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.2009 Jan;20(1):419-27.
doi: 10.1091/mbc.e08-08-0792. Epub 2008 Nov 5.

PCAF acetylates {beta}-catenin and improves its stability

Affiliations

PCAF acetylates {beta}-catenin and improves its stability

Xinjian Ge et al. Mol Biol Cell.2009 Jan.

Abstract

beta-Catenin plays an important role in development and tumorigenesis. However, the effect of a key acetyltransferase p300/CBP-associated factor (PCAF) on beta-catenin signaling is largely unknown. In this study, we found PCAF could increase the beta-catenin transcriptional activity, induce its nuclear translocation, and up-regulate its protein level by inhibiting its ubiquitination and improving its stability. Further studies showed that PCAF directly binds to and acetylates beta-catenin. The key ubiquitination sites Lys-19 and Lys-49 of beta-catenin were shown as the critical residues for PCAF-induced acetylation and stabilization. Knockdown of PCAF in colon cancer cells markedly reduced the protein level, transcriptional activity, and acetylation level of beta-catenin; promoted cell differentiation; inhibited cell migration; and repressed xenografted tumorigenesis and tumor growth in nude mice. All these data demonstrate that PCAF acetylates beta-catenin and regulates its stability, and they raise the prospect that therapies targeting PCAF may be of clinical use in beta-catenin-driven diseases, such as colon cancer.

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Figures

Figure 1.
Figure 1.
PCAF enhances β-catenin transcriptional activity, induces its nuclear translocation and up-regulates its protein level. (A) PCAF activated β-catenin transcriptional activity in a dose-dependent manner. HEK293T cells were transfected with Super8×TOPFlash and the indicated amount of PCAF-FLAG or ΔHAT2-PCAF-FLAG for luciferase assay. **p < 0.01, versus the cells transfected with empty vector;#p < 0.05,##p < 0.01, versus the cells transfected with equal amount of PCAF-FLAG. In this and all other figures, error bars represent SD (B–D) PCAF synergized with β-catenin or T41A-β-catenin to activate Super8×TOPFlash. **p < 0.01; NS, no significant difference. (E) PCAF induced nuclear translocation of β-catenin. HeLa cells were transfected with indicated constructs. After transfection for 28 h, the cells shown (bottom) were treated with 20 mM LiCl. After transfection for 40 h, cells were fixed and immunostained with anti-Myc or anti-FLAG antibody. Nuclei were visualized by DAPI staining. Bar, 10 μm. (F) Up-regulation of endogenous β-catenin by increased expression of PCAF. After transfection with PCAF-FLAG or ΔHAT2-PCAF-FLAG in HEK293T cells for 40 h, total β-catenin, PCAF, and tubulin level in the extract were detected by Western blot. (G and H) PCAF increased β-catenin protein level when cotransfected with β-catenin-Myc or T41A-β-catenin-Myc. After transfection with indicated constructs in HEK293T cells for 40 h, β-catenin, PCAF, and tubulin in cell lysates were detected with anti-Myc, anti-PCAF, or anti-tubulin antibody.
Figure 2.
Figure 2.
PCAF improves the stability of β-catenin. (A) PCAF did not affect β-catenin mRNA level. After transfected with indicated constructs for 40 h, cells were harvested for RT-PCR. (B) Quantification of mRNA levels showed in (A). **p< 0.01 versus vector;#p < 0.05,##p < 0.01 versus PCAF. (C) PCAF significantly inhibited degradation of β-catenin. HEK293T cells were transfected with PCAF-FLAG or empty vector. Thirty hours later, the cells were treated with 100 μg/ml cycloheximide (CHX) for indicated times and then harvested for Western blot. (D) Quantification of endogenous β-catenin level in (C). (E) MG-132, a proteasome inhibitor, failed to up-regulate β-catenin protein level in the presence of overexpressed exogenous PCAF. After transfection with PCAF-FLAG or empty vector for 30 h, HEK293T cells were treated with or without 25 μM MG-132 for 3 or 5 h. (F) PCAF inhibited the ubiquitination of endogenous β-catenin. After transfection with PCAF-FLAG for 30 h, HEK293T cells were treated with 10 μM MG-132 for 8 h and then harvested for immunoprecipitation. The immunoprecipitated protein level was balanced to detect its ubiquitination level.
Figure 3.
Figure 3.
PCAF interacts with β-catenin, and this interaction is enhanced by activation of Wnt signaling. (A and B) PCAF interacts with β-catenin in vitro. Purified GST-PCAF and GST-β-catenin was mixed, and then immunoprecipitation was carried out with an anti-PCAF antibody (A) or anti-β-catenin antibody (B). (C) PCAF was coimmunoprecipitated with β-catenin. After transfection with indicated constructs for 30 h, cells were harvested for immunoprecipitation with anti-HA antibody. Western blots were performed with the indicated antibodies. (D) Quantification of coimmunoprecipitated PCAF and ΔHAT1,2-PCAF level in (C). **p < 0.01. (E) Endogenous PCAF was coimmunoprecipitated with anti-β-catenin antibody, and this effect was enhanced by Wnt3a treatment. After treated with Wnt3a conditioned medium (Wnt3a-CM) or the control medium for 12 h, HEK293T cells were harvested for immunoprecipitation. (F) Quantification of coimmunoprecipitated PCAF level in (E). **p < 0.01. (G) Endogenous β-catenin was coimmunoprecipitated by anti-PCAF antibody, and this effect was enhanced by LiCl treatment. After treated with or without 20 mM LiCl for 12 h. HEK293T cells were harvested for immunoprecipitation. (H) Quantification of coimmunoprecipitated β-catenin level in (G). *p < 0.05. The immunoprecipitated protein level was balanced to detect the relative level of coimmunoprecipitated proteins.
Figure 4.
Figure 4.
PCAF acetylates β-catenin in vitro and in vivo. (A) PCAF acetylated β-catenin in vitro. The acetylation of GST-β-catenin by PCAF was measured by in vitro acetylation assay and finally detected by Western blot. (B) PCAF acetylated exogenous β-catenin. After transfected with T41A-β-catenin-FLAG and PCAF-FLAG or ΔHAT2-PCAF-FLAG for 40 h, HEK293T cells were harvested for immunoprecipitation. The same membrane was stripped and reblotted with anti-β-catenin antibody. (C) PCAF acetylated endogenous β-catenin. After transfected with PCAF-FLAG or ΔHAT2-PCAF-FLAG for 40 h, HEK293T cells were harvested for immunoprecipitation with anti-β-catenin antibody. Western blots were performed with the indicated antibodies. The immunoprecipitated protein level was balanced to detect its acetylation level.
Figure 5.
Figure 5.
K19 and K49 of β-catenin are critical residues for its acetylation and stabilization induced by PCAF. (A) PCAF up-regulated the protein level of WT, K19R, or K49R-β-catenin but not that of K19,49R-β-catenin. After transfection with WT-β-catenin or its mutants and/or PCAF-Myc to HEK293T cells for 40 h, total β-catenin, PCAF, and tubulin level were detected by Western blot. (B) Quantification of β-catenin level in (A). **p< 0.01, versus the vector in the same group. (C) PCAF up-regulated the protein level of T41A-β-catenin but not that of K19,49R-β-catenin. (D) PCAF could not induce the acetylation of K19,49R-β-catenin. After transfection with WT-β-catenin or its mutants and/or PCAF-Myc for 40 h, HEK293T cells were harvested for immunoprecipitation. (E) Quantification of acetylation level of β-catenin in (D). *p< 0.05, versus WT-β-catenin with PCAF.#p < 0.05 versus K19R or K49R-β-catenin with PCAF. (F) PCAF could not inhibit the ubiquitination of K19,49R-β-catenin. HEK293T cells were transfected with K19,49R-β-catenin and/or PCAF-Myc for 30 h and then treated with 10 μM MG-132 for 8 h and harvested for immunoprecipitation. The immunoprecipitated protein level was balanced to detect its acetylation or ubiquitination level.
Figure 6.
Figure 6.
Knockdown of PCAF reduces the protein level, transcriptional activity, and acetylation level of β-catenin. (A) Knockdown of PCAF reduced the protein level of exogenous β-catenin. After transfection with β-catenin-FLAG (β-cat) or T41A-β-catenin-FLAG (T41A), and luc-RNAi or PCAF-RNAi in for 72 h, HEK293 cells were harvested, and β-catenin, PCAF, and tubulin in the cell lysates were detected by Western blot with anti-FLAG, anti-PCAF, or anti-tubulin antibody. (B) Knockdown of PCAF down-regulated endogenous β-catenin protein level. After transfected with luc-RNAi or PCAF-RNAi for 72 h, HEK293 cells were harvested, and endogenous β-catenin, PCAF, and tubulin in cell lysates were detected by Western blot. (C) Knockdown of PCAF inhibited β-catenin transcriptional activity measured by luciferase assay with Super8×TOPFlash. *p< 0.05, **p< 0.01, versus luc-RNAi. (D) Knockdown of PCAF decreased the acetylation level of β-catenin. HEK293 cells were transfected with luc-RNAi or PCAF-RNAi for 72 h. Immunoprecipitated proteins were detected by Western blot with anti-acetyl-Lys antibody. The same membrane was stripped and reblotted with anti-β-catenin antibody. The immunoprecipitated protein level was balanced to detect its acetylation level. (E) Knockdown of PCAF could not decrease the protein level of K19,49R-β-catenin. After transfecting HEK293 cells with WT-β-catenin or K19,49R-β-catenin, and luc-RNAi or PCAF-RNAi for 72 h, total β-catenin, PCAF, and tubulin in cell lysates were detected by Western blot.
Figure 7.
Figure 7.
Knockdown of PCAF in colon cancer cells promotes cell differentiation, blocks cell migration and tumor growth. (A) PCAF RNAi induced the differentiation of LoVo cells. After transfection with plasmids expressing luc-RNAi, PCAF-RNAi or β-catenin-RNAi for 3 d, differentiated LoVo cells were measured by alkaline phosphatase activity assay. **p< 0.01 versus luc-RNAi. (B) PCAF RNAi induced the expression of villin, a marker of colonic cell differentiation and down-regulated the protein level of β-catenin and c-Myc. After infection with lentivirus expressing luc-RNAi or PCAF-RNAi for 72 h, PCAF, villin, β-catenin, and tubulin in LoVo cells were detected by Western blot. (C) PCAF-RNAi slowed down the migration of LoVo cells. After infection with lentivirus expressing luc-RNAi or PCAF-RNAi for 7 d, LoVo cells were used for wound assay. GFP represented the cells expressing luc-RNAi or PCAF-RNAi. Bar, 200 μm. (D) PCAF RNAi reduced the tumor number. LoVo cells expressing luc-RNAi or PCAF-RNAi were injected subcutaneously into nude mice. The mice were killed 2 mo later to analyze the tumor number and tumor size. The chi-square test was used for the analysis of detection frequency of tumors in nude mice. *p< 0.05 versus luc-RNAi. (E) Gross appearance of xenografts and excised tumors. Bar, 1 cm. (F) PCAF RNAi reduced the tumor size. *p< 0.05 versus luc-RNAi. (G) Effect of PCAF-RNAi to down-regulate PCAF in the tumors was confirmed by Western blot. (H) Quantification of western blot results from G. **p< 0.01.
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