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J. Biol. Chem., Vol. 281, Issue 6, 3642-3650, February 10, 2006

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

Gipsy Majumdar, Adrienne Harrington¹, James Hungerford, Antonio Martinez-Hernandez^¶||, Ivan C. Gerling², Rajendra Raghow^**3, and Solomon Solomon^**4

From the Research, Medical, and ^¶ Pathology Services, Veterans Affairs Medical Center Memphis and the Departments of Medicine, ^||Pathology, and ^**Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee 38104

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.

Received for publication, October 14, 2005 , and in revised form, November 30, 2005.

^* This work was supported by Merit Review grants from the Department of Veterans Affairs (to S. S. and R. R.) and the National Institutes of Health (NIH) (to S. S. and R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Medical Student Short Term Research Training Grant T35-DK-07405-20.

² Supported by NIH Grants DK 62103 and RR 15373 and American Diabetes Association Innovative Award 7/1/05.

³ A Senior Research Career Scientist of the Department of Veteran Affairs.

⁴ To whom correspondence and reprint requests should be addressed: Research Service (151), VAMC, 1030 Jefferson Ave., Memphis, TN 38104. Tel.: 901-577-7274; Fax: 901-577-7273; E-mail: ssolomon{at}utmem.edu.

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