Stable overexpression of AMF1 decreases growth rate of U2OS cells. A total of 2 × 10⁵ U2OS/AMF1 or U2OS/β-Gal cells were cultured in Dulbecco's modified Eagle medium (GIBCO/BRL) supplemented with 10% fetal bovine serum. Cells from three pairs of U2OS/AMF1 and U2OS/β-Gal dishes were counted at each time point of 24, 48, 72, and 96 h, and values were plotted. Error bars indicate the variation of cell numbers from three identical dishes. The procedures were repeated three times with similar results.

In vivo and in vitro complex formation of AMF1 and p53. (A) AMF1 expression in human U2OS cell lines. Equal amounts of total cellular protein extracts from parental U2OS (lane 1), U2OS/β-Gal (lane2), and U2OS/AMF1 (lane 3) cells were subjected to Western blotting with polyclonal rabbit serum against AMF1. (B) U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 8 h, extracts were prepared and incubated with Ni-NTA resin in the presence (lane 3) or absence (lanes 1 and 2) of 10 mM EDTA. After extensive washing, six-histidine-tagged proteins were eluted and concentrated. Copurification of p53 with 6H-AMF1 (lane 1) or 6H–β-Gal (lane 2) was probed by Western blotting with MAb DO-1. (C) In vitro binding of AMF1 to p53. (Upper panel) Sf9 cells were infected by recombinant baculoviruses expressing AU1-AMF1, p53, or bovine papillomavirus E1 proteins, harvested 40 h postinfection, and lysed as described previously (54). Cell extract with AU1-AMF1 was incubated with extract containing E1 (lane 2) or p53 (lane 3). P53 was immunoprecipitated with MAb pAb421 and protein A-conjugated Sepharose beads. After washing, the immunoprecipitates were analyzed by Western blotting with polyclonal antibody against AMF1. Input cell extract (10%) containing AU1-AMF1 was loaded in lane 1. (Lower panel) GST (lane 5) or GST-AMF1 fusion (lane 6) was incubated with 6H-p53 and washed extensively. Bound proteins were resolved by SDS-PAGE followed by Western blotting with MAb DO-1 against p53. Input 6H-p53 (5%) was loaded in lane 4.

Mapping the AMF1 binding domain on p53. (A) Coprecipitation of wild-type and deletion mutant p53 with GST-AMF1 fusion protein. GST-AMF1 was expressed in E. coli and affinity purified with glutathione-Sepharose. Wild-type and mutant p53 were prepared by in vitro translation. (Upper panel) Input p53 proteins (10%); (lower panel) p53 proteins bound on GST-AMF1. (B) Schematic representation of wild-type p53 (49) and results for AMF1 binding assays. Numbers represent amino acid residues.

N terminus of AMF1 interacts with p53. Six-histidine-tagged wild-type and mutant AMF1 proteins were prepared by in vitro translation. These proteins (named by the number of amino acids contained) were incubated with Sf9 cell extract containing either papillomavirus E1 (lanes 2, 5, 8, and 11) or p53 (lanes 3, 6, 9, and 12), in the presence of MAb pAb421 against p53. Immunocomplexes were precipitated with protein A-Sepharose. After washing, AMF1 proteins remaining on the beads were run on an SDS–15% polyacrylamide gel together with 10% volume of each input (lanes 1, 4, 7, 10). The gel was analyzed with a Bio-Rad GS-250 molecular imager.

AMF1 stimulates transcriptional activation by p53. (A) Transient transactivation in U2OS cells. A total of 500 ng of p53-dependent luciferase reporters PG13 or pWAF1-luciferase (19) was cotransfected into U2OS cells with 0, 1, or 4 μg of AMF1-expressing vector pDB327. At 32 h after transfection, luciferase activities were measured and are presented as the increase in activation over that of the reporter alone. Each sample was analyzed in triplicate, and standard deviations are shown (error bars). (B) Transient transactivation in Saos-2 cells. The same procedure as that for panel A was used, except that 50 ng of vector pC53SN was cotransfected to provide wild-type p53 activity in the cell.

Self-association of AMF1 in vivo and in vitro. (A) Coprecipitation of endogenous AMF1 with 6H-AMF1 from U2OS cells. Cell extracts of parental U2OS and U2OS/AMF1 were mixed and incubated with Ni-NTA resin in the presence (lane 2) or absence (lane 3) of 10 mM EDTA. Parental U2OS cell extract alone was also incubated with Ni-NTA resin without EDTA as a control (lane 1). After washing, proteins bound on the resin were eluted and concentrated. AMF1 proteins were detected by Western blotting. (B) In vitro association of HA-AMF1 with 6H-AMF1. Both proteins were prepared by in vitro translation and labeled with [³⁵S]methionine. 6H-AMF1 was incubated with anti-HA MAb 12CA5 (Boehringer Mannheim) and protein A-Sepharose beads with (lane 4) or without (lane 3) HA-AMF1. Protein complexes were resolved on an SDS–15% polyacrylamide gel and analyzed with a Bio-Rad GS-250 molecular imager. Input HA-AMF1 (20%) (lane 1) and input 6H-AMF1 (10%) (lane 2) are shown.

Mapping the oligomerization domain on AMF1. (A) Interaction of VP16-AMF1 with LexA-AMF1 fusion proteins. Galactose-inducible expression vectors containing the LexA-AMF1 deletion fusions were transformed along with VP16-AMF1, AMF1, or the VP16 AD into DBY1 containing a lexA operator-lacZ reporter. Colonies were transferred to galactose-X-Gal plates and color formation was monitored. All constructs except LexA-AMF1(14–76) activate transcription of pSH18-34. A plus sign indicates earlier and more intense color formation in the presence of VP16-AMF1 than in the presence of the VP16 AD or AMF1. (B) In vitro binding of deletion mutant AMF1 and wild-type AMF1. Three of the AMF1 deletion mutants, AMF1(14–212), AMF1(14–130), and AMF1(14–76), were subcloned into vector pGEX2T (Pharmacia), and expressed in E. coli BL21::DE3(pLysS). Purified GST and GST-AMF1 fusion proteins were incubated with ³⁵S-labeled in vitro-translated full-length AMF1. Bound AMF1 was resolved by SDS–15% PAGE and analyzed with a Bio-Rad GS-250 molecular imager. The right side lane shows 10% of input AMF1.

Expression of AMF1(1–103) interferes with wild-type AMF1 function in vivo. (A) AMF1(1–103) inhibits p53 transcriptional activation in Saos-2 cells. A total of 500 ng of p53-dependent luciferase reporter PG13 was cotransfected into Saos-2 cells with or without vectors expressing p53, AMF1(1–327) (wild type), and AMF1(1–103) as indicated. A total of 100 ng of pC53SN plasmid was used. Total DNA for each transfection was made up to 6.6 μg by using vector plasmid pCG. Luciferase activities were measured at 32 h after transfection and are presented as the increase in activation over reporter alone. Each sample was analyzed in triplicate, and standard deviations are shown (error bars). (B) In vitro binding of AMF1(1–103) to HA-AMF1. Both proteins were prepared by in vitro translation. Experimental procedures were the same as in Fig. 5B. Each input (10%) is shown in the first two lanes. Lanes 3 and 4 demonstrate that AMF1(1–103) can be coimmunoprecipitated by HA-AMF1 but not the anti-HA MAb.

Overexpression of AMF1 affects cellular responses under etoposide treatment. (A) Overexpression of AMF1 in U2OS holds more cells in G₁ phase and inhibits etoposide-induced S-G₂ arrest and apoptosis. U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 0, 24, and 48 h. Cells were harvested, processed, and subjected to flow cytometric analysis. Cell cycle stages are represented by the cellular DNA content, which was analyzed by PI staining and fluorescence-activated cell sorting. Boundaries for G₁ and S-G₂ phases are labeled on the top pair of graphs. (B) Western blotting analysis of AMF1, p53, and p21^WAF1/CIP1 in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. Protein concentrations in cell extracts were determined, and equal amounts were loaded in each lane as judged by the level of α-tubulin. Intensity of bands at 0 h shows the baseline level of each protein. (C) RT-PCR analysis of p21^WAF1/CIP1 transcripts in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. The 370-bp product was amplified using p21 oligonucleotides. As a control, the cDNA encoding GAPDH was amplified with specific oligonucleotides.

Overexpression of AMF1 sensitizes U2OS cells to UV irradiation-induced apoptosis. (A) U2OS/AMF1 and U2OS/β-Gal cells were exposed to UV irradiation (100 J/m²) at 70% confluence. Cells were harvested after 48 h of incubation, processed, and subjected to flow cytometric analysis. Boundaries for sub-G₁, indicative of apoptosis, are marked on top of each graph. (B) Series of UV dosages were tested under the same conditions as in panel A. The percentage of cells with a sub-G₁ DNA content is graphed. Data were derived from three separate experiments. Error bars, standard errors.