O-GlcNAc stabilizes SMAD4 by inhibiting GSK-3β-mediated proteasomal degradation

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-β (TGF-β). TGF-β is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3β which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-β-responsive SMAD binding element (SBE). This study’s findings imply that cellular O-GlcNAc may regulate the TGF-β/SMAD signaling pathway by stabilizing SMAD4.

Methods for Studying Site-Specific O-GlcNAc Modifications: Successes, Limitations, and Important Future Goals

“…In a study by Kim et al, this technique was used to probe the PTM’s effect on the proteasomal clearance of SMAD4. 31 Through mass spectrometry analysis, four residues of the protein were found to be O-GlcNAc modification sites, and these sites were mutated to alanines or valines using site-directed mutagenesis. Subsequent Western blotting analysis validated that each of the mutants were less O-GlcNAc-modified than the wild-type, and a quadruple mutation abolished almost all O-GlcNAcylation, validating that all four sites are O-GlcNAcylated.…”

O-GlcNAcylation: An Emerging Protein Modification Regulating the Hippo Pathway

“…Hence, O -GlcNAcylation may indirectly enhance YAP/TAZ activity by controlling Wnt signaling. Likewise, the O -GlcNAcylation of Smad4, an important regulator of the TGFβ signaling pathway, at Thr63 prevents the GSK3β-mediated proteasomal degradation of Smad4, inducing the TGFβ signaling pathway [ 125 ]. SnoN, a target gene of TGFβ signaling, stabilizes TAZ by preventing phosphorylation by LATS [ 68 ].…”

Progenitor/Stem Cells in Vascular Remodeling during Pulmonary Arterial Hypertension

“…This dynamic posttranslational modification leads to changes in protein function, trafficking, and localization and regulates stem cell self-renewal, pluripotency, and differentiation, in part, via epigenetic regulations (for review, see [46]). In particular, O-GlcNAcylation stabilizes HIF-1α [155], SMAD4, a major regulator of TGF-β and BMP signaling pathways [156], or the EndMT regulator SNAIL1 [157]. Therefore, modifications in O-GlcNAc transferase activity and substrate level (glucose) may alter stem/progenitor cell function and participation in vascular remodeling.…”