Limits...
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 ΔNp63α protein is acetylated in human keratynocytes. (A) Western Blot (WB) analysis of whole HaCaT cell extracts treated with increasing amounts of TSA (5 ng/ml and 10 ng/ml) for 5 h or VPA (0.5 mm and 1 mm) for 3 h. (B) Whole cell extracts from HaCaT cells treated with 5 ng/ml of Trichostatin (TSA) for 5 h were analyzed by immunoprecipitation of endogenous ΔNp63α with an anti-p63 antibody followed by WB analysis with an anti-acetylated lysines. U2OS cells, not expressing p63, were used as negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492388&req=5

DDV151F1: The ΔNp63α protein is acetylated in human keratynocytes. (A) Western Blot (WB) analysis of whole HaCaT cell extracts treated with increasing amounts of TSA (5 ng/ml and 10 ng/ml) for 5 h or VPA (0.5 mm and 1 mm) for 3 h. (B) Whole cell extracts from HaCaT cells treated with 5 ng/ml of Trichostatin (TSA) for 5 h were analyzed by immunoprecipitation of endogenous ΔNp63α with an anti-p63 antibody followed by WB analysis with an anti-acetylated lysines. U2OS cells, not expressing p63, were used as negative control.

Mentions: In order to assess whether p63 could be acetylated in human cells, we treated the human keratinocytes HaCaT cell line, expressing endogenous ΔNp63α, with Valproic-Acid (VPA), which selectively inhibits class I deacetylases, or with Trichostatin-A (TSA) which inhibits class I and II deacetylases. VPA and TSA treatments resulted in an increase in ΔNp63α abundance (Fig. 1A). Similar effects of ΔNp63α accumulation were also obtained when ΔNp63α was ectopically overexpressed in U2OS cells, a human osteosarcoma cell line devoid of endogenous p63 expression (Supplementary Material, Fig. S1). Then, we performed immunoprecipitation of endogenous ΔNp63α from total protein extracts of HaCaT cells treated with TSA. The level of ΔNp63α acetylation was detected using an antibody against acetylated lysines: we observed that ΔNp63α is found acetylated at a basal level, as previously reported (39), and that its acetylation increased upon TSA treatment (Fig. 1B). These results show that the ΔNp63α protein is acetylated in human cells and that the acetylation levels of ΔNp63α correlate with its accumulation in human cells following deacetyl-transferases inhibition.Figure 1.


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 ΔNp63α protein is acetylated in human keratynocytes. (A) Western Blot (WB) analysis of whole HaCaT cell extracts treated with increasing amounts of TSA (5 ng/ml and 10 ng/ml) for 5 h or VPA (0.5 mm and 1 mm) for 3 h. (B) Whole cell extracts from HaCaT cells treated with 5 ng/ml of Trichostatin (TSA) for 5 h were analyzed by immunoprecipitation of endogenous ΔNp63α with an anti-p63 antibody followed by WB analysis with an anti-acetylated lysines. U2OS cells, not expressing p63, were used as negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDV151F1: The ΔNp63α protein is acetylated in human keratynocytes. (A) Western Blot (WB) analysis of whole HaCaT cell extracts treated with increasing amounts of TSA (5 ng/ml and 10 ng/ml) for 5 h or VPA (0.5 mm and 1 mm) for 3 h. (B) Whole cell extracts from HaCaT cells treated with 5 ng/ml of Trichostatin (TSA) for 5 h were analyzed by immunoprecipitation of endogenous ΔNp63α with an anti-p63 antibody followed by WB analysis with an anti-acetylated lysines. U2OS cells, not expressing p63, were used as negative control.
Mentions: In order to assess whether p63 could be acetylated in human cells, we treated the human keratinocytes HaCaT cell line, expressing endogenous ΔNp63α, with Valproic-Acid (VPA), which selectively inhibits class I deacetylases, or with Trichostatin-A (TSA) which inhibits class I and II deacetylases. VPA and TSA treatments resulted in an increase in ΔNp63α abundance (Fig. 1A). Similar effects of ΔNp63α accumulation were also obtained when ΔNp63α was ectopically overexpressed in U2OS cells, a human osteosarcoma cell line devoid of endogenous p63 expression (Supplementary Material, Fig. S1). Then, we performed immunoprecipitation of endogenous ΔNp63α from total protein extracts of HaCaT cells treated with TSA. The level of ΔNp63α acetylation was detected using an antibody against acetylated lysines: we observed that ΔNp63α is found acetylated at a basal level, as previously reported (39), and that its acetylation increased upon TSA treatment (Fig. 1B). These results show that the ΔNp63α protein is acetylated in human cells and that the acetylation levels of ΔNp63α correlate with its accumulation in human cells following deacetyl-transferases inhibition.Figure 1.

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