Insulin dynamically regulates calmodulin gene expression by sequential O-glycosylation and phosphorylation of Sp1 and its subcellular compartmentalization in liver cells

Gipsy Majumdar, Adrienne Harrington, James Hungerford, Antonio Martinez-Hernandez, Ivan Gerling, Rajendra Raghow, Solomon Solomon

Research output: Contribution to journalArticle

66 Citations (Scopus)

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 (∼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.

Original languageEnglish (US)
Pages (from-to)3642-3650
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number6
DOIs
StatePublished - Feb 10 2006

Fingerprint

Glycosylation
Phosphorylation
Calmodulin
Gene expression
Liver
Insulin
Gene Expression
Mass spectrometry
Mass Spectrometry
Post Translational Protein Processing
Genes
Cells
Confocal microscopy
Streptozocin
Confocal Microscopy
Serine
Ionization
Hepatocellular Carcinoma
Desorption
Cytoplasm

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Insulin dynamically regulates calmodulin gene expression by sequential O-glycosylation and phosphorylation of Sp1 and its subcellular compartmentalization in liver cells. / Majumdar, Gipsy; Harrington, Adrienne; Hungerford, James; Martinez-Hernandez, Antonio; Gerling, Ivan; Raghow, Rajendra; Solomon, Solomon.

In: Journal of Biological Chemistry, Vol. 281, No. 6, 10.02.2006, p. 3642-3650.

Research output: Contribution to journalArticle

Majumdar, Gipsy ; Harrington, Adrienne ; Hungerford, James ; Martinez-Hernandez, Antonio ; Gerling, Ivan ; Raghow, Rajendra ; Solomon, Solomon. / Insulin dynamically regulates calmodulin gene expression by sequential O-glycosylation and phosphorylation of Sp1 and its subcellular compartmentalization in liver cells. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 6. pp. 3642-3650.
@article{4a0cd28daee14113b9cace75d5e614e4,
title = "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 (∼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.",
author = "Gipsy Majumdar and Adrienne Harrington and James Hungerford and Antonio Martinez-Hernandez and Ivan Gerling and Rajendra Raghow and Solomon Solomon",
year = "2006",
month = "2",
day = "10",
doi = "10.1074/jbc.M511223200",
language = "English (US)",
volume = "281",
pages = "3642--3650",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

}

TY - JOUR

T1 - Insulin dynamically regulates calmodulin gene expression by sequential O-glycosylation and phosphorylation of Sp1 and its subcellular compartmentalization in liver cells

AU - Majumdar, Gipsy

AU - Harrington, Adrienne

AU - Hungerford, James

AU - Martinez-Hernandez, Antonio

AU - Gerling, Ivan

AU - Raghow, Rajendra

AU - Solomon, Solomon

PY - 2006/2/10

Y1 - 2006/2/10

N2 - 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 (∼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.

AB - 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 (∼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.

UR - http://www.scopus.com/inward/record.url?scp=33645646866&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645646866&partnerID=8YFLogxK

U2 - 10.1074/jbc.M511223200

DO - 10.1074/jbc.M511223200

M3 - Article

C2 - 16332679

AN - SCOPUS:33645646866

VL - 281

SP - 3642

EP - 3650

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 6

ER -