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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

The K193E mutation alters ΔNp63α transcriptional activity in a promoter-specific manner. (A) Luciferase assay performed on U2OS cells transiently co-transfected with the −1200 bp DLX5 promoter (200 ng) in the presence of ΔNp63α or ΔNp63α-K193E (50 ng) with increasing amounts of p300 (10 and 20 ng) expression vectors. Each histogram bar represents the mean of three indipendent transfection duplicates. Standard deviation are indicated. (B) Luciferase assay performed in U2OS cells transiently co-transfected with the DLX6, and EGFR reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. (C) Luciferase assay performed on U2OS cells transiently co-transfected with the p57kip2, and ADA reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. For (A–C) cells were lysed 24 h after transfection and luciferase activity was determined. The basal activity of the reporter plasmid was set to 1. Data are presented as fold activation/repression relative to the sample without effector. Each histogram bar represents the mean of three independent transfection duplicates. Standard deviations are indicated.
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DDV151F5: The K193E mutation alters ΔNp63α transcriptional activity in a promoter-specific manner. (A) Luciferase assay performed on U2OS cells transiently co-transfected with the −1200 bp DLX5 promoter (200 ng) in the presence of ΔNp63α or ΔNp63α-K193E (50 ng) with increasing amounts of p300 (10 and 20 ng) expression vectors. Each histogram bar represents the mean of three indipendent transfection duplicates. Standard deviation are indicated. (B) Luciferase assay performed in U2OS cells transiently co-transfected with the DLX6, and EGFR reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. (C) Luciferase assay performed on U2OS cells transiently co-transfected with the p57kip2, and ADA reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. For (A–C) cells were lysed 24 h after transfection and luciferase activity was determined. The basal activity of the reporter plasmid was set to 1. Data are presented as fold activation/repression relative to the sample without effector. Each histogram bar represents the mean of three independent transfection duplicates. Standard deviations are indicated.

Mentions: In order to verify whether p300 could act as a ΔNp63α co-activator we performed luciferase-reporter assays with the DLX5 promoter. Interestingly, we observed that p300 co-transfection greatly enhanced the transcriptional activity of ΔNp63α, while the transcriptional activity of the ΔNp63α-K193E mutant could not be enhanced by p300 overexpression (Fig. 5A).Figure 5.


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)

The K193E mutation alters ΔNp63α transcriptional activity in a promoter-specific manner. (A) Luciferase assay performed on U2OS cells transiently co-transfected with the −1200 bp DLX5 promoter (200 ng) in the presence of ΔNp63α or ΔNp63α-K193E (50 ng) with increasing amounts of p300 (10 and 20 ng) expression vectors. Each histogram bar represents the mean of three indipendent transfection duplicates. Standard deviation are indicated. (B) Luciferase assay performed in U2OS cells transiently co-transfected with the DLX6, and EGFR reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. (C) Luciferase assay performed on U2OS cells transiently co-transfected with the p57kip2, and ADA reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. For (A–C) cells were lysed 24 h after transfection and luciferase activity was determined. The basal activity of the reporter plasmid was set to 1. Data are presented as fold activation/repression relative to the sample without effector. Each histogram bar represents the mean of three independent transfection duplicates. Standard deviations are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492388&req=5

DDV151F5: The K193E mutation alters ΔNp63α transcriptional activity in a promoter-specific manner. (A) Luciferase assay performed on U2OS cells transiently co-transfected with the −1200 bp DLX5 promoter (200 ng) in the presence of ΔNp63α or ΔNp63α-K193E (50 ng) with increasing amounts of p300 (10 and 20 ng) expression vectors. Each histogram bar represents the mean of three indipendent transfection duplicates. Standard deviation are indicated. (B) Luciferase assay performed in U2OS cells transiently co-transfected with the DLX6, and EGFR reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. (C) Luciferase assay performed on U2OS cells transiently co-transfected with the p57kip2, and ADA reporter promoters (200 ng) in the presence of increasing amounts of ΔNp63α or ΔNp63α-K193E (50 and 100 ng) plasmids. For (A–C) cells were lysed 24 h after transfection and luciferase activity was determined. The basal activity of the reporter plasmid was set to 1. Data are presented as fold activation/repression relative to the sample without effector. Each histogram bar represents the mean of three independent transfection duplicates. Standard deviations are indicated.
Mentions: In order to verify whether p300 could act as a ΔNp63α co-activator we performed luciferase-reporter assays with the DLX5 promoter. Interestingly, we observed that p300 co-transfection greatly enhanced the transcriptional activity of ΔNp63α, while the transcriptional activity of the ΔNp63α-K193E mutant could not be enhanced by p300 overexpression (Fig. 5A).Figure 5.

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