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Molecular and Cellular Biology, May 2007, p. 3542-3555, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.01595-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The MDM2 Ubiquitination Signal in the DNA-Binding Domain of p53 Forms a Docking Site for Calcium Calmodulin Kinase Superfamily Members

Ashley L. Craig,¹ Jennifer A. Chrystal,¹ Jennifer A. Fraser,¹ Nathalie Sphyris,¹ Yao Lin,¹ Ben J. Harrison,¹ Mary T. Scott,^1, Irena Dornreiter,² and Ted R. Hupp^1*

University of Edinburgh, Cancer Research Centre, CRUK p53 Signal Transduction Group, South Crewe Road, Edinburgh, United Kingdom EH4 2XR,¹ Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie, Universitat Hamburg, Hamburg, Germany²

Received 26 August 2006/ Returned for modification 16 October 2006/ Accepted 31 January 2007

Genetic and biochemical studies have shown that Ser²⁰ phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser²⁰ phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser²⁰ kinases. Phosphorylation of a p53 transactivation domain fragment at Ser²⁰ by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser²⁰ kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser²⁰ phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser²⁰ kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser²⁰, (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser²⁰ phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the p53 spliced variant lacking the BOX-V motif is refractory to Ser²⁰ phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.

Published ahead of print on 5 March 2007.

Present address: CRUK Beatson Laboratories, Glasgow, Scotland.

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