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Acetylation of the cell-fate factor dachshund determines p53 binding and signaling modules in breast cancer.

Chen K, Wu K, Gormley M, Ertel A, Wang J, Zhang W, Zhou J, Disante G, Li Z, Rui H, Quong AA, McMahon SB, Deng H, Lisanti MP, Wang C, Pestell RG - Oncotarget (2013)

Bottom Line: DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding.DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628.DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.

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NAD-dependent binding of DACH1 to p53(A) A peptide encoding the candidate DACH1 acetylation site (626 VQKRLKKEKKAKRKLQEAL 644) was incubated with p300 as an enzyme source in the presence of acetyl CoA. Acetylation products were purified with reverse-phase HPLC followed by MALDI-TOF mass spectrometry (A) and then sequenced by Edman degradation. In (A), the resulting DACH1 peptide (DACH1aa 626-644) mass spectrum is shown with mass/charge expressed in atomic mass units (amu). The major peaks labeled X corresponds to the expected mass of DACH1 whereas the large peak Y, is larger by 42 mass units, representing a single acetylated peptide. In (B) the bars represent the amount of phenylthiohydantoin-acetyl-lysine present in the corresponding position. The major acetylated product corresponds to residue K628. The inset shows an alignment of the DACH1 sequence and the acetylated residue motif seen in other acetylated transcription factors and the corresponding consensus sequence KX(x)KK. (C) IP-WB of HEK293T cells transfected with expression vectors encoding p53 or DACH1 and subsequently treated with Nicotinamide (NAM 20 mM) and/or Trichostatin A (TSA 2μm) for 8hrs. (D) Western blot of lysates corresponding to IP-Western in (C). (E) Relative binding of DACH1 to p53 shown as mean ± SEM for 3 separate experiments. IP-Western blot of MDA-MB-453 cells treated with TSA (2 μM) or NAM (20 mM) showing (G) relative binding of p53 to DACH1 (data are mean ± SEM for 3 separate experiments).
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Figure 3: NAD-dependent binding of DACH1 to p53(A) A peptide encoding the candidate DACH1 acetylation site (626 VQKRLKKEKKAKRKLQEAL 644) was incubated with p300 as an enzyme source in the presence of acetyl CoA. Acetylation products were purified with reverse-phase HPLC followed by MALDI-TOF mass spectrometry (A) and then sequenced by Edman degradation. In (A), the resulting DACH1 peptide (DACH1aa 626-644) mass spectrum is shown with mass/charge expressed in atomic mass units (amu). The major peaks labeled X corresponds to the expected mass of DACH1 whereas the large peak Y, is larger by 42 mass units, representing a single acetylated peptide. In (B) the bars represent the amount of phenylthiohydantoin-acetyl-lysine present in the corresponding position. The major acetylated product corresponds to residue K628. The inset shows an alignment of the DACH1 sequence and the acetylated residue motif seen in other acetylated transcription factors and the corresponding consensus sequence KX(x)KK. (C) IP-WB of HEK293T cells transfected with expression vectors encoding p53 or DACH1 and subsequently treated with Nicotinamide (NAM 20 mM) and/or Trichostatin A (TSA 2μm) for 8hrs. (D) Western blot of lysates corresponding to IP-Western in (C). (E) Relative binding of DACH1 to p53 shown as mean ± SEM for 3 separate experiments. IP-Western blot of MDA-MB-453 cells treated with TSA (2 μM) or NAM (20 mM) showing (G) relative binding of p53 to DACH1 (data are mean ± SEM for 3 separate experiments).

Mentions: Previous studies have demonstrated the physical association of DACH1 with the acetyl-transferase CBP [20], and in chromatin immunoprecipitation (ChIP) and WB, the association of DACH1 with SIRT1 and HDAC [6]. Collectively, these studies suggested a role for DACH1 in binding to decetylases. In order to determine whether DACH1 could serve as a substrate for acetylates and decetylases, we conducted a mass spectrometry analysis of the DACH1 protein. LC/MS analysis of DACH1 after trypsin digestion identified peptides with high mascot scores. The panel of peptides mapped acetylation sites to residues Lysine 628. In order to determine whether lysine residue K628 within the 628-633 motif was acetylated, Edman-degradation analysis was conducted revealing acetylation, primarily of Lysine 628 (Fig. 3A). The DACH1 Lys 628 resembles a core acetylation motif found in p53 and nuclear receptors (Fig. 3B inset).


Acetylation of the cell-fate factor dachshund determines p53 binding and signaling modules in breast cancer.

Chen K, Wu K, Gormley M, Ertel A, Wang J, Zhang W, Zhou J, Disante G, Li Z, Rui H, Quong AA, McMahon SB, Deng H, Lisanti MP, Wang C, Pestell RG - Oncotarget (2013)

NAD-dependent binding of DACH1 to p53(A) A peptide encoding the candidate DACH1 acetylation site (626 VQKRLKKEKKAKRKLQEAL 644) was incubated with p300 as an enzyme source in the presence of acetyl CoA. Acetylation products were purified with reverse-phase HPLC followed by MALDI-TOF mass spectrometry (A) and then sequenced by Edman degradation. In (A), the resulting DACH1 peptide (DACH1aa 626-644) mass spectrum is shown with mass/charge expressed in atomic mass units (amu). The major peaks labeled X corresponds to the expected mass of DACH1 whereas the large peak Y, is larger by 42 mass units, representing a single acetylated peptide. In (B) the bars represent the amount of phenylthiohydantoin-acetyl-lysine present in the corresponding position. The major acetylated product corresponds to residue K628. The inset shows an alignment of the DACH1 sequence and the acetylated residue motif seen in other acetylated transcription factors and the corresponding consensus sequence KX(x)KK. (C) IP-WB of HEK293T cells transfected with expression vectors encoding p53 or DACH1 and subsequently treated with Nicotinamide (NAM 20 mM) and/or Trichostatin A (TSA 2μm) for 8hrs. (D) Western blot of lysates corresponding to IP-Western in (C). (E) Relative binding of DACH1 to p53 shown as mean ± SEM for 3 separate experiments. IP-Western blot of MDA-MB-453 cells treated with TSA (2 μM) or NAM (20 mM) showing (G) relative binding of p53 to DACH1 (data are mean ± SEM for 3 separate experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757249&req=5

Figure 3: NAD-dependent binding of DACH1 to p53(A) A peptide encoding the candidate DACH1 acetylation site (626 VQKRLKKEKKAKRKLQEAL 644) was incubated with p300 as an enzyme source in the presence of acetyl CoA. Acetylation products were purified with reverse-phase HPLC followed by MALDI-TOF mass spectrometry (A) and then sequenced by Edman degradation. In (A), the resulting DACH1 peptide (DACH1aa 626-644) mass spectrum is shown with mass/charge expressed in atomic mass units (amu). The major peaks labeled X corresponds to the expected mass of DACH1 whereas the large peak Y, is larger by 42 mass units, representing a single acetylated peptide. In (B) the bars represent the amount of phenylthiohydantoin-acetyl-lysine present in the corresponding position. The major acetylated product corresponds to residue K628. The inset shows an alignment of the DACH1 sequence and the acetylated residue motif seen in other acetylated transcription factors and the corresponding consensus sequence KX(x)KK. (C) IP-WB of HEK293T cells transfected with expression vectors encoding p53 or DACH1 and subsequently treated with Nicotinamide (NAM 20 mM) and/or Trichostatin A (TSA 2μm) for 8hrs. (D) Western blot of lysates corresponding to IP-Western in (C). (E) Relative binding of DACH1 to p53 shown as mean ± SEM for 3 separate experiments. IP-Western blot of MDA-MB-453 cells treated with TSA (2 μM) or NAM (20 mM) showing (G) relative binding of p53 to DACH1 (data are mean ± SEM for 3 separate experiments).
Mentions: Previous studies have demonstrated the physical association of DACH1 with the acetyl-transferase CBP [20], and in chromatin immunoprecipitation (ChIP) and WB, the association of DACH1 with SIRT1 and HDAC [6]. Collectively, these studies suggested a role for DACH1 in binding to decetylases. In order to determine whether DACH1 could serve as a substrate for acetylates and decetylases, we conducted a mass spectrometry analysis of the DACH1 protein. LC/MS analysis of DACH1 after trypsin digestion identified peptides with high mascot scores. The panel of peptides mapped acetylation sites to residues Lysine 628. In order to determine whether lysine residue K628 within the 628-633 motif was acetylated, Edman-degradation analysis was conducted revealing acetylation, primarily of Lysine 628 (Fig. 3A). The DACH1 Lys 628 resembles a core acetylation motif found in p53 and nuclear receptors (Fig. 3B inset).

Bottom Line: DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding.DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628.DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.

Show MeSH
Related in: MedlinePlus