Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein.

Abstract

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992. We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.Proteins that control important cellular processes are commonly short lived (30). This requires a means of recognizing them and tagging them for destruction. A few protein pairs have been identified, such that the first binds to and apparently targets the second for degradation, e.g., the human papillomavirus oncoprotein E6 and the tumor suppressor p53 (5,33,39). How these interactions direct proteolysis has not been determined. We (18) and others (4,7,15,25,26) have shown that ornithine decarboxylase (ODC), a short-lived enzyme, and antizyme, a tightly binding protein inhibitor of ODC, represent such a pair: antizyme accelerates the intracellular destruction of ODC. This interaction subserves a form of feedback regulation: ODC initiates the synthesis of polyamines, excess polyamines induce the production of antizyme (7, 15), and antizyme both inhibits ODC activity and leads to its destruction (26). Evidence for this chain of events comes from correlations among cellular polyamine pool size, antizyme level, and the rate of ODC turnover (25), and, more recently, the observation that forced expression of cloned antizyme accelerates the turnover of ODC (27). Conversely, if ODC is structurally altered so as to disrupt the site of antizyme binding, the ability of antizyme to inhibit enzymatic activity in vitro is abolished and the enzyme is no longer regulated by polyamines in vivo (18). Although it has been maintained that intracellular polyamines decrease ODC by reducing the translation of its mRNA, we have considered and negated these claims (9,18,38).Mouse ODC is a homodimer of subunits that each contains 461 amino acids. Turnover can proceed along two different pathways, constitutive and polyamine dependent. The C terminus is both necessary and sufficient to make an ODC protein constitutively unstable.

Cloning of Antizyme Inhibitor, a Highly Homologous Protein to Ornithine Decarboxylase

“…Rat ODC contains two PEST regions (20) in an internal region and near the carboxyl-terminal, respectively. The carboxyl-terminal region of ODC (422-461) including the second PEST region (423-449) has been assigned as an element necessary for the rapid degradation of ODC (6). Antizyme inhibitor lacks this region and does not contain PEST sequences.…”

“…The turnover of ODC is very rapid and highly regulated (3,4). The degradation of ODC catalyzed by the 26 S proteasome is accelerated by ODC antizyme (5,6), an inhibitory protein induced by polyamines (7). Strict regulation of ODC appears to be important for cell growth, because overproduction of ODC is associated with neoplastic transformation (8,9), whereas overproduction of antizyme inhibits cell growth (10,11).…”

Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells

“…If antizyme does have a direct affinity for the polyamine transporter as well as ODC, then it would appear that the dynamics of this partitioning of a labile regulatory protein (antizyme) between interactions with a stable membrane protein (transporter) and a labile cytoplasmic enzyme (ODC) would be quite complex. Further, the binding of antizyme to ODC is thought to alter the structure of ODC sufficiently to expose a region near the C-terminal end that stimulates degraReceived 30 December 1993/28 January 1994; accepted 28 January 1994 dation by the 26 S proteasome [10,11,29]. If antizyme also binds a component of the polyamine transporter, then it will be of interest to ascertain whether this interaction has any effect on the half-life of this protein that has previously been shown to be rather stable [17].…”

Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A

“…Amino acid residues R 131 and G 145 of Az1 are essential for the degradation of Aurora-A It has been demonstrated that N terminus of ODC interacts with Az1 (Li and Coffino, 1993) and that the element contained within amino acids 130-145 of rat Az1 is essential for targeting ODC for proteasomal degradation (Chen et al, 2002). To define whether this targeting domain of Az1 is involved in the targeting of Aurora-A for degradation, Az1 mutant, which lacks the N-terminal 120 amino acids (DN120-Az1) and Az1, which lacks only the amino acids 130-145 (D130-145-Az1) (Figure 4a), were made and used to study in vivo degradation of Aurora-A in HeLa cells as described earlier.…”

“…Az increases ODC degradation by enhancing ODC association with proteasome, rather than accelerating the rate of proteasomal processing (Zhang et al, 2003). The attachment of Az causes conformational changes in ODC, thereby exposing its C-terminal degradation signal for recognition by 26S proteasome (Li and Coffino, 1993). Unlike Ub, Az is usually spared from destruction and released from the ODCAz complex at the proteasome (Murakami et al, 1992).…”