1. Volume 64, Issue 4, Pages 803-814 (November 2016)
TRAF6 Restricts p53 Mitochondrial Translocation, Apoptosis, and Tumor Suppression 
Xian Zhang, Chien-Feng Li, Ling Zhang, Ching-Yuan Wu, Lixia Han, Guoxiang Jin, Abdol Hossein Rezaeian, Fei Han, Chunfang Liu, Chuan Xu, Xiaohong Xu, Chih-Yang Huang, Fuu-Jen Tsai, Chang-Hai Tsai, Kounosuke Watabe, Hui-Kuan Lin 
Molecular Cell 
Volume 64, Issue 4, Pages (November 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 803-814DOI: (10.1016/j.molcel.2016.10.002)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 p53 Is Ubiquitinated by TRAF6 through K63 Linkage
(A) U2OS cells were transfected with indicated plasmids for in vivo ubiquitination assay. See also Experimental Procedures.
(B) In vivo ubiquitination assay was performed for U2OS cells transfected with indicated plasmids.
(C) U2OS cells were transfected with indicated plasmids for in vivo ubiquitination.
(D) TRAF6 ubiquitinates p53 in vitro. See also Experimental Procedures.
(E) Immunoprecipitation assay was performed for U2OS cells.
(F) Immunoprecipitation assay was performed for the cytosol fraction of WT and Traf6−/− primary MEFs.
(G) Cellular fractionation assay was performed for U2OS cells treated with CDDP for indicated times.
(H) Immunoprecipitation assay was performed for U2OS cells treated with CDDP for indicated times. Phosphorylation was detected by the anti-phosphor-TQ/SQ motif, which is the consensus phosphorylation site for ATM and ATR kinase.
(I) Immunoprecipitation assay was performed for 293T cells expressing WT and mutant TRAF6 as indicated. CDDP and/or ATM inhibitor was also treated as indicated.
(J) Cellular fractionation was performed for 293T cells expressing WT and mutant TRAF6 with or without CDDP treatment.
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4. Figure 2
TRAF6 Inhibits p53 Translocation to Mitochondria and Apoptosis
(A) WT and Traf6−/− total thymus cells were analyzed by TUNEL assay. See also Experimental Procedures.
(B) WT and Traf6−/− total thymus cells were analyzed by western blot.
(C) WT and Traf6−/− mice were treated with pifithrin-μ or pifithrin-α and apoptosis of total thymus cells was analyzed by TUNEL assay.
(D) Mitochondrial fractionation was performed for WT and Traf6−/− thymus treated with pifithrin-μ or pifithrin-α. See also Experimental Procedures.
(E) TUNEL assay was performed for U2OS cells with indicated knockdown and examined by fluorescence microscopy.
(F and G) Mitochondria were isolated from total thymus cells (F) or U2OS cells (G). U2OS cells were treated with or without CDDP for 6 hr.
(H) Mitochondria fractionation was performed for the WT and Traf6−/− mice thymus treated with or without IR.
(I) TRAF6 deficiency induced mitochondrial fragmentation was p53 dependent. Immunofluorescence assay was performed for U2OS cells with indicated knockdown.
All data are represented as mean ± SEM. ∗p < 0.01, Student’s t test.
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5. Figure 3
K63-Linked Ubiquitination of p53 Inhibited the Mitochondrial Translocation of p53
(A) In vivo ubiquitination assay was performed for U2OS cells transfected with indicated plasmids.
(B) Mitochondria were isolated from H1299 cells transfected with p53 WT or K24R.
(C) Immunoprecipitation assay was performed for control and TRAF6-knockdown U2OS.
(D and E) Mitochondria were isolated from total thymus cells (D) or U2OS cells (E) and crosslinked as described in Experimental Procedures.
(F) U2OS cells were treated with or without CDDP for 6 hr before BAK oligomerization assay.
(G) Mitochondria were isolated from TRAF6-knockdown U2OS cells restored with TRAF6 WT or C70A and crosslinked by BMH.
(H) Immunoprecipitation assay was performed for H1299 cells transfected with p53 WT and K24R.
(I) Mitochondria were isolated from H1299 cells transfected with p53 WT and K24R and crosslinked by BMH.
(J and K) Ubiquitinated p53 failed to interact with mitochondria and trigger BAK oligomerization in vitro. See also Figure S3H and Experimental Procedures.
(L) In vitro binding assay was performed for recombinant MCL-1, BAK, and GST-p53 derived from in vitro ubiquitination assay.
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6. Figure 4
TRAF6 Inhibited p53 WT but Not p53 K24R-Induced Tumor Suppression
(A–C) Colony-forming assay (A), xenograft tumor growth assay (B), and western blot assay (C) were performed for H1299 cells expressing indicated exogenous proteins.
(D) Representative rectal cancers after concurrent CCRT showing low (left) and high (right) TRAF6 immunoreactivity were significantly associated with a higher tumor regression grade (TRG) and increased TUNEL and acspase-3 expression (left) and vice versa (right).
(E and F) In post-CCRT rectal cancers, TRAF6 expression is significantly and negatively related to cell death as determined by TUNEL and active caspase-3 staining.
(G and H) Survival analysis plotted by using Kaplan-Meier methods discloses TRAF6 expression in post-CCRT biopsy specimens is significantly predictive for disease-free and metastasis-free survival.
All data are represented as mean ± SEM. ∗p < 0.01, Student’s t test.
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7. Figure 5
TRAF6 Is Required for Genotoxic Stress-Induced p53 Target Gene Expression
(A) WT and Traf6−/− mice aged 2.5 weeks were treated with IR (15 Gy) for 10 hr before thymus was isolated and total RNA or total protein was extracted. Genes with greater than 1.5-fold change in mRNA expression were used to generate the heatmap.
(B and C) The expression of p53 target genes, p21 and GADD45, was validated by qPCR and western blot in the similar sample from Figure 5D.
(D and E) Representative rectal cancers showing low (left) and high (right) TRAF6 immunoreactivity in pre-treatment (Pre-Tx) biopsy specimens were linked to low p21 (left) and high p21 (right) expression, respectively. In the pre-treatment (Pre-Tx) biopsy specimens, TRAF6 immunoexpression is significantly and positively associated with p21 expression.
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8. Figure 6 TRAF6 Is Required for p300 Recruitment and p53 Acetylation
(A) Predicted regulators for TRAF6-dependent transcription regulation.
(B) Thymus prepared as described in Figure 5D was analyzed by western blot.
(C) Immunoprecipitation assay was performed for WT and Traf6−/− MEFs treated with or without IR (10 Gy) for 4 hr.
(D) Immunoprecipitation assay was performed for the nuclear fraction of WT and Traf6−/− primary MEFs to access p53 ubiquitination.
(E) Immunoprecipitation assay was performed for the H1299 cells transfected with p53 WT or K24R to access its binding with p300.
(F) Western blot was performed for H1299 cells transfected with p53 WT or K24R to detect p21 and GADD45.
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9. Figure 7 Schematic Model
Before genotoxic stress, p53 in the cytosol is bond by TRAF6 and K63-linked ubiquitinated by TRAF6. This ubiquitination inhibited the binding between p53 and BAK/MCL-1, thus keeping p53 away from mitochondria. In the nucleus, the level of TRAF6 and K63-linked ubiquitination of p53 is low, which keeps p53 activity at basal level. Upon genotoxic stress, p53 level is drastically increased, while TRAF6 level is reduced in cytosol, which results in the more non-ubiquitinated p53 and allows p53 translocation to mitochondria for BAK oligomerization and subsequent apoptosis. Meanwhile, genotoxic stress enhances the level of TRAF6 in the nucleus and drives the K63-linked ubiquitination of p53, which facilitates the recruitment of p300 for p53 transactivation. Thus, p53 target genes, including p21 and GADD45 were actively transcribed and exert their functions in cell-cycle control and cell survival. In the TRAF6-deficient condition, p53 accumulated in the mitochondria triggers Bak oligomerization and apoptosis under the unstressed condition. Upon genotoxic stress, TRAF6-deficient cells failed to activate p53 and express p21 and GADD45 and are more sensitive to genotoxic stress.
Molecular Cell  , DOI: ( /j.molcel )
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