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 (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