Interaction between tumor suppressor adenomatous polyposis coli and topoisomerase IIalpha: implication for the G2/M transition - PubMed (original) (raw)

Interaction between tumor suppressor adenomatous polyposis coli and topoisomerase IIalpha: implication for the G2/M transition

Yang Wang et al. Mol Biol Cell. 2008 Oct.

Abstract

The tumor suppressor adenomatous polyposis coli (APC) is implicated in regulating multiple stages of the cell cycle. APC participation in G1/S is attributed to its recognized role in Wnt signaling. APC function in the G2/M transition is less well established. To identify novel protein partners of APC that regulate the G2/M transition, APC was immunoprecipitated from colon cell lysates and associated proteins were analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). Topoisomerase IIalpha (topo IIalpha) was identified as a potential binding partner of APC. Topo IIalpha is a critical regulator of G2/M transition. Evidence supporting an interaction between endogenous APC and topo IIalpha was obtained by coimmunoprecipitation, colocalization, and Förster resonance energy transfer (FRET). The 15-amino acid repeat region of APC (M2-APC) interacted with topo IIalpha when expressed as a green fluorescent protein (GFP)-fusion protein in vivo. Although lacking defined nuclear localization signals (NLS) M2-APC predominantly localized to the nucleus. Furthermore, cells expressing M2-APC displayed condensed or fragmented nuclei, and they were arrested in the G2 phase of the cell cycle. Although M2-APC contains a beta-catenin binding domain, biochemical studies failed to implicate beta-catenin in the observed phenotype. Finally, purified recombinant M2-APC enhanced topo IIalpha activity in vitro. Together, these data support a novel role for APC in the G2/M transition, potentially through association with topo IIalpha.

PubMed Disclaimer

Figures

Figure 1.

Endogenous topo IIα associates with endogenous full-length APC. (A) Endogenous full-length APC coimmunoprecipitated with topo IIα by using anti-topo IIα rabbit sera, but not using rabbit IgG. P, precipitated proteins; S, nonprecipitated supernatant proteins. Representative blot from eight independent experiments. (B) Full-length APC immunoprecipitated with affinity-purified polyclonal anti-APC sera (left and middle). Endogenous topo IIα (top) coimmunoprecipitated with the endogenous full-length APC, whereas topo IIβ did not (bottom). Quantification of the band intensity from three independent experiments revealed 1.8% of the total topo IIα coprecipitated with full-length APC (right). Representative blots from seven independent experiments. (C) Colocalization of endogenous APC and topo IIα in HCT 116βw cells by using APC antibody (ab-7), polyclonal topo IIα antibody, and confocal microscopy. (a) Confocal image shows APC (green) and topo IIα (red) colocalized in the nucleus (yellow in merge). Bar, 5 μm. (b) Confocal images through the entire cell thickness were collected for APC (green) and topo IIα (red) and are shown as a projection of the 3-D Z-series images. (c) Overlapping pixels for the Z-series images in b are projected and shown in yellow. (d) Graph shows the average number of overlapping pixels calculated from six individual Z-series images; 1.9% of the topo IIα pixels overlap with APC, whereas 10% of the APC pixels overlap with topo IIα. (D) Measurements of FRET between APC-Alexa 488 and topo IIα-Alexa 568 were performed using the method of donor fluorescence sensitization after acceptor photobleaching in fixed samples of immunofluorescently labeled HCT116βw cells. Endogenous APC was labeled using either anti-APC ab-1 or anti-APC ab-7 followed by goat anti-mouse Alexa 488 secondary antibody. Endogenous topo IIα was labeled using anti-topo IIα antibody followed by goat anti-rabbit Alexa 568 secondary antibody. Energy transfer efficiencies (E) between immunolabeled Alexa 488-APC and Alexa 568-topo IIα were ∼20.8% (gray bar, left, n = 10) within photobleached regions. This value is significantly positive (*p = 0.00021) compared with E measured without photobleaching (E = −0.9%, n = 10, white bar) or to energy transfer (E = −6.6%, n = 3, gray bar, right) between photobleached GFP (**p = 0.0000000056), which is abundantly expressed in the nucleus and immunolabeled Alexa 488-APC.

Figure 2.

The 15-amino acid and 20-amino acid repeat regions of APC each colocalize with topo IIα and localize to the nucleus. (A) Schematic diagram of APC with domains implicated in nuclear function marked. Two NLSs are designated by thin pink lines in both full-length APC and M3-APC. Five APC fragments expressed as GFP fusions are shown. (B) Colocalization of the GFP-fused APC fragments with endogenous topo IIα in HCT116βw cells. Of the five APC fragments, both M2 and M3 displayed significant nuclear localization and partial colocalization with topo IIα. Bar, 5 μm.

Figure 3.

The 15-amino acid repeat region of APC protein alters nuclear morphology. (A) Confocal immunofluorescence microscopy revealed abnormal nuclei (condensed or fragmented) in M2-APC–expressing HCT116βw cells. Bar, 5 μm. (B) Nuclear phenotype of HCT116βw cells expressing GFP-M2-APC (light bar), GFP-M3-APC (dark bar), or GFP (white bar) at 24 and 48 h after transfection. More than 80% of the M2-APC–expressing cells displayed abnormal nuclear morphology by 48 h. (C) Nuclear phenotype of SW480 cells and HCA7 cells expressing GFP-M2-APC (light bar) or GFP (white bar) at 48 h after transfection. (B and C) Graphs represent analysis of 100 cells for each transfection in three independent experiments, with error bars indicating SD.

Figure 4.

Expression of the 15-amino acid repeat region of APC results in G2 accumulation. (A) Histograms showing representative FACS displays of cell cycle distribution assessed by propidium iodide staining. UN, untransfected cells. For both GFP- and GFP-M2-APC–expressing cells, only GFP-positive cells are displayed. (B) FACS data from three independent experiments. The fraction of cells in G2/M doubled, and the S phase decreased by half for cells expressing GFP-M2-APC compared with the untransfected control cells or cells expressing only GFP. Values for G0/G1, S, and G2/M, respectively, are 35.6 ± 2.1, 40.9 ± 2.4, and 23.5 ± 3.3% (for untransfected); 37.7 ± 1.3, 41.0 ± 9.6, and 21.3 ± 8.5% (for GFP); and 28.0 ± 5.6, 18.1 ± 5.6, and 53.9 ± 8.9% (for M2-APC). For each transfection, 15,000 GFP-positive cells were analyzed. (C) Mitotic events assessed after DAPI staining. One hundred randomly chosen M2-APC– or GFP-expressing cells were analyzed from three independent experiments at 24, 48, and 72 h after transfection. None of the M2-APC–expressing cells appeared to be mitotic. A small number of the GFP-expressing cells were mitotic. (D) Representative immunofluorescence confocal microscopy of mitotic marker phospho-histone H3 from three independent experiments. No M2-APC–expressing cells were positive for phospho-histone H3, whereas a few GFP-expressing cells were positive. Arrowhead indicates a cell that is positive for both GFP and phospho-histone H3. Bar, 10 μm.

Figure 5.

Expression of the 15-amino acid repeat region of APC does not alter β-catenin expression or localization. (A) Confocal immunofluorescence microscopy reveals similar β-catenin localization in M2-APC– and GFP-expressing HCT116βw cells. Note that the nucleus (right) is abnormal in the M2-APC–expressing cell. Bar, 5 μm. (B) Less than 1% of the total β-catenin coimmunoprecipitated with GFP-M2-APC by using a GFP antibody (M2), and none coimmunoprecipitated with GFP alone (GFP). Ten percent input is 25 μg of total protein. Transfection efficiency was ∼50%. (C) Nearly 10% of the total β-catenin coimmunoprecipitated with endogenous APC. Ten percent input is 30 μg of total protein. Representative blots from five independent experiments. (D) Western blot reveals comparable levels of total β-catenin in M2-APC- (M2) and GFP-expressing (GFP) cells. Equivalent amounts of total protein from whole cell lysates were loaded. (B and D) Representative blots from three independent experiments. (E) Cells were cotransfected with GFP-M2-APC or GFP, SuperTOP-flash or FOP-flash reporters, and pRL-TK Renilla luciferase plasmid. Luciferase activities were determined 24 h after transfection and normalized against both pRL-TK Renilla activity and FOP-flash reporter activity. Values are mean ± SD for triplicate samples from a representative experiment.

Figure 6.

The 15-amino acid repeat region of APC associates with topo IIα and enhances topo IIα activity. (A) Increased expression of topo IIα in M2-APC–expressing cells compared with GFP-expressing cells. Equivalent total protein loaded in each lane. (B) Topo IIα coimmunoprecipitates with M2-APC, but not with GFP. Ten percent input is 25 μg of total protein. The star marks migration of antibody heavy chain. (A and B) Representative blots from five independent experiments. (C and D) Representative topo IIα DNA relaxation assays from five independent experiments. R, relaxed plasmid DNA; S, supercoiled plasmid. (C) Purified recombinant human topo IIα (0.35 μM) slightly relaxed supercoiled plasmid DNA (lane 2). Addition of purified recombinant M2-APC (0.35 or 1.75 μM) to the reaction resulted in progressively enhanced topo IIα plasmid relaxation activity (lanes 3 and 4). M2-APC did not have plasmid relaxation activity in the absence of topo IIα (lane 5). (D) Using a higher concentration of topo IIα (0.7 μM), the addition of BSA (0.7 μM) did not enhance the plasmid relaxation activity, but rather slightly inhibited it (compare lanes 2 and 3). M2-APC (0.7 μM) enhanced the plasmid relaxation activity (lane 5).

References

1. Agostinho M., Rino J., Braga J., Ferreira F., Steffensen S., Ferreira J. Human topoisomerase IIalpha: targeting to subchromosomal sites of activity during interphase and mitosis. Mol. Biol. Cell. 2004;15:2388–2400. - PMC - PubMed
1. Anderson C. B., Neufeld K. L., White R. L. Subcellular distribution of Wnt pathway proteins in normal and neoplastic colon. Proc. Natl. Acad. Sci. USA. 2002;99:8683–8688. - PMC - PubMed
1. Aoki K., et al. Chromosomal instability by beta-catenin/TCF transcription in APC or beta-catenin mutant cells. Oncogene. 2007;26:3511–3520. - PubMed
1. Azuma Y., Arnaoutov A., Dasso M. SUMO-2/3 regulates topoisomerase II in mitosis. J. Cell Biol. 2003;163:477–487. - PMC - PubMed
1. Bhat U. G., Raychaudhuri P., Beck W. T. Functional interaction between human topoisomerase IIalpha and retinoblastoma protein. Proc. Natl. Acad. Sci. USA. 1999;96:7859–7864. - PMC - PubMed

Interaction between tumor suppressor adenomatous polyposis coli and topoisomerase IIalpha: implication for the G2/M transition - PubMed (original) (raw)