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

Sundeepg Reddy,Sarahm Mcllheran,Barbaraj Cochran,Laural Worth,Lydiaa Bishop,Patriciaj Brown,Victoriap Knutson,Marik Haddox

doi:10.1074/jbc.271.40.24945

Sundeepg Reddy, Sarahm Mcllheran + Show 6 more

Open Access

https://doi.org/10.1074/jbc.271.40.24945

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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 32P-labeled serine. ODC was metabolically radiolabeled with 32Pi 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 32P-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 Vmax of 20.54 +/- 1.65 micromol/min/mg, whereas the phosphorylated form had a Vmax of 30.61 +/- 2.6 micromol/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.

Highlights

Ʈ To whom correspondence should be addressed: Dept. of Pharmacology, The Medical School, The University of Texas, P
In this report we demonstrate by comparative two-dimensional phosphopeptide mapping and phosphoamino acid analysis that in the RAW264 cell line Ornithine decarboxylase (ODC) is phosphorylated on at least three amino acid residues in addition to that phosphorylated by casein kinase II (CKII)
When the same cell extracts were assayed for ODC activity, no activity was detectable in extracts from unstimulated RAW264 cells, whereas in the stimulated cells, the ODC activity was 3.2 nmol/min/mg

Summary

Introduction

Ʈ To whom correspondence should be addressed: Dept. of Pharmacology, The Medical School, The University of Texas, P. ODC Phosphorylation Increases Enzyme Activity and Stability question whether ODC was subject to phosphorylation in RAW264 cells at additional sites to that phosphorylated by CKII.

Results

Conclusion