MULTISITE PHOSPHORYLATION OF ORNITHINE DECARBOXYLASE IN TRANSFORMED MACROPHAGES RESULTS IN INCREASED INTRACELLULAR ENZYME STABILITY AND CATALYTIC EFFICIENCY

Ornithine decarboxylase (ODC) is the initial inducible enzyme in the p olyamine biosynthetic pathway, In the transformed macrophage-derived R AW264 cell line, ODC was overproduced and existed in both unphosphoryl ated and phosphorylated forms, To date, the only protein kinase known to phosphorylate mammalian ODC is casein kinase II (CKII), ODC was pho sphorylated in vitro by CKII and subjected to exhaustive sequential pr oteolysis with trypsin and V8 protease. Two-dimensional peptide mappin g showed only a single phosphopeptide; two dimensional phosphoamino ac id analysis of the phosphopeptide revealed only P-32-labeled serine. O DC was metabolically radiolabeled with P-32(i) in RAW264 cells and als o subjected to proteolysis, two-dimensional peptide mapping, and phosp hoamino acid analysis. Two phosphopeptides were generated from the met abolically radiolabeled ODC, including one that migrated similarly to the peptide phosphorylated by CKII in vitro, Each of the in situ radio labeled ODC peptides contained both P-32-labeled serine and threonine residues. Thus, in RAW264 cells, ODC is phosphorylated on at least one serine residue in addition to that phospho rylated by CKII and on at least two threonine residues. Phosphorylated ODC had an increased stab ility 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 w ere independently purified to homogeneity. Kinetic analysis revealed t hat 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 mu mol/min/mg, whereas the phosphory lated form had a V-max of 30.61 +/- 2.6 mu mol/min/mg (p = 0.005). Pho sphorylation of ODC by CKII has no effect on enzyme activity. Taken to gether, these findings demonstrate that regulation of ODC activity is governed by as yet unidentified protein kinases.