Mouse ornithine decarboxylase is phosphorylated by casein kinase-II at a predominant single location (serine 303).
Ornithine decarboxylase (ODC), a key enzyme in the biosynthetic pathway of polyamines in mammalian cells is characterized by an extremely short half-life and by a rapid induction following stimulation with growth-promoting agents. Inspection of its deduced amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase II (CK-II). In the present study we demonstrate that ODC serves as a substrate for phosphorylation by CK-II in vitro and that it is phosphorylated in intact mammalian cells. One-dimensional phosphopeptide analysis demonstrated that all the phosphopeptides generated by V8 protease digestion of in vivo phosphorylated ODC correspond to the major phosphopeptides of ODC phosphorylated in vitro by CK-II. Phosphopeptide analysis of wild-type ODC and of a mutant in which serine 303 was converted to alanine demonstrated that the latter lacks the phosphopeptides that correspond to those detected in ODC phosphorylated in vivo. In addition, no incorporation of phosphate into the alanine 303 mutant was observed when it was expressed in transfected cos cells. Based on these observations, we conclude that in mammalian cells serine 303 is the major (if not the only) phosphorylated residue of ODC and that CK-II or another cellular kinase with very similar sequence specificity is responsible for manifestation of this modification. The unphosphorylated alanine 303 mutant retained enzymatic activity, which decayed at a similar rate to that of the wild-type enzyme. We therefore conclude that phosphorylation is not essential for maintaining enzymatic activity or regulating ODC turnover.
Rosenberg-Hasson
Y
Y
Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Strumpf
D
D
Kahana
C
C
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014407:Tumor Cells, Cultured
D011494:Protein Kinases; D047388:Casein Kinases; D009955:Ornithine Decarboxylase