Multisite Phosphorylation of Ornithine Decarboxylase in Transformed Macrophages Results in Increased Intracellular Enzyme Stability and Catalytic Efficiency

Abstract

Ornithine decarboxylase (ODC) is the initial inducible enzyme in the polyamine biosynthetic pathway. In the transformed macrophage-derived RAW264 cell line, ODC was overproduced and existed in both unphosphorylated and phosphorylated forms. To date, the only protein kinase known to phosphorylate mammalian ODC is casein kinase II (CKII). ODC was phosphorylated in vitro by CKII and subjected to exhaustive sequential proteolysis with trypsin and V8 protease. Two-dimensional peptide mapping showed only a single phosphopeptide; two-dimensional phosphoamino acid analysis of the phosphopeptide revealed only 32 P-labeled serine. ODC was metabolically radiolabeled with 32 P i in RAW264 cells and also subjected to proteolysis, two-dimensional peptide mapping, and phosphoamino acid analysis. Two phosphopeptides were generated from the metabolically radiolabeled ODC, including one that migrated similarly to the peptide phosphorylated by CKII in vitro. Each of the in situ radiolabeled ODC peptides contained both 32 P-labeled serine and threonine residues. Thus, in RAW264 cells, ODC is phosphorylated on at least one serine residue in addition to that phosphorylated by CKII and on at least two threonine residues. Phosphorylated ODC had an increased stability to intracellular proteolysis compared with unphosphorylated ODC, their half-lives being 49.2 ؎ 3.78 and 23.9 ؎ 2.6 min (p ‫؍‬ 0.001), respectively. The phosphorylated and unphosphorylated forms of ODC were independently purified to homogeneity. Kinetic analysis revealed that the catalytic efficiency of the phosphorylated form of ODC was 50% greater than that of the unphosphorylated form; the unphosphorylated ODC had a V max of 20.54 ؎ 1.65 mol/min/mg, whereas the phosphorylated form had a V max of 30.61 ؎ 2.6 mol/min/mg (p ‫؍‬ 0.005). Phosphorylation of ODC by CKII has no effect on enzyme activity. Taken together, these findings demonstrate that regulation of ODC activity is governed by as yet unidentified protein kinases.Ornithine decarboxylase (ODC, EC 4.1.1.17) 1 is the initial rate-limiting enzyme in polyamine biosynthesis. Putrescine, the product of ODC catalysis, and the subsequent metabolic pathway products spermidine and spermine are small aliphatic nitrogenous bases that fulfill structural and regulatory roles in protein and nucleic acid biosynthesis and function (1). Cellular ODC activity increases rapidly and transiently in response to trophic, proliferative, or toxic stimuli (2). Both somatic cell genetic and pharmacologic experimental approaches have demonstrated that expression of ODC is essential for cell growth. Regulation of enzyme expression at the levels of DNA transcription, mRNA translation, and protein turnover have been described (for reviews, see Refs. 3 and 4). However, little is known about potential posttranslational regulation of ODC.Multiple forms of ODC protein occur in a variety of cells and tissues. Anion exchange chromatography resolves several peaks of ODC activity in extracts prepared from calf liver (5), rat thymus and kidney (6), rat he...

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

“…45 In RAW 264 cells, there are phosphothreonine residues found in phosphorylated ODC, that is, in situ phosphorylation of ODC threonine residues is catalyzed by an unidentified protein kinase(s) other than CKII. 40 The activity of CKII in rat lymphoid Nb2 cells was 2-fold higher than in the control 24 h later following prolactin treatment. 48 The slow stimulation of CKII by prolactin in Nb2 cells suggests that this kinase is not specifically induced and is not responsible for inducing ODC activity in prolactin action.…”

“…Neither mutation of ODC serine 303 [42][43][44] to alanine had an effect on ODC activity, 45 nor did phosphorylation of ODC by CKII. 40,46,47 In addition, serine is the only ODC amino acid residue modified by CKII in vitro. 45 In RAW 264 cells, there are phosphothreonine residues found in phosphorylated ODC, that is, in situ phosphorylation of ODC threonine residues is catalyzed by an unidentified protein kinase(s) other than CKII.…”

“…39 Phosphorylated ODC is more stable and has 50% greater catalytic efficiency than unphosphorylated forms in RAW264 cells. 40 The protein kinase known to phosphorylate ODC is casein kinase II (CKII). 41 A consensus sequence for CKII-catalyzed phosphorylation is conserved in ODC amino acid sequence around serine 303.…”

Amino- and Carboxy-Terminal PEST Domains Mediate Gastrin Stabilization of Rat l-Histidine Decarboxylase Isoforms

“…This model assumes a constant rate of conversion between isoforms and the potential for independent degradation of each of the isoforms. At the present time, however, we cannot determine categorically whether selective conversion of isoforms occurs or whether parameters known to regulate stability (such as covalent modification or dimerization [2,36,17]) might represent an additional level of regulation.…”

“…There is increasing consensus that protein degradation can play a key role in the control of protein function, with parameters such as covalent modification (36), intracellular localization (50), and regulated cleavage (7,31) all capable of influencing turnover rates. In many cases this degradation is mediated by a multicatalytic proteinase referred to as the proteasome.…”