ASPP1 and ASPP2: common activators of p53 family members

Abstract

We recently showed that ASPP1 and ASPP2 stimulate the apoptotic function of p53. We show here that ASPP1 and ASPP2 also induce apoptosis independently of p53. By binding to p63 and p73 in vitro and in vivo, ASPP1 and ASPP2 stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 or p21(WAF-1/CIP1). The expression of ASPP1 and ASPP2 also enhances the apoptotic function of p63 and p73 by selectively inducing the expression of endogenous p53 target genes, such as PIG3 and PUMA, but not mdm2 or p21(WAF-1/CIP1). Removal of endogenous p63 or p73 with RNA interference demonstrated that (16) the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated mainly by p63 and p73. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members. All these results suggest that ASPP1 and ASPP2 could suppress tumor growth even in tumors expressing mutant p53.

FIG. 1.

ASPP1 and ASPP2 induce apoptosis independently of p53 in Saos-2 (A) and H1299 (B) cells. In response to treatment with cisplatin (3.5 μg/ml for 16 h), the number of apoptotic cells induced by the expression of ASPP1 and ASPP2 was increased further (C and D). The transfected cells were gated based on the expression of CD20 (16). The percentage of apoptotic cells was measured by the accumulation of cells with a sub-G₁ DNA content. The bar graphs show the percentage of apoptotic cells 36 h after transfection and were derived from at least two independent experiments. (E) Expression levels of endogenous p63γ, p73α, ASPP1, and ASPP2 in response to cisplatin treatment (+) in Saos-2 and H1299 cells. IVT, in vitro translation.

FIG. 2.

ASPP1 and ASPP2 interact with p63 and p73 in vitro and in vivo. (A) Sequence comparison of the DNA binding domains (DBDs) of p53, p63, and p73 reveals that the majority of the residues involved in ASPP binding are conserved. p53, p63, and p73 sequences were obtained from GenBank and aligned using CLUSTAL W. The ASPP contact residues of human p53 (Hp53) are indicated with numbered arrows as follows: 1, H178; 2, H179; 3, R181; 4, S183; 5, S241; 6, M243; 7, N247; 8, R248; and 9, R273. ASPP1 and ASPP2 interact with p53 and its family members in vitro (B and C). p53, p63γ, and p73α were in vitro translated and labeled with [³⁵S]methionine. V5-tagged ASPP1 and ASPP2 proteins were in vitro translated with cold methionine and immunoprecipitated with anti-V5 antibody. The immunoprecipitates were fractionated on SDS-10% polyacrylamide gels. The presence of radiolabeled p53, p63γ, or p73α complexed with ASPP1 or ASPP2 was detected by autoradiography, and the amount of ASPP1 and ASPP2 immunoprecipitated was detected using anti-V5 antibody by Western blotting.

FIG. 3.

ASPP1 and ASPP2 interact with p63γ and p73α in vivo. Samples (2 mg) of H1299 or Saos-2 cell lysates were immunoprecipitated with rabbit anti-ASPP1 ASPP1.88 and anti-ASPP2 BP77 antibodies, respectively. The immunoprecipitates were separated on SDS-polyacrylamide gels, and the presence of p63γ and p73α on the immunoblots was detected by mouse anti-p63 4A4 and anti-p73 ER-15 monoclonal antibodies, respectively. The presence of ASPP1 or ASPP2 was detected with mouse monoclonal antibody LX011 or DX5410, respectively. In vitro-translated (IVT) p63γ and p73α lysates were used as positive controls for the detection of antibody. IP, immunoprecipitation; Ab, antibody.

FIG.4.

ASPP1 and ASPP2 can specifically stimulate the transactivation function of p53 family members on the promoters of proapoptotic genes, such as Bax, PIG3, and PUMA but not mdm2 and p21^WAF-1/CIP1. (A and B) The bar graphs show the effects of ASPP1 and ASPP2 on the transactivation (TA) function of p53, p63γ, or p73α on the PIG3 promoter. luc, luciferase. (C) The fold increase in p53, p63γ, or p73α transactivation activity by either ASPP1 or ASPP2 on five luciferase (luc) reporters of p53 target genes, Bax, PIG3, PUMA, mdm2, and p21^WAF-1/CIP1, is shown. The fold increase was calculated as follows: (activity of p53 family members plus ASPP)/(activity of p53 family members alone). The expression levels of various transfected proteins, ASPP1 (V5 tagged), ASPP2, p53, p63, and p73, were detected using 40 μg of the respective lysates using antibodies V5, DX.5410, DO1, 4A4, and ER-15, respectively. The results were derived from at least three independent experiments.

FIG. 5.

Increasing amounts of ASPP1 and ASPP2 induce the expression of two endogenous p53 target genes, PIG3 and PUMA, at both RNA (A) and protein (B) levels in Saos-2 and 1299 cells independently of p53. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 6.

Fluorescence-activated cell sorting analysis showing that expression of ASPP1 and ASPP2 stimulate the apoptotic function of p53, p63γ, and p73α. The transfected cells were gated and analyzed as described in the legend to Fig. 1. The percentage of apoptotic cells 36 h after transfection is shown. The values were derived from two independent experiments.

FIG.7.

Saos-2 and H1299 cells were transfected with plasmid expressing a cell surface marker CD20 together with p53, p63γ, or p73α in the presence or absence of ASPP1, ASPP2, p63 RNAi, p73 RNAi, or p53 RNAi as indicated (A, C, and D). The ability of p63 and p73 RNAi to inhibit the expression of p63 and p73 is shown in panel B. The vector used to express RNAi of p63, p73, or p53 is pSuper, and cisplatin treatment was performed as described in the legend to Fig. 1. The bar graph represents the percentage of apoptotic cells 36 h after transfection and was derived from two independent experiments. cmv, cytomegalovirus; IVT, in vitro translation.