Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

M Hjerrild, D Milne, N Dumaz, T Hay, O G Issinger, D Meek

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 2001-Apr-15
OriginalsprogEngelsk
TidsskriftBiochemical Journal
Vol/bind355
Udgave nummerPt 2
Sider (fra-til)347-56
ISSN0264-6021
DOI
StatusUdgivet - 2001

Fingeraftryk

Proto-Oncogene Proteins c-mdm2
Casein Kinase II
Phosphorylation
Serine
Cultured Cells
Clone Cells
Cells
Dichlororibofuranosylbenzimidazole
Degradation
Phosphopeptides
Proteins
Ubiquitin-Protein Ligases
Oncogene Proteins
Ionizing radiation
Proteasome Endopeptidase Complex
Fractionation
Ubiquitin
Chromatography
Ultraviolet radiation
Protein Kinases

Citer dette

@article{ce78be806fcd11db81a9000ea68e967b,
title = "Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.",
abstract = "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.",
keywords = "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",
author = "M Hjerrild and D Milne and N Dumaz and T Hay and Issinger, {O G} and D Meek",
year = "2001",
doi = "10.1042/bj3550347",
language = "English",
volume = "355",
pages = "347--56",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "Pt 2",

}

Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells. / Hjerrild, M; Milne, D; Dumaz, N; Hay, T; Issinger, O G; Meek, D.

I: Biochemical Journal, Bind 355, Nr. Pt 2, 2001, s. 347-56.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

AU - Hjerrild, M

AU - Milne, D

AU - Dumaz, N

AU - Hay, T

AU - Issinger, O G

AU - Meek, D

PY - 2001

Y1 - 2001

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Animals

KW - Base Sequence

KW - Casein Kinase II

KW - Cell Line

KW - Cells, Cultured

KW - DNA Primers

KW - Humans

KW - Mice

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Nuclear Proteins

KW - Protein-Serine-Threonine Kinases

KW - Proto-Oncogene Proteins

KW - Proto-Oncogene Proteins c-mdm2

KW - Serine

KW - Tumor Suppressor Protein p53

U2 - 10.1042/bj3550347

DO - 10.1042/bj3550347

M3 - Journal article

VL - 355

SP - 347

EP - 356

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - Pt 2

ER -