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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 acetyl-transferase domain of p300 is required to induce ΔNp63α protein stabilization. (A) WB analysis of whole HaCaT cell extracts transiently transfected with increasing amounts (20 ng and 40 ng) of shp300 or shLuc expression vectors (B) WB analysis of HaCaT whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 encoding plasmids (p300 (WT) or p300-LY-RR, mutated in the HAT domain (10 and 20 ng). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 expression vectors (10 and 20 ng).
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DDV151F2: The acetyl-transferase domain of p300 is required to induce ΔNp63α protein stabilization. (A) WB analysis of whole HaCaT cell extracts transiently transfected with increasing amounts (20 ng and 40 ng) of shp300 or shLuc expression vectors (B) WB analysis of HaCaT whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 encoding plasmids (p300 (WT) or p300-LY-RR, mutated in the HAT domain (10 and 20 ng). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 expression vectors (10 and 20 ng).

Mentions: Acetylation of p53 and p73 proteins is required for their stabilization and transcriptional activation in response to DNA damage (31–33,35–38) and the p300 acetyl-transferase is known to be involved in this process (29,35,36,38). To determine whether p300 could acetylate ΔNp63α, we silenced endogenous p300 in HaCaT cells by transfecting a p300-specific shRNA plasmid. Depletion of p300 was clearly detected and, concomitant with p300 reduction, a significant decrease of ΔNp63α was also observed (Fig. 2A). Conversely, when p300 protein levels were increased by transient overexpression in HaCaT or U2OS cells, ΔNp63α protein was stabilized in a dose-dependent manner (Fig. 2B and C). In contrast, a construct expressing a mutated variant of p300, with a mutation affecting the Histone Acetyl-Transferase domain (LY-RR) (36), failed to stabilize endogenous ΔNp63α in HaCaT cells (Fig. 2B). These data clearly indicate that p300 and its acetyl-transferase activity are required for ΔNp63α protein levels regulation.Figure 2.


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 acetyl-transferase domain of p300 is required to induce ΔNp63α protein stabilization. (A) WB analysis of whole HaCaT cell extracts transiently transfected with increasing amounts (20 ng and 40 ng) of shp300 or shLuc expression vectors (B) WB analysis of HaCaT whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 encoding plasmids (p300 (WT) or p300-LY-RR, mutated in the HAT domain (10 and 20 ng). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 expression vectors (10 and 20 ng).
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DDV151F2: The acetyl-transferase domain of p300 is required to induce ΔNp63α protein stabilization. (A) WB analysis of whole HaCaT cell extracts transiently transfected with increasing amounts (20 ng and 40 ng) of shp300 or shLuc expression vectors (B) WB analysis of HaCaT whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 encoding plasmids (p300 (WT) or p300-LY-RR, mutated in the HAT domain (10 and 20 ng). (C) WB analysis of U2OS whole cell extracts transiently co-transfected with equal amount of ΔNp63α expression vectors (30 ng) and increasing amounts of p300 expression vectors (10 and 20 ng).
Mentions: Acetylation of p53 and p73 proteins is required for their stabilization and transcriptional activation in response to DNA damage (31–33,35–38) and the p300 acetyl-transferase is known to be involved in this process (29,35,36,38). To determine whether p300 could acetylate ΔNp63α, we silenced endogenous p300 in HaCaT cells by transfecting a p300-specific shRNA plasmid. Depletion of p300 was clearly detected and, concomitant with p300 reduction, a significant decrease of ΔNp63α was also observed (Fig. 2A). Conversely, when p300 protein levels were increased by transient overexpression in HaCaT or U2OS cells, ΔNp63α protein was stabilized in a dose-dependent manner (Fig. 2B and C). In contrast, a construct expressing a mutated variant of p300, with a mutation affecting the Histone Acetyl-Transferase domain (LY-RR) (36), failed to stabilize endogenous ΔNp63α in HaCaT cells (Fig. 2B). These data clearly indicate that p300 and its acetyl-transferase activity are required for ΔNp63α protein levels regulation.Figure 2.

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