p53 Is Phosphorylated in Vitro and in Vivo by an Ultraviolet Radiation-induced Protein Kinase Characteristic of the c-Jun Kinase, JNK1

doi:10.1074/jbc.270.10.5511

p53 Is Phosphorylated in Vitro and in Vivo by an Ultraviolet Radiation-induced Protein Kinase Characteristic of the c-Jun Kinase, JNK1 (*)

From the Biomedical Research Center, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom and the Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 4HN, United Kingdom

^§ The recipient of a Medical Research Council Senior Non-clinical Fellowship. To whom correspondence should be addressed. Tel.: 44-382-660111 ext. 3517 (office) or 3514 (laboratory); Fax: 44-382-69993.

Abstract

The p53 tumor suppressor protein is thought to play a major role in the defense of the cell against agents that damage DNA. In this report, we describe the identification and characterization of a protein kinase that phosphorylates mouse p53 at a single site, serine 34, a major site of phosphorylation in the cell. The protein kinase is activated strikingly following treatment of cells with ultraviolet radiation, has a native molecular weight of approximately 45,000, and can be resolved from mitogen-activated protein (MAP) kinase by chromatography on Superose 6 and DEAE-cellulose. The p53 kinase activity co-purifies with UV-activated c-Jun kinase activity on heparin-Sepharose and on a c-Jun (but not a v-Jun-) affinity column. Treatment of the partially purified kinase with CL100, a protein phosphatase that specifically dephosphorylates MAP kinase homologues, inhibits its activity. Taken together, the data suggest that this p53 kinase is likely to be activated by phosphorylation and may be a member of the stress-activated protein kinase subfamily of MAP kinases. UV irradiation of SV3T3 cells leads to increased phosphorylation of p53 at serine 34, indicating that phosphorylation of p53 by this kinase is likely to be physiological. Phosphorylation of p53 by this protein kinase may be a key event in a signal transduction mechanism that coordinately controls key nuclear proteins in response to oxidative stress or DNA damaging agents.

Footnotes

↵* This work was supported by the Medical Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MAP

mitogen-activated protein

GST-p53

glutathione S-transferase-p53

HPLC

high performance liquid chromatography

SAP

stress-activated protein

FP

fusion protein.
↵²S. M. Keyes and A. Gartner, unpublished data.
- Received October 17, 1994.
- Revision received December 15, 1994.