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PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression.

Wan J, Zhan J, Li S, Ma J, Xu W, Liu C, Xue X, Xie Y, Fang W, Chin YE, Zhang H - Nucleic Acids Res. (2015)

Bottom Line: We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1.Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2).Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embrology, Peking University Health Science Center, Beijing 100191, China.

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EZH2 acetylation regulates its phosphorylation and stability (A) HEK-293T cells were transfected respectively with EZH2-wt, EZH2-8KR and EZH2-8KQ. Cell lysates were immunoblotted and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (B) HEK-293T cells were transfected with FLAG-EZH2 constructs with various mutations, followed by SDS-PAGE and analyzed by western blot and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (C) H1299 cells were transfected with empty vector, EZH2-wt or EZH2-K348Q mutant. Lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by immunoblotted with indicated Abs. (D) EZH2 hypoacetylated mimic EZH2-K348R raises the phosphorylation at T345 and T487. EZH2-wt or the acetylation-deficient mutant EZH2-K348R were co-transfected with PCAF and analyzed by western blot with indicated Abs. (E) GST-fusion proteins containing EZH2-wt and EZH2-K348Q N-terminal domain (1-522) were affinity-purified and measured by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (F) GST-fusion proteins containing EZH2-wt and EZH2-K348R N-terminal domain (1-522) were affinity-purified and measured firstly by in vitro acetylation assay, then by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the indicated Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (G) H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h, followed by SDS-PAGE and western blot with indicated Abs. (H and I) K348 acetylation increases the protein stability of EZH2. HEK-293T cells were co-transfected with FLAG-PCAF plus FLAG-EZH2-wt or FLAG-EZH2-wt, FLAG-EZH2-K348R and FLAG-EZH2-K348Q alone. Thirty-six hours post transfection, cells were treated with 100 mg/ml cycloheximide for the indicated times, followed by western blot with an anti-FLAG Ab (H). The bands of FLAG-EZH2 proteins were quantified by software Image J and plotted (I). Results presented are expressed as mean ± SD from three independent experiments.
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Figure 5: EZH2 acetylation regulates its phosphorylation and stability (A) HEK-293T cells were transfected respectively with EZH2-wt, EZH2-8KR and EZH2-8KQ. Cell lysates were immunoblotted and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (B) HEK-293T cells were transfected with FLAG-EZH2 constructs with various mutations, followed by SDS-PAGE and analyzed by western blot and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (C) H1299 cells were transfected with empty vector, EZH2-wt or EZH2-K348Q mutant. Lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by immunoblotted with indicated Abs. (D) EZH2 hypoacetylated mimic EZH2-K348R raises the phosphorylation at T345 and T487. EZH2-wt or the acetylation-deficient mutant EZH2-K348R were co-transfected with PCAF and analyzed by western blot with indicated Abs. (E) GST-fusion proteins containing EZH2-wt and EZH2-K348Q N-terminal domain (1-522) were affinity-purified and measured by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (F) GST-fusion proteins containing EZH2-wt and EZH2-K348R N-terminal domain (1-522) were affinity-purified and measured firstly by in vitro acetylation assay, then by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the indicated Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (G) H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h, followed by SDS-PAGE and western blot with indicated Abs. (H and I) K348 acetylation increases the protein stability of EZH2. HEK-293T cells were co-transfected with FLAG-PCAF plus FLAG-EZH2-wt or FLAG-EZH2-wt, FLAG-EZH2-K348R and FLAG-EZH2-K348Q alone. Thirty-six hours post transfection, cells were treated with 100 mg/ml cycloheximide for the indicated times, followed by western blot with an anti-FLAG Ab (H). The bands of FLAG-EZH2 proteins were quantified by software Image J and plotted (I). Results presented are expressed as mean ± SD from three independent experiments.

Mentions: Given that EZH2 phosphorylation at T345 and T487 are important for its function (21–23,34), it is necessary to clarify whether EZH2 acetylation affects its phosphorylation. To this end, we generated an EZH2-8KQ mutant with all the eight lysines (K) mutated to glutamine (Q), a mimic of hyperacetylated EZH2. Lysates from cells transfected with different mutants were analyzed by western blot with the anti-EZH2-T345-P and anti-EZH2-T487-P antibodies. Intriguingly, phosphorylation of EZH2-8KQ at T345 and T487 were markedly decreased compared to the EZH2-wt (Figure 5A). In contrast, the acetylation-deficient mimic EZH2-8KR retained a similar phosphorylation level to the EZH2-wt (Figure 5A). Furthermore, to pinpoint which acetylation site is critical to the decrease of EZH2 phosphorylation at T345 and T487, we generated eight mutants with all the single lysine (K) mutated to glutamine (Q). By transfection of these eight EZH2 acetylation mutants in cells we found that phosphorylation at T345 and T487 of EZH2-K348Q mutant was markedly reduced compared to the EZH2-wt, while other mutants did not show changes in EZH2 phosphorylation (Figure 5B and Supplementary Figure S2A for quantification), indicating that EZH2 acetylation at K348 may regulate the decrease of EZH2 phosphorylation at T345 and T487. The importance of EZH2-K348 acetylation on EZH2 phosphorylation was re-confirmed in H1299 cells (Figure 5C). Oppositely, phosphorylation of EZH2-K348R at T345 and T487 were increased compared with EZH2-wt in the presence of PCAF (Figure 5D). To avoid any possible cellular background interference, we set up an in vitro phosphorylation assay using purified CDK1, EZH2-wt, EZH2-K348Q and EZH2-K348R proteins. Consistent with the in vivo results, we found that phosphorylation of EZH2-K348Q at T345 and T487 by CDK1 were greatly decreased compared to the EZH2-wt (Figure 5E) and phosphorylation of EZH2-K348R by CDK1 at T345 and T487 were increased compared with EZH2-wt after acetylated by PCAF in vitro (Figure 5F). Furthermore, phosphorylation of EZH2 at T345 and T487 were reduced when cells treated with nicotinamide (Figure 5G). However, neither T345A nor T487A phosphorylation-deficient mutants affected the EZH2-K348 acetylation (Supplementary Figure S2B), suggesting that EZH2 phosphorylation at T345 and T487 does not affect its acetylation at K348. We also examined different PTMs status of EZH2 in lung adenocarcinoma patients by immunohistochemical analysis (IHC). Importantly, we found that there exists a negative correlation between K348 acetylated EZH2 and T487 phosphorylated EZH2 in a preliminary experiment, while not the same tendency was found for the T345 phosphorylation of EZH2 (Supplementary Figure S2C). Collectively, these data clearly demonstrated that EZH2-K348 acetylation impairs its phosphorylation at T345 and T487, but not vice versa.


PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression.

Wan J, Zhan J, Li S, Ma J, Xu W, Liu C, Xue X, Xie Y, Fang W, Chin YE, Zhang H - Nucleic Acids Res. (2015)

EZH2 acetylation regulates its phosphorylation and stability (A) HEK-293T cells were transfected respectively with EZH2-wt, EZH2-8KR and EZH2-8KQ. Cell lysates were immunoblotted and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (B) HEK-293T cells were transfected with FLAG-EZH2 constructs with various mutations, followed by SDS-PAGE and analyzed by western blot and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (C) H1299 cells were transfected with empty vector, EZH2-wt or EZH2-K348Q mutant. Lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by immunoblotted with indicated Abs. (D) EZH2 hypoacetylated mimic EZH2-K348R raises the phosphorylation at T345 and T487. EZH2-wt or the acetylation-deficient mutant EZH2-K348R were co-transfected with PCAF and analyzed by western blot with indicated Abs. (E) GST-fusion proteins containing EZH2-wt and EZH2-K348Q N-terminal domain (1-522) were affinity-purified and measured by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (F) GST-fusion proteins containing EZH2-wt and EZH2-K348R N-terminal domain (1-522) were affinity-purified and measured firstly by in vitro acetylation assay, then by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the indicated Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (G) H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h, followed by SDS-PAGE and western blot with indicated Abs. (H and I) K348 acetylation increases the protein stability of EZH2. HEK-293T cells were co-transfected with FLAG-PCAF plus FLAG-EZH2-wt or FLAG-EZH2-wt, FLAG-EZH2-K348R and FLAG-EZH2-K348Q alone. Thirty-six hours post transfection, cells were treated with 100 mg/ml cycloheximide for the indicated times, followed by western blot with an anti-FLAG Ab (H). The bands of FLAG-EZH2 proteins were quantified by software Image J and plotted (I). Results presented are expressed as mean ± SD from three independent experiments.
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Figure 5: EZH2 acetylation regulates its phosphorylation and stability (A) HEK-293T cells were transfected respectively with EZH2-wt, EZH2-8KR and EZH2-8KQ. Cell lysates were immunoblotted and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (B) HEK-293T cells were transfected with FLAG-EZH2 constructs with various mutations, followed by SDS-PAGE and analyzed by western blot and probed with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. (C) H1299 cells were transfected with empty vector, EZH2-wt or EZH2-K348Q mutant. Lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by immunoblotted with indicated Abs. (D) EZH2 hypoacetylated mimic EZH2-K348R raises the phosphorylation at T345 and T487. EZH2-wt or the acetylation-deficient mutant EZH2-K348R were co-transfected with PCAF and analyzed by western blot with indicated Abs. (E) GST-fusion proteins containing EZH2-wt and EZH2-K348Q N-terminal domain (1-522) were affinity-purified and measured by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the anti-EZH2-T345-P and anti-EZH2-T487-P Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (F) GST-fusion proteins containing EZH2-wt and EZH2-K348R N-terminal domain (1-522) were affinity-purified and measured firstly by in vitro acetylation assay, then by in vitro kinase assay. Reaction mixtures were resolved by SDS-PAGE and analyzed by western blot with the indicated Abs. The purified GST fusion proteins were detected by Coomassie blue staining. (G) H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h, followed by SDS-PAGE and western blot with indicated Abs. (H and I) K348 acetylation increases the protein stability of EZH2. HEK-293T cells were co-transfected with FLAG-PCAF plus FLAG-EZH2-wt or FLAG-EZH2-wt, FLAG-EZH2-K348R and FLAG-EZH2-K348Q alone. Thirty-six hours post transfection, cells were treated with 100 mg/ml cycloheximide for the indicated times, followed by western blot with an anti-FLAG Ab (H). The bands of FLAG-EZH2 proteins were quantified by software Image J and plotted (I). Results presented are expressed as mean ± SD from three independent experiments.
Mentions: Given that EZH2 phosphorylation at T345 and T487 are important for its function (21–23,34), it is necessary to clarify whether EZH2 acetylation affects its phosphorylation. To this end, we generated an EZH2-8KQ mutant with all the eight lysines (K) mutated to glutamine (Q), a mimic of hyperacetylated EZH2. Lysates from cells transfected with different mutants were analyzed by western blot with the anti-EZH2-T345-P and anti-EZH2-T487-P antibodies. Intriguingly, phosphorylation of EZH2-8KQ at T345 and T487 were markedly decreased compared to the EZH2-wt (Figure 5A). In contrast, the acetylation-deficient mimic EZH2-8KR retained a similar phosphorylation level to the EZH2-wt (Figure 5A). Furthermore, to pinpoint which acetylation site is critical to the decrease of EZH2 phosphorylation at T345 and T487, we generated eight mutants with all the single lysine (K) mutated to glutamine (Q). By transfection of these eight EZH2 acetylation mutants in cells we found that phosphorylation at T345 and T487 of EZH2-K348Q mutant was markedly reduced compared to the EZH2-wt, while other mutants did not show changes in EZH2 phosphorylation (Figure 5B and Supplementary Figure S2A for quantification), indicating that EZH2 acetylation at K348 may regulate the decrease of EZH2 phosphorylation at T345 and T487. The importance of EZH2-K348 acetylation on EZH2 phosphorylation was re-confirmed in H1299 cells (Figure 5C). Oppositely, phosphorylation of EZH2-K348R at T345 and T487 were increased compared with EZH2-wt in the presence of PCAF (Figure 5D). To avoid any possible cellular background interference, we set up an in vitro phosphorylation assay using purified CDK1, EZH2-wt, EZH2-K348Q and EZH2-K348R proteins. Consistent with the in vivo results, we found that phosphorylation of EZH2-K348Q at T345 and T487 by CDK1 were greatly decreased compared to the EZH2-wt (Figure 5E) and phosphorylation of EZH2-K348R by CDK1 at T345 and T487 were increased compared with EZH2-wt after acetylated by PCAF in vitro (Figure 5F). Furthermore, phosphorylation of EZH2 at T345 and T487 were reduced when cells treated with nicotinamide (Figure 5G). However, neither T345A nor T487A phosphorylation-deficient mutants affected the EZH2-K348 acetylation (Supplementary Figure S2B), suggesting that EZH2 phosphorylation at T345 and T487 does not affect its acetylation at K348. We also examined different PTMs status of EZH2 in lung adenocarcinoma patients by immunohistochemical analysis (IHC). Importantly, we found that there exists a negative correlation between K348 acetylated EZH2 and T487 phosphorylated EZH2 in a preliminary experiment, while not the same tendency was found for the T345 phosphorylation of EZH2 (Supplementary Figure S2C). Collectively, these data clearly demonstrated that EZH2-K348 acetylation impairs its phosphorylation at T345 and T487, but not vice versa.

Bottom Line: We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1.Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2).Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embrology, Peking University Health Science Center, Beijing 100191, China.

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Related in: MedlinePlus