Editors' ChoicePosttranslational Modifications

TRAF6 targets p53

Sci. Signal.  22 Nov 2016:
Vol. 9, Issue 455, pp. ec275
DOI: 10.1126/scisignal.aal4327

TRAF6 is an E3 ubiquitin ligase that attaches K63-linked ubiquitin to proteins, thereby affecting protein function, interactions, and localization. The protein p53 is multifunctional and extensively posttranslationally modified and is activated in response to genotoxic stress to stimulate the expression of genes that cause cell cycle arrest. Cytosolic p53 associates with the proapoptotic protein Bak at the mitochondria to trigger apoptosis. In an attempt to understand how cells prevent inappropriate association of p53 with mitochondria, Zhang et al. found that the E3 ubiquitin ligase TRAF6 mediated the K63-linked ubiquitylation of p53 in the cytosol and nucleus, thereby promoting the p53-mediated cell cycle arrest response and preventing the p53-mediated apoptotic response. K63-linked ubiquitylated p53 was detected in the cytosol of nonstressed cells, and mitochondrially associated p53 did not have this modification. The amount of K63-linked ubiquitylated p53 decreased upon genotoxic stress in conjunction with an increase in the abundance of p53 at mitochondria. Overexpression of various E3 ubiquitin ligases identified TRAF6 as responsible for this modification. Genotoxic stress induced the redistribution of TRAF6 from the cytosol to the nucleus through a process that was blocked by inhibition of the kinases ATM and ATR and depended on phosphorylation of two residues (Ser13 and Thr330) in TRAF6. K63-linked ubiquitylation of p53 was markedly reduced in Traf6–/– mouse embryonic fibroblasts (MEFs), and the thymus, spleen, and lungs of Traf6–/– mice had a higher percentage of apoptotic cells than did tissues from wild-type mice even in the absence of irradiation (to induce DNA damage and genotoxic stress). Additionally, gene expression profiling of the thymus from irradiated Traf6–/– and wild-type mice indicated that p53-dependent gene expression was altered in the Traf6–/– mice and included impaired induction of cell cycle arrest genes encoding p21 and Gadd45. In Traf6–/– MEFs, the interaction between p53 and the acetyltransferase p300 was reduced, resulting in less p53 acetylation, a modification required for p53 transcriptional activity. Mitochondria isolated from TRAF6-deficient cells (genetic knockout or knockdown) had increased amounts of p53, similar to those associated with mitochondria from wild-type cells that had been exposed to cisplatin. In vivo and in vitro ubiquitylation assays with mutant versions of p53 identified Lys24 as the site of TRAF6-mediated ubiquitylation. Compared with cells overexpressing wild-type p53, cells expressing the p53 L24R mutant had increased mitochondrial association of p53, an increase in the Bak-p53 interaction, and an increase in the ability of p53 to promote oligomerization of Bak and cytochrome c release from mitochondria. Overexpression of wild-type p53 or the L24R mutant inhibited in vitro colony formation and in vivo tumor growth in xenograft models. In human tumor samples, higher TRAF6 abundance negatively correlated with markers of apoptosis; high abundance of TRAF6 predicted poor survival in colorectal cancer patients treated with chemotherapy and radiation. The data indicate that TRAF6 performs key regulatory functions related to p53: TRAF6 prevented inappropriate mitochondrial association and apoptosis in the cytosol in unstressed cells and then translocated to the nucleus in response to DNA damage–induced phosphorylation to ubiquitylate nuclear p53, thus promoting its interaction with and acetylation by p300 to enable transcription-mediated cell cycle arrest.

X. Zhang, C.-F. Li, L. Zhang, C.-Y. Wu, L. Han, G. Jin, A. H. Rezaeian, F. Han, C. Liu, C. Xu, X. Xu, C.-Y. Huang, F.-J. Tsai, C.-H. Tsai, K. Watabe, H.-K. Lin, TRAF6 restricts p53 mitochondrial translocation, apoptosis, and tumor suppression. Mol. Cell 64, 803–814 (2016). [PubMed]

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