D for a brief time only. Daxx co-precipitated from cells not treated with MG132 is for that reason only weakly visible. (e) MCF7 cells were transfected with manage siRNA or Pdcd4-specific siRNA. The cells were D-Lysine monohydrochloride supplier analyzed soon after two days by western blotting for the expression of Daxx, Pdcd4 and b-actin. (f ) HeLa wildtype cells or even a clone of HeLa cells stably expressing Pdcd4-specific quick hairpin RNA (HeLa-K11) were analyzed as described in (e).knockdown experiments employing transient transfection of Pdcd4-specific small interfering RNA (siRNA) (Figure 3e) or stable expression of Pdcd4-specific short hairpin RNA (Figure 3f). In each cases, there was a slight boost on the quantity of Daxx, supporting the notion that Pdcd4 decreases the half-life of at the least a fraction of Daxx. Pdcd4 disrupts the interaction of Daxx with protein kinase Hipk2 and inhibits Ser-46 phosphorylation of p53 Daxx has been shown to act as a scaffold that stimulates the phosphorylation of p53 by the protein kinase Hipk2.49 Hipk2 interacts with the amino-terminal half of Daxx and phosphorylates the tumor suppressor protein p53 at Ser-46 in response to DNA damage.58,59 We as a result wondered no matter whether the interaction of Pdcd4 with Daxx would influence the phosphorylation of p53 at Ser-46. To find out if Pdcd4 affects the binding of Hipk2 to Daxx, we performed a co-precipitation experiment, using cells transfected with expression vectors for HA-Hipk2 and green fluorescent protein (GFP)-Daxx with each other with escalating amounts of a FlagPdcd4 expression vector. We then analyzed the quantity of Hipk2 that was co-precipitated with Daxx. Figure 4a shows that Hipk2013 Macmillan Publishers Limitedwas efficiently co-precipitated by means of Daxx (lane three), whereas no coprecipitation was observed inside the absence of Daxx (lane 2), indicating that the co-precipitation was particular and that a significant amount of Hipk2 was related with Daxx. The coprecipitation of Hipk2 was strongly diminished by increasing amounts of Pdcd4 (lanes 4 and 5), demonstrating that Pdcd4 interferes with all the formation of the Daxx ipk2 complex. The data shown in Figure 4a are consistent using the idea that Pdcd4 disrupts the Daxx ipk2 interaction and, as a consequence, suppresses the phosphorylation of p53 in the Ser-46. To investigate whether the manipulation with the Pdcd4 expression level impacts the phosphorylation of p53 also in cells not overexpressing Pdcd4, Daxx or Hipk2, we performed a Pdcd4 knockdown experiment and analyzed the degree of the phosphorylation of p53. If Pdcd4 suppresses the phosphorylation, we expected the Ser-46 phosphorylation of p53 to boost right after knock down of Pdcd4. To address this issue, we employed an antiserum whose specificity for phosphorylated Ser-46 of p53 was confirmed by its capability to detect p53 in etoposide-treated but not in -untreated cells (Supplementary Figure 2). Figure 4b shows that Pdcd4 knockdown indeed increased the phosphorylation of p53 at Ser-46. This experiment, therefore, supports a model inOncogenesis (2013), 1 HMGelHa-Flag-PdcdK-+MGwcd4.siR N APdcd4 axx interaction N Kumar et alFigure four. Pdcd4 inhibits Ser-46 phosphorylation of p53. (a) QT6 cells have been transfected together with the indicated combinations of expression vectors for HA-Hipk2, GFP-Daxx and Flag-Pdcd4, as indicated beneath the lanes. Cells have been lysed right after 24 h and TCEs have been either analyzed directly by SDS AGE and western blotting with all the indicated antibodies or were initial immunoprecipitated with antibodies against GFP (second.
