The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser20 inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr18 (adjacent to the DO-1 epitope) and phospho-Ser20 (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser20 detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr18 was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr18 and Ser20 in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser20 or phospho-Thr18 containing p53 peptides are as effective as the phospho-Ser15 peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser20 and Thr18 that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser20 and Thr18 kinase signalling cascades to modulate p53 activity.
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Research Article|
August 10 1999
Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers
Ashley L. CRAIG;
Ashley L. CRAIG
*Department of Molecular and Cellular Pathology, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
†Department of Molecular Oncology, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
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Lindsay BURCH;
Lindsay BURCH
*Department of Molecular and Cellular Pathology, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
†Department of Molecular Oncology, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
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Borek VOJTESEK;
Borek VOJTESEK
‡Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 656 53 Brno, Czech Republic
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Jaroslava MIKUTOWSKA;
Jaroslava MIKUTOWSKA
‡Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 656 53 Brno, Czech Republic
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Alastair THOMPSON;
Alastair THOMPSON
§Department of Surgery; Dundee Cancer Institute, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
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Ted R. HUPP
Ted R. HUPP
1
*Department of Molecular and Cellular Pathology, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
†Department of Molecular Oncology, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
1To whom correspondence should be addressed (e-mail t.r.hupp@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
November 02 1998
Revision Received:
May 04 1999
Accepted:
June 02 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (1): 133–141.
Article history
Received:
November 02 1998
Revision Received:
May 04 1999
Accepted:
June 02 1999
Citation
Ashley L. CRAIG, Lindsay BURCH, Borek VOJTESEK, Jaroslava MIKUTOWSKA, Alastair THOMPSON, Ted R. HUPP; Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers. Biochem J 15 August 1999; 342 (1): 133–141. doi: https://doi.org/10.1042/bj3420133
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