications. Therefore, regulation of protein Ser/Thr phosphorylation and glycosylation at proximal sites can be interdependent and provides a potential mechanism of counter regulation.Biochemical and Biophysical Research Communications 01/2007; 351(3):708-12. DOI:10.1016/j.bbrc.2006.10.092 · 2.28 Impact Factor