Role for 53BP1 Tudor Domain Recognition of p53 Dimethylated at Lysine 382 in DNA Damage Signaling

Ioulia Kachirskaia,Xiaobing Shi,Hiroshi Yamaguchi,Kan Tanoue,Hong Wen,Evelyn W Wang,Ettore Appella,Or Gozani

doi:10.1074/jbc.m806020200

Ioulia Kachirskaia, Xiaobing Shi + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m806020200

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Abstract

Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.

Highlights

Lysine methylation is a principal mechanism involved in chromatin regulation via modification of histone proteins [1]
We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1
Stability upon DNA Damage—To dissect potential roles for ciated p53 species that is dimethylated at Lys382

Summary

MATERIALS AND METHODS

Constructs and Reagents—Human 53BP1 cDNA and hemagglutinin-53BP1 constructs were gifts from Jiri Lukas and Phillip Carpenter, respectively. ␮g of biotinylated peptides was incubated with 1 ␮g of protein in binding buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors) for 4 h at 4 °C with rotation. ␮g of GST-p53 or 1 ␮g of p53 peptides was incubated with 1 ␮g of recombinant histone methyltransferase and 2 ␮Ci of S-adenosylmethionine (Amersham Biosciences) in reaction buffer containing 50 mM Tris-HCl, pH 8.0, 10% glycerol, 20 mM KCl, 5 mM. Immunoprecipitation and Western Immunoblotting—Endogenous p53 or ectopically expressed FLAG-tagged p53 were immunoprecipitated with agarose-conjugated p53 or FLAG antibodies from whole cell extracts in cell lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors). Gene expression was calculated and normalized to glyceraldehyde-3phosphate dehydrogenase levels by the comparative Cycle threshold method

RESULTS

DISCUSSION