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FGF8, c-Abl and p300 participate in a pathway that controls stability and function of the ΔNp63α protein.

Restelli M, Molinari E, Marinari B, Conte D, Gnesutta N, Costanzo A, Merlo GR, Guerrini L - Hum. Mol. Genet. (2015)

Bottom Line: Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions.Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway.Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Università degli Studi di Milano, 20133 Milano, Italy.

No MeSH data available.


Related in: MedlinePlus

p300 interacts with ΔNp63α in human cells and catalizes in vitro acetylation of lysine K193. (A) U2OS whole cell extracts transiently co-transfected with ΔNp63α and p300 were analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. U2OS cells, not transfected with p63 encoding plasmid were used as negative control. (B) In vitro acetylation assay performed according to the HAT assay kit protocol (Active Motif) with an H4 peptide and p53 peptides as positive controls, H4 plus anacardic acid 15 μM (an inhibitor of acetyl-transferase activity used as a negative control) and p63 peptides (peptide sequences are indicated). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193E, ΔNp63α-K193R expression vectors (30 ng) and increasing amounts of p300 encoding plasmid (10 and 20 ng). (D) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193R and p300 expression vectors (30 ng and 5 ng respectively). 24 h after transfection protein half-life was measured by treating cells with 10 μg/ml of CHX.
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DDV151F3: p300 interacts with ΔNp63α in human cells and catalizes in vitro acetylation of lysine K193. (A) U2OS whole cell extracts transiently co-transfected with ΔNp63α and p300 were analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. U2OS cells, not transfected with p63 encoding plasmid were used as negative control. (B) In vitro acetylation assay performed according to the HAT assay kit protocol (Active Motif) with an H4 peptide and p53 peptides as positive controls, H4 plus anacardic acid 15 μM (an inhibitor of acetyl-transferase activity used as a negative control) and p63 peptides (peptide sequences are indicated). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193E, ΔNp63α-K193R expression vectors (30 ng) and increasing amounts of p300 encoding plasmid (10 and 20 ng). (D) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193R and p300 expression vectors (30 ng and 5 ng respectively). 24 h after transfection protein half-life was measured by treating cells with 10 μg/ml of CHX.

Mentions: Accordingly, when we overexpressed ΔNp63α with p300 in U2OS cells and treated the cells with the protein synthesis inhibitor Cycloheximide (CHX), we observed an increase of ΔNp63α protein half-life (Fig. 3D). We also determined the effect of p300 silencing on ΔNp63α protein half-life in HaCaT cells, by transfecting p300-specific shRNA plasmid and treated the cells with CHX. As shown in Supplementary Material, Figure S2, the levels of ΔNp63α protein decreased upon p300 silencing with only a modest decrease of ΔNp63α half-life upon CHX addition.Figure 3.


FGF8, c-Abl and p300 participate in a pathway that controls stability and function of the ΔNp63α protein.

Restelli M, Molinari E, Marinari B, Conte D, Gnesutta N, Costanzo A, Merlo GR, Guerrini L - Hum. Mol. Genet. (2015)

p300 interacts with ΔNp63α in human cells and catalizes in vitro acetylation of lysine K193. (A) U2OS whole cell extracts transiently co-transfected with ΔNp63α and p300 were analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. U2OS cells, not transfected with p63 encoding plasmid were used as negative control. (B) In vitro acetylation assay performed according to the HAT assay kit protocol (Active Motif) with an H4 peptide and p53 peptides as positive controls, H4 plus anacardic acid 15 μM (an inhibitor of acetyl-transferase activity used as a negative control) and p63 peptides (peptide sequences are indicated). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193E, ΔNp63α-K193R expression vectors (30 ng) and increasing amounts of p300 encoding plasmid (10 and 20 ng). (D) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193R and p300 expression vectors (30 ng and 5 ng respectively). 24 h after transfection protein half-life was measured by treating cells with 10 μg/ml of CHX.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492388&req=5

DDV151F3: p300 interacts with ΔNp63α in human cells and catalizes in vitro acetylation of lysine K193. (A) U2OS whole cell extracts transiently co-transfected with ΔNp63α and p300 were analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. U2OS cells, not transfected with p63 encoding plasmid were used as negative control. (B) In vitro acetylation assay performed according to the HAT assay kit protocol (Active Motif) with an H4 peptide and p53 peptides as positive controls, H4 plus anacardic acid 15 μM (an inhibitor of acetyl-transferase activity used as a negative control) and p63 peptides (peptide sequences are indicated). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193E, ΔNp63α-K193R expression vectors (30 ng) and increasing amounts of p300 encoding plasmid (10 and 20 ng). (D) WB analysis of U2OS whole cell extracts transiently co-transfected with ΔNp63α, ΔNp63α-K193R and p300 expression vectors (30 ng and 5 ng respectively). 24 h after transfection protein half-life was measured by treating cells with 10 μg/ml of CHX.
Mentions: Accordingly, when we overexpressed ΔNp63α with p300 in U2OS cells and treated the cells with the protein synthesis inhibitor Cycloheximide (CHX), we observed an increase of ΔNp63α protein half-life (Fig. 3D). We also determined the effect of p300 silencing on ΔNp63α protein half-life in HaCaT cells, by transfecting p300-specific shRNA plasmid and treated the cells with CHX. As shown in Supplementary Material, Figure S2, the levels of ΔNp63α protein decreased upon p300 silencing with only a modest decrease of ΔNp63α half-life upon CHX addition.Figure 3.

Bottom Line: Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions.Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway.Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Università degli Studi di Milano, 20133 Milano, Italy.

No MeSH data available.


Related in: MedlinePlus