O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic β cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Aktl. MALDI-TOF/TOF mass spectrometry analysis of Aktl immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc -modification analysis of wildtype and mutant Aktl revealed that S473 was targeted by recombinant OGT. A S473A Aktl mutant demonstrated reduced basal and glucos amine-induced Akt1 O-GlcNAc modification compared with wildtype Aktl. Furthermore, wildtype Aktl, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Aktl Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate. (C) 2008 Elsevier Inc. All rights reserved.