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J. Biol. Chem., Vol. 283, Issue 42, 28563-28573, October 17, 2008

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A Central Role for CK1 in Catalyzing Phosphorylation of the p53 Transactivation Domain at Serine 20 after HHV-6B Viral Infection^*

Nicola J. MacLaine, Bodil Øster, Bettina Bundgaard, Jennifer A. Fraser, Carolyn Buckner¹, Pedro A. Lazo^¶, David W. Meek^||, Per Höllsberg², and Ted R. Hupp³

From the Institute of Genetics and Molecular Medicine, Division of Cancer Biology, CRUK p53 Signal Transduction Group, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, United Kingdom, the Institute of Medical Microbiology and Immunology, University of Aarhus, Aarhus DK 800, Denmark, ^¶Programa de Oncología Translacional, Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas, Universidad de Salamanca, Salamanca 37071, Spain, and ^||Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 4HN, United Kingdom

The tumor suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at Ser²⁰ whose modification stabilizes the binding of the transcriptional co-activator p300 and whose mutation in murine transgenics induces B-cell lymphoma. Although the checkpoint kinase CHK2 is implicated in promoting Ser²⁰ site phosphorylation after irradiation, the enzyme that triggers this phosphorylation after DNA viral infection is undefined. Using human herpesvirus 6B (HHV-6B) as a virus that induces Ser²⁰ site phosphorylation of p53 in T-cells, we sought to identify the kinase responsible for this virus-induced p53 modification. The p53 Ser²⁰ kinase was fractionated and purified using cation, anion, and dye-ligand exchange chromatography. Mass spectrometry identified casein kinase 1 (CK1) and vaccinia-related kinase 1 (VRK1) as enzymes that coeluted with virus-induced Ser²⁰ site kinase activity. Immunodepletion of CK1 but not VRK1 removed the kinase activity from the peak fraction, and bacterially expressed CK1 exhibited Ser²⁰ site kinase activity equivalent to that of the virus-induced native CK1. CK1 modified p53 in a docking-dependent manner, which is similar to other known Ser²⁰ site p53 kinases. Low levels of the CK1 inhibitor D4476 selectively inhibited HHV-6B-induced Ser²⁰ site phosphorylation of p53. However, x-ray-induced Ser²⁰ site phosphorylation of p53 was not blocked by D4476. These data highlight a central role for CK1 as the Ser²⁰ site kinase for p53 in DNA virus-infected cells but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser²⁰.

Received for publication, June 9, 2008 , and in revised form, July 29, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the Biotechnology and Biological Sciences Research Council-Radical Solutions for Researching the Proteome (RASOR) Interdisciplinary Research Collaboration in Proteomic Technologies.

² Supported by grants from the Lundbeck Foundation, Edith Stern, and the Faculty of Health Science (Program on Molecular Medicine), University of Aarhus, Denmark.

³ Supported by Cancer Research UK. To whom correspondence should be addressed. Tel.: 44-1317773538; Fax: 44-1317773583; E-mail: ted.hupp{at}ed.ac.uk.

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