Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.
Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteasome. Stress signals which impinge on p53, leading to its ... activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.
Mesh Terms:
Amino Acid Sequence, Animals, Base Sequence, Casein Kinase II, Cell Line, Cells, Cultured, DNA Primers, Humans, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Proteins, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Serine, Tumor Suppressor Protein p53
Amino Acid Sequence, Animals, Base Sequence, Casein Kinase II, Cell Line, Cells, Cultured, DNA Primers, Humans, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Proteins, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Serine, Tumor Suppressor Protein p53
Biochem. J.
Date: Apr. 15, 2001
PubMed ID: 11284721
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