Glycoproteome profiling of transforming growth factor‐β (TGFβ) signaling: Nonglycosylated cell death‐inducing DFF‐like effector A inhibits TGFβ1‐dependent apoptosis

Transforming growth factor‐β (TGF&β) is a potent regulator of cell growth, differentiation, and apoptosis. TGFβ binds to specific serine/threonine kinase receptors, which leads to activation of Smad‐dependent and Smad‐independent signaling pathways. O ‐Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFβ signaling. To explore the effect of TGFβ1 on protein O ‐glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2‐DE and MALDI‐TOF‐MS, we identified 21 HPA‐precipitated proteins, which were affected by treatment of cells with TGFβ1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFβ1 inhibited the appearance of cell death‐inducing DFF‐like effector A (CIDE‐A) in 2‐D gels with HPA‐precipitated proteins. CIDE‐A is a cell death activator which promotes DNA fragmentation. We observed that TGFβ1 did not affect expression of CIDE‐A, but inhibited its glycosylation. We found that deglycosylation of CIDE‐A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE‐A inhibited TGFβ1‐dependent cell death. Thus, inhibition of the glycosylation of CIDE‐A may be a mechanism to protect cells from apoptosis.