Proteasome-mediated CCAAT/enhancer-binding protein δ (C/EBPδ) degradation is ubiquitin-independent

C/EBPδ (CCAAT/enhancer-binding protein δ) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPδ gene transcription is highly induced in G0 growth-arrested mammary epithelial cells but the C/EBPδ protein exhibits a t1⁄2 of only ∼120 min. The goal of the present study was to investigate the role of C/EBPδ modification by ubiquitin and C/EBPδ proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPδ ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPδ ubiquitination is not required for proteasome-mediated C/EBPδ degradation and the presence of ubiquitin does not increase C/EBPδ degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPδ protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPδ ubiquitination we treated G0 growth-arrested mammary epithelial cells with DNA-damageand oxidativestress-inducing agents and found that C/EBPδ ubiquitination is induced in response to H2O2. However, C/EBPδ protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPδ is ubiquitin-independent.