The O-GlcNAcylation of β-actin Ser199 controls nuclear speckle localization and is dysregulated in diabetes

Actin is a pivotal cytoskeletal protein that also regulates chromatin remodeling, transcription, and RNA processing within the nucleus. These nuclear functions are regulated by post-translational modifications (PTMs), but the roles of specific PTMs of nuclear actin remain poorly understood. Of these, the O-GlcNAcylation of Ser199 (gS199) is of particular interest, because this residue can also be phosphorylated (pS199) and is adjacent to the Thr201-203 cluster, a known promoter of filament elongation. In this study, we aimed to elucidate the role of Ser199 O-GlcNAcylation in nuclear actin organization and function. We demonstrate that O-GlcNAcylation at Ser199 actin is associated with actin localization to nuclear speckles and suppresses filament formation. In vivo and in vitro assays revealed that gS199- and pS199-actin have a punctate distribution within the nucleus and colocalize with the speckle marker SRSF2 (SC35). Immunoelectron microscopy showed that this localization was markedly enhanced under diabetic conditions. Furthermore, the introduction of an anti-gS199-actin antibody induced nuclear filament formation, directly linking Ser199 O-GlcNAcylation to the inhibition of actin polymerization. Immunoprecipitation and mass spectrometry identified glyceraldehyde 3-phosphate dehydrogenase and histone H1.4 as nuclear binding partners of modified Ser199-actin. These findings suggest a mechanism by which Ser199 O-GlcNAcylation restricts actin polymerization, anchors actin to nuclear speckles, and thereby influences RNA processing. Dysregulation of this pathway in diabetes may destabilize nuclear speckle organization and contribute to the transcriptional defects that underlie diabetic complications.