Identification of Nuclear Export Signals in Antizyme-1

Abstract

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259 -275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.Polyamines are ubiquitous organic cations with tightly regulated cellular concentrations and are essential for cell growth and development (1, 2). Antizyme (AZ) 1 was first described as an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis (3). When cellular polyamine levels rise, AZ is induced and then binds to the monomeric form of ODC (4, 5), which makes the enzyme a preferential substrate for proteolysis by the 26 S proteasome without ubiquitination (6). AZ also inhibits the uptake of extracellular polyamines (7,8). There are at least three independent antizyme isoforms conserved among mammals. AZ1 and AZ2 have a wide tissue distribution, whereas AZ3 is testis-specific (9, 10). Expression of AZs is induced by a unique translational frameshift mechanism (11). For AZ1, when cellular polyamine levels are low, the polypeptide corresponding to amino acid residues 1-68 is produced and translation is terminated at the following UGA codon (11). When cellular polyamine levels are high, ϩ1 frameshifting occurs after the 68th codon is decoded, resulting in the full-length antizyme protein (amino acid residues 1-227). All of the known regulatory activities of AZ1 are present in amino acid residues 69 -227 (12). A second AUG exists in AZ1 mRNA at the 34th codon, and this second AUG is also utilized as initiation codon (11, 13). The two resulting translation products have different half-lives (14). Furthermore, it has been suggested that the N-terminal region of the translation product from the first AUG contains a putative mitochondrial targeting signal and thus the distribution of AZ1 may depend on this region (15). It is curre...

Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification

“…We did not, however, observe a preferential association of GFP-antizyme with one centriole which might be due to non-physiological expression of the fusion protein. Consistent with previous reports (Murai et al, 2003), the GFP-antizyme 1 signal was otherwise mostly cytoplasmic and not nuclear.…”

“…Treatment with the microtubule-depolymerizing drug nocodazole did not displace the centrosomal antizyme 1 signal demonstrating that the localization of antizyme 1 is microtubule independent (Supplementary data S1). In addition to its centrosomal localization, antizyme 1 was also detected in the nucleus (Figures 1a and d and Supplementary data S1) as reported previously (Murai et al, 2003). The nuclear localization of antizyme 1 was, however, only detected in a fraction of U2OS and NIH-3T3 cells (Figures 1a and d).…”

A Novel CRM1-mediated Nuclear Export Signal Governs Nuclear Accumulation of Glyceraldehyde-3-phosphate Dehydrogenase following Genotoxic Stress

“…In accordance with our results with alanine scanning, this substitution resulted in nuclear accumulation of K259N GAPDH (Fig. 6, G and H) (7,(23)(24)(25)(26).…”

Antizyme is necessary for conversion of pancreatic tumor cells into glucagon-producing differentiated cells

“…The previous report showed that AZ1 is primarily localized in the nucleus where it transports ODC from the cytosol for degradation by nuclear proteasome (Gritli-Linde et al 2001, Murai et al 2003, Schipper et al 2004. By contrast, we found that AZ1 was mainly localized in the cytosol during conversion of AsPC-1 cells to the differentiated state (Fig.…”

“…Rat AZ1 has an w85% homology to human AZ1. It expresses a full-length AZ1 without the frameshift because of the deletion of one thymine nucleotide (Murai et al 2003). Transfection was carried out with 4.0 mg plasmid DNA/well using Lipofectamine 2000.…”