Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis

Abstract

The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in both the Ad12 55-kDa protein and p53 that underpin their colocalization in the cytoplasmic body and show that the N-terminal transactivation domain (TAD) of p53 is essential for retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27 or even point mutation L22Q/W23S abolished the localization of p53 to the cytoplasmic body, whereas other parts of TAD and the C-terminal domain of p53 are dispensable. This cytoplasmic body is distinct from aggresome associated with overexpression of some proteins, since it neither altered vimentin intermediate filaments nor associated with centrosome or ubiquitin. Formation of this structure is sensitive to mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A near the C terminus of the Ad12 55-kDa protein eliminated the formation of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa protein were shown to be critical for its ability to inhibit p53. Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis. Finally, we show that E1B can still sequester p53 that contains the mitochondrial import sequence, thereby potentially preventing the localization of p53 to mitochondria. Thus, cytoplasmic sequestration of p53 by the E1B 55-kDa protein plays an important role in restricting p53 activities.

FIG. 1.

Colocalization of Ad12 E1B 55-kDa protein and p53 in the cytoplasmic body. (A) p53 aa 11 to 27 are required for localization of p53 to the cytoplasmic body. Vectors for fusion of GFP and the Ad12 55-kDa protein and various p53 constructs as indicated were transfected into p53-null Saos2 cells. p53 was detected using goat polyclonal antibody raised against full-length p53 (Santa Cruz Biotechnology) and rabbit anti-goat immunoglobulin G-rhodamine conjugate. The p53 mutants are as follows: K8R, lysine residues at 319 to 321, 372 to 373, 381 to 382, and 386 were converted to arginine; 1-375, aa 1 to 375; 1-355, aa 1 to 355; 1-315, aa 1 to 315; 83-393, aa 83 to 393; 83-355, aa 83 to 355; D11-27, aa 11 to 27 are deleted; D61-75, aa 61 to 75 are deleted; 22/23, L22Q/W23S. The cells were counterstained with DAPI. (B) p73 does not localize to the E1B cytoplasmic body. Flag-p73α or Flag-p73β vector was cotransfected with plasmid for the GFP fusion with Ad12 E1B, and the transfected cells were stained with rabbit anti-Flag polyclonal antibody (Sigma) and rhodamine-conjugated secondary antibody. (C) Summary of results shown in panels A and B. p53 and its mutants, as well as p73α and p73β are schematically drawn, and whether they localize to the E1B cytoplasmic body is indicated with + and −, denoting the presence or absence of colocalization, respectively. The individual domains in both p53 and p73 are illustrated: DBD, DNA-binding domain; OD, oligomerization domain; RD, regulatory domain; PxxP, proline-rich motif; SP, spacer sequence with unknown function; SAM, sterile-α domain. The numbers denote the amino acid position. The black box in the C terminus of p73β indicates distinct amino acids.

FIG. 2.

The E1B cytoplasmic body is distinct from aggresomes. (A) Intracellular distributions of wild-type Ad12 E1B 55-kDa protein in relation to various cellular proteins. G401 cells that constitutively express the Ad12 E1B 55-kDa protein were stained with rabbit polyclonal antibody raised against the N-terminal domain of Ad12 E1B (14). Antibodies to other proteins were as described in Materials and Methods. The nuclei were visualized by DAPI staining. (B) Staining pattern of GFP-E1B, vimentin, and the Golgi marker giantin. Saos2 cells were transfected with GFP-E1B, and the transfected cells were stained with antibodies to vimentin and giantin and an appropriate secondary antibody, respectively. The cells were counterstained with DAPI.

FIG. 3.

An S476/7A mutation in the Ad12 55-kDa protein abolishes the formation of the E1B cytoplasmic body. (A) Subcellular distributions of p53 and GFP fusions with the Ad12 E1B 55-kDa protein and its mutants in Saos2 cells. Various mutants of the Ad12 55-kDa protein were fused with GFP, and their expression vectors were transfected alone or together with p53 plasmid into Saos2 cells. p53 was revealed with antibody to p53 (DO-1) and rabbit anti-mouse immunoglobulin G-rhodamine conjugate. The E1B mutants are as follows: 136-482, aa 136 to the C terminus; 341-482, aa 341 to the C terminus; 432-482, aa 432 to the C terminus; 101/4A, substitutions with alanine at L101 and L104; 101/4/6/8A, substitutions with alanine at L101, L104, and L108 as well as V106; 476/7A, S476 and S477 mutated to alanine; 476/7/481A, S476, S477, and D481 mutated to alanine; 481A, D481 mutated to alanine. The nuclei were revealed by DAPIstaining. (B) Summary of the results shown in panel A. The GFP fusion of wt Ad12 E1B 55-kDa protein and its various mutants are schematically depicted. Their ability to form a cytoplasmic body is indicated by + or − signs.

FIG. 4.

S476/7A mutation of the Ad12 E1B 55-kDa protein relieves its inhibition of p53-mediated transactivation. (A) Sequence alignment of the C-terminal sequences of Ad2 and Ad12 E1B 55-kDa proteins. The numbers above or below the sequence indicate the position of the amino acid residues. Identical residues are indicated by a white box, and similar residues are indicated by a gray box. (B) Point mutations near the C terminus of the Ad12 E1B 55-kDa oncoprotein abolish its inhibition of p53 transactivation. Wild-type E1B (lanes 3 and 4) and point mutants (S476A/S477A [lanes 5 and 6], S476A/S477A/D481A [lanes 7 and 8], and D481A [lanes 9 and 10]) were transfected into Saos2 cells alone with reporters (PG13-Luc and pRL-SV40) or together with p53 and reporters, as indicated, and dual luciferase assays were performed 48 h after transfection. The expression of p53 and E1B in transfected cells is shown on the right. The lane numbers in the left and right panels are the same. The middle panel shows relative reporter activities from cells transfected with wt Ad12 E1B and its mutants in the absence of p53 expression in an enlarged format to facilitate a comparison of the activities in each transfection. Error bars represent one standard deviation of two independent transfections.

FIG. 5.

Effect of the Ad12 55-kDa protein and its mutant on p53-imediated suppression of colony formation. (A) Representative images of colony formation assays. Saos2 cells were transfected with the indicated plasmids and plated in medium containing puromycin. E1B AA is the S476/7A mutant of the Ad12 E1B 55-kDa protein. (B) Quantification of the data from the colony formation assays. Error bars represent one standard deviation of three independent assays.

FIG. 6.

Formation of the E1B cytoplasmic body is required to inhibit p53-dependent apoptosis. (A) Representative micrographs of Saos2 cells transfected with the indicated plasmids. Saos2 cells were transfected with expression vectors for p53 and wt Ad12 E1B 55-kDa protein fusion with GFP or with E1B mutants as specified. Cells were treated with 5-fluorouracil 24 h after transfection and were grown for an additional 24 h before being fixed. The cells were examined under a fluorescence microscope. Apoptotic cells with shrunken morphology and a fragmented nucleus are indicated by white arrows. The mutants of the Ad12 E1B 55-kDa protein are as described in the legend to Fig. 3. The nuclei were visualized by DAPI staining. (B) Quantification of apoptotic cells. Error bars represent one standard deviation of two independent assays. (C and D) Effects of the wt Ad12 E1B 55-kDa protein and its mutants on p53-mediated cell death as assessed by flow cytometry. In panel C, vectors for expressing the GFP fusion of wt E1B and mutants as indicated were transfected alone or together with the p53 plasmid into Saos2 cells, and at 24 h posttransfection the cells were treated with 0.4 mM 5-fluorouracil for 48 h and processed for analysis by flow cytometry. In panel D, expression vectors for the indicated proteins were cotransfected with vector expressing GFP and cells were treated and analyzed in the same way as in panel C. The percentage of cells with sub-G₁ DNA content in various transfections is plotted.

FIG. 6.

Formation of the E1B cytoplasmic body is required to inhibit p53-dependent apoptosis. (A) Representative micrographs of Saos2 cells transfected with the indicated plasmids. Saos2 cells were transfected with expression vectors for p53 and wt Ad12 E1B 55-kDa protein fusion with GFP or with E1B mutants as specified. Cells were treated with 5-fluorouracil 24 h after transfection and were grown for an additional 24 h before being fixed. The cells were examined under a fluorescence microscope. Apoptotic cells with shrunken morphology and a fragmented nucleus are indicated by white arrows. The mutants of the Ad12 E1B 55-kDa protein are as described in the legend to Fig. 3. The nuclei were visualized by DAPI staining. (B) Quantification of apoptotic cells. Error bars represent one standard deviation of two independent assays. (C and D) Effects of the wt Ad12 E1B 55-kDa protein and its mutants on p53-mediated cell death as assessed by flow cytometry. In panel C, vectors for expressing the GFP fusion of wt E1B and mutants as indicated were transfected alone or together with the p53 plasmid into Saos2 cells, and at 24 h posttransfection the cells were treated with 0.4 mM 5-fluorouracil for 48 h and processed for analysis by flow cytometry. In panel D, expression vectors for the indicated proteins were cotransfected with vector expressing GFP and cells were treated and analyzed in the same way as in panel C. The percentage of cells with sub-G₁ DNA content in various transfections is plotted.

FIG. 7.

The Ad12 E1B 55-kDa protein does not affect apoptosis induced by L22Q/W23S mutant p53. Saos2 cells were transfected with vector for GFP alone or together with indicated plasmids, treated with 5-fluorouracil 24 h later, and harvested for flow cytometry analysis 72 h posttransfection, as was done for Fig. 6C and D. The GFP-positive cells were analyzed, and cells with sub-G₁ DNA content were considered apoptotic. (Top) Representative cell cycle profiles of transfected cells. Cells with G₁ and sub-G₁ DNA content are indicated. (Bottom) Percentage of sub-G₁ cells in various transfections.

FIG. 8.

The Ad12 E1B 55-kDa protein can sequester p53 with the mitochondrial import leader sequence. Saos2 cells were transfected with vector producing mito-l-p53 alone or together with plasmid for the wt Ad12 E1B 55-kDa protein fused with GFP. The mito-l-p53 construct contains the Flag epitope tag followed by the mitochondrial import leader sequence from the human ornithine transcarbamylase. Cells were fixed 16 h after transfection and stained with rabbit polyclonal anti-Flag antibody and goat anti-rabbit immunoglobulin G-rhodamine conjugate. The nuclei were visualized by DAPI staining.