Damage-mediated Phosphorylation of Human p53 Threonine 18 through a Cascade Mediated by a Casein 1-like Kinase
EFFECT ON Mdm2 BINDING*
- Kazuyasu Sakaguchiद,
- Shin'ichi Saito‡§,
- Yuichiro Higashimoto‡,
- Siddhartha Roy‖,
- Carl W. Anderson**‡ and
- Ettore Appellaत
- From the ‡NCI, National Institutes of Health, Bethesda, Maryland 20892, the ‖Department of Biophysics, Bose Institute, P-1/12 C.I.T. Scheme VII M, Calcutta, 700 054, India, and the **Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Abstract
The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr18 but not by phosphorylation at other N-terminal sites, including Ser15 and Ser37. Thr18 was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser15. Thr18 was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser15. Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser15 followed by phosphorylation of Thr18.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ The first two authors contributed equally to this work.
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↵¶ Supported in part by The Japan Securities Scholarship Foundation. Present address: Laboratory of Structure-Function Biochemistry, Dept. of Molecular Chemistry, Graduate School of Science, Kyushu University, Fukuoka 812-8581, Japan.
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↵‡ Supported at Brookhaven National Laboratory in part by National Institutes of Health Grant GM52825 and by a Cooperative Research and Development Agreement funded by the Laboratory Technology Research Program in the Office of Science of the U.S. Department of Energy.
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↵§§ To whom correspondence should be addressed: Chemical Immunology Section, Lab. of Cell Biology, Bldg. 37, Rm. 1B03, National Cancer Institute, National Institutes of Health, 37 Convent Dr. MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-402-4177; Fax: 301-402-0450; E-mail: appellae@pop.nci.nih.gov.
- Abbreviations:
- IR
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ionizing radiation
- ALLN
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N-acetyl-Leu-Leu-Nle-CHO
- CK1
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casein kinase 1
- DNA-PK
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DNA-activated protein kinase
- F2Pab
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(2-amino-4-phosphono)-4,4-difluorobutanoic acid)
- Fmoc
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9-fluorenylmethoxycarbonyl
- MES
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4-morpholineethanesulfonic acid
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- Received August 31, 1999.
- Revision received January 10, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
