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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

FGF8 positively regulates ΔNp63α protein stability inducing its interaction with c-Abl and promoting ΔNp63α acetylation. (A) WB analysis of HaCaT whole cell extracts treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (B) WB analysis of HaCaT whole cell extracts stably transfected with an shRNA against c-Abl or shLuc plasmids, treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (C) WB analysis of HaCaT cells treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) or pre-treated for 30 min with Imatinib (10 μM) followed by FGF8 treatment for 3 h. (D) U2OS whole cell extracts transiently co-transfected with either ΔNp63α or ΔNp63α-3Y (10 μg) and p300 (5 μg), and then analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. (E) HaCaT whole cell extracts treated with FGF8 (0.5 ng/ml) or DMSO for 3 h were analyzed by immunoprecipitation with anti-p63 antibodies followed by WB analysis with the indicated antibodies. U2OS cells, not expressing p63 were used as negative control. (F) WB analysis of U2OS whole cell extracts transiently transfected with ΔNp63α or ΔNp63α-K193E encoding plasmids (30 ng). 24 h after transfection U2OS cells were treated with increasing amounts of FGF8 for 2 h (0.5 ng/ml and 1 ng/ml). (G) WB analysis of total proteins extracts from forelimbs isolated from wild-type mouse embryos at E11.5, cultured whole-mount for 48 h in the absence or presence of recombinant FGF8 (0.5 μg/ml and 1 μg/ml).
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DDV151F4: FGF8 positively regulates ΔNp63α protein stability inducing its interaction with c-Abl and promoting ΔNp63α acetylation. (A) WB analysis of HaCaT whole cell extracts treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (B) WB analysis of HaCaT whole cell extracts stably transfected with an shRNA against c-Abl or shLuc plasmids, treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (C) WB analysis of HaCaT cells treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) or pre-treated for 30 min with Imatinib (10 μM) followed by FGF8 treatment for 3 h. (D) U2OS whole cell extracts transiently co-transfected with either ΔNp63α or ΔNp63α-3Y (10 μg) and p300 (5 μg), and then analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. (E) HaCaT whole cell extracts treated with FGF8 (0.5 ng/ml) or DMSO for 3 h were analyzed by immunoprecipitation with anti-p63 antibodies followed by WB analysis with the indicated antibodies. U2OS cells, not expressing p63 were used as negative control. (F) WB analysis of U2OS whole cell extracts transiently transfected with ΔNp63α or ΔNp63α-K193E encoding plasmids (30 ng). 24 h after transfection U2OS cells were treated with increasing amounts of FGF8 for 2 h (0.5 ng/ml and 1 ng/ml). (G) WB analysis of total proteins extracts from forelimbs isolated from wild-type mouse embryos at E11.5, cultured whole-mount for 48 h in the absence or presence of recombinant FGF8 (0.5 μg/ml and 1 μg/ml).

Mentions: First, we treated HaCaT cells with increasing amounts of soluble FGF8 that resulted in efficient ΔNp63α protein stabilization as expected (Fig. 4A).Figure 4.


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)

FGF8 positively regulates ΔNp63α protein stability inducing its interaction with c-Abl and promoting ΔNp63α acetylation. (A) WB analysis of HaCaT whole cell extracts treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (B) WB analysis of HaCaT whole cell extracts stably transfected with an shRNA against c-Abl or shLuc plasmids, treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (C) WB analysis of HaCaT cells treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) or pre-treated for 30 min with Imatinib (10 μM) followed by FGF8 treatment for 3 h. (D) U2OS whole cell extracts transiently co-transfected with either ΔNp63α or ΔNp63α-3Y (10 μg) and p300 (5 μg), and then analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. (E) HaCaT whole cell extracts treated with FGF8 (0.5 ng/ml) or DMSO for 3 h were analyzed by immunoprecipitation with anti-p63 antibodies followed by WB analysis with the indicated antibodies. U2OS cells, not expressing p63 were used as negative control. (F) WB analysis of U2OS whole cell extracts transiently transfected with ΔNp63α or ΔNp63α-K193E encoding plasmids (30 ng). 24 h after transfection U2OS cells were treated with increasing amounts of FGF8 for 2 h (0.5 ng/ml and 1 ng/ml). (G) WB analysis of total proteins extracts from forelimbs isolated from wild-type mouse embryos at E11.5, cultured whole-mount for 48 h in the absence or presence of recombinant FGF8 (0.5 μg/ml and 1 μg/ml).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492388&req=5

DDV151F4: FGF8 positively regulates ΔNp63α protein stability inducing its interaction with c-Abl and promoting ΔNp63α acetylation. (A) WB analysis of HaCaT whole cell extracts treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (B) WB analysis of HaCaT whole cell extracts stably transfected with an shRNA against c-Abl or shLuc plasmids, treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) for 3 h. (C) WB analysis of HaCaT cells treated with increasing amounts of FGF8 (0.5 ng/ml and 1 ng/ml) or pre-treated for 30 min with Imatinib (10 μM) followed by FGF8 treatment for 3 h. (D) U2OS whole cell extracts transiently co-transfected with either ΔNp63α or ΔNp63α-3Y (10 μg) and p300 (5 μg), and then analyzed by immunoprecipitation with an anti-p300 antibody followed by WB analysis with an anti-p63 antibody. (E) HaCaT whole cell extracts treated with FGF8 (0.5 ng/ml) or DMSO for 3 h were analyzed by immunoprecipitation with anti-p63 antibodies followed by WB analysis with the indicated antibodies. U2OS cells, not expressing p63 were used as negative control. (F) WB analysis of U2OS whole cell extracts transiently transfected with ΔNp63α or ΔNp63α-K193E encoding plasmids (30 ng). 24 h after transfection U2OS cells were treated with increasing amounts of FGF8 for 2 h (0.5 ng/ml and 1 ng/ml). (G) WB analysis of total proteins extracts from forelimbs isolated from wild-type mouse embryos at E11.5, cultured whole-mount for 48 h in the absence or presence of recombinant FGF8 (0.5 μg/ml and 1 μg/ml).
Mentions: First, we treated HaCaT cells with increasing amounts of soluble FGF8 that resulted in efficient ΔNp63α protein stabilization as expected (Fig. 4A).Figure 4.

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