Degradation of Antizyme Inhibitor, an Ornithine Decarboxylase Homologous Protein, Is Ubiquitin-dependent and Is Inhibited by Antizyme

Zippi Bercovich,Chaim Kahana

doi:10.1074/jbc.m410234200

Abstract

Ornithine decarboxylase (ODC) is the most notable example of a protein degraded by the 26 S proteasome without ubiquitination. Instead, ODC is targeted to degradation by direct binding to a polyamine-induced protein termed antizyme (Az). Antizyme inhibitor (AzI) is an ODC-related protein that does not retain enzymatic activity yet binds Az with higher affinity than ODC. We show here that like ODC, AzI is also a short-lived protein that undergoes proteasomal degradation. However, in contrast to ODC degradation, the degradation of AzI is ubiquitin-dependent and does not require interaction with Az. Moreover, Az binding actually stabilizes AzI by inhibiting its ubiquitination. Substituting the C terminus of AzI with that of ODC, which together with Az constitutes the complete degradation signal of ODC, does not subvert AzI degradation from the ubiquitin-dependent mode to the Az-dependent mode, suggesting dominance of the ubiquitination signal. Our results suggest opposing roles of Az in regulating the degradation of AzI and ODC.

Highlights

The polyamines spermine, spermidine, and their precursor putrescine are natural organic cations that play a crucial role in regulating fundamental cellular processes such as proliferation, differentiation, transformation, and apoptosis [1,2,3,4,5,6,7,8]
In contrast to Ornithine decarboxylase (ODC) degradation, which is greatly stimulated by Az, Antizyme inhibitor (AzI) degradation is inhibited by interaction with Az
AzI Degradation Requires Ubiquitination—Since we demonstrate here that AzI degradation does not require interaction with Az and that its C-terminal segment does not serve as a degradation signal, we set out to investigate the possibility that AzI is degraded via the ubiquitin system, as is the degradation of the majority of cellular proteins

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Culture—The cell lines used were: 293 human embryo kidney (293 HEK), NIH3T3 mouse fibroblasts, and A31N-ts, a BALB/c mouse cell line that harbors a temperature-sensitive E1 ubiquitin-activating enzyme, and its parental cell line A31N [25]. Immunoblot Analysis—Cell extracts were prepared by lysing phosphate-buffered saline-washed cells in radioimmune precipitation lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8, 1 mM dithiothreitol, and 1 ␮g/ml each of leupeptin, aprotinin, and pepstatin (Sigma mixture)). In Vivo Ub Conjugation Assay—Twenty hours after co-transfection with the constructs encoding the tested protein and HA-Ub, the cells were treated for 4 h with MG132 (100 ␮M) and harvested, and cellular extracts were prepared in radioimmune precipitation lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8, 1 mM dithiothreitol, and 1 ␮g/ml each of leupeptin, aprotinin, pepstatin (Sigma mixture)). The immunoprecipitated material was resolved by electrophoresis in an SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-HA antibodies (Sigma)

RESULTS

Findings

DISCUSSION