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  1. J. Hungerford,
  2. G. Majumdar,
  3. R. Raghow,
  4. A. Martinez-Hernandez,
  5. I. Gerling,
  6. S. Solomon
  1. Research and Medical Services VA Medical Center and Departments of Medicine, Pathology, and Pharmacology, University of Tennessee, Memphis, TN


Insulin activates calmodulin (CaM) gene transcription, which leads to activation of low Km cAMP phosphodiesterase. This results in reversal of diabetic ketoacidosis. We have previously shown by 32 P labeling experiments and Western blots with antibodies highly specific for Sp1, O-GlcNAc, and phosphoserine that insulin first stimulates synthesis of Sp1 and then O-glycosylates it (early), followed by phosphorylation (later). Transcription of the CaM gene then occurs. H-411E liver cells in tissue culture were incubated with insulin (10,000 μU/mL) and processed at 0, 30, and 240 min. After incubation, cell extracts were prepared and run on 7.5% SDS polyacrylamide gel. The Sp1 band, localized by SYPRO stain and Western blot using specific anti-Sp1 antibody, was trypsin digested and then analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS). The data revealed that at least 3 peptide fragments containing serine/threonine sites are modified either by O-glycosylation or phosphorylation over the period of 4 hr of insulin exposure. The peptides are (1) 612-616; (2) 641-645; and (3) 699-704. The serine sites of these peptides were unmodified at 0 min and O-glycosylated at 30 min and later that same site was converted to a phosphate, eg, one of the three peptide fragments with mass 563.24 kDa (serine 613) is glycosylated at 30 min with a peptide mass of 766.31 kDa (563.24 + 203.19) and then at 240 min deglycosylated and phosphorylated with a peptide of mass of 644.26 (563.24 + 80.9) appearing. Insulin treatment at 0 min shows that the 4 serine sites in these 3 peptides initially are unmodified, but after 30 min all of these serine sites (100%) are O-GlcNAced. Following 4 hr insulin treatment, 3 out of 4 (75%) of the serine sites are phosphorylated.

Conclusions MALDI TOF MS experiments support a yin-yang hypothesis, ie, the existence of a reciprocal relationship between O-glycosylation and phosphorylation of Sp1 and its role in translating insulin's effect on CaM gene transcription.

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