Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response

Abstract

The "apoptotic ring" is characterized by the phosphorylation of histone H2AX at serine 139 (γ-H2AX) by DNA-dependent protein kinase (DNA-PK). The γ-H2AX apoptotic ring differs from the nuclear foci patterns observed in response to DNA-damaging agents. It contains phosphorylated DNA damage response proteins including activated Chk2, activated ATM, and activated DNA-PK itself but lacks MDC1 and 53BP1, which are required to initiate DNA repair. Because DNA-PK can phosphorylate heat shock protein 90α (HSP90α) in biochemical assays, we investigated whether HSP90α is involved in the apoptotic ring. Here we show that HSP90α is phosphorylated by DNA-PK on threonines 5 and 7 early during apoptosis and that both phosphorylated HSP90α and DNA-PK colocalize in the apoptotic ring. We also show that DNA-PK is a client of HSP90α and that HSP90α is required for full DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. In contrast, HSP90 inhibition by geldanamycin markedly enhances TRAIL-induced DNA-PK and H2AX activation. Together, our results reveal that HSP90α is a substrate and chaperone of DNA-PK in the apoptotic response. The response of phosphorylated HSP90α to TRAIL and its localization to the γ-H2AX ring represent epigenetic features of apoptosis that offer insights for studying and monitoring nuclear apoptosis.

Fig. 1.

Extensive and rapid HSP90α phosphorylation at threonines 5 and 7 in apoptotic cells. (A) HCT116 and HeLa cells were treated with camptothecin (CPT, 1 μM, 10 μM, 3 h) or TRAIL (0.1 μg/mL, 3 h). Jurkat cells were treated with FasL (0.1 μg/mL, 6 h), TRAIL (0.1 μg/mL, 6 h), or staurosporine (STS, 0.1 μM, 6 h). P-HSP90α-T5/7, total HSP90α, and γ-H2AX were analyzed by Western blotting. GAPDH was used as a loading control. (B) TRAIL induces rapid HSP90α phosphorylation. HCT116 cells were treated with 0.1 μg/mL TRAIL for the indicated times. P-HSP90α-T5/7, total HSP90α, and γ-H2AX were analyzed by Western blotting. GAPDH was used as a loading control.

Fig. 2.

TRAIL induces rapid HSP90α phosphorylation and localization of HSP90α to the apoptotic ring. (A) P-HSP90α-T5/7 and γ-H2AX confocal immunofluorescence staining in HCT116 cells treated with TRAIL. P-HSP90α-T5/7 was labeled in green and γ-H2AX in red; nuclei were stained blue with DAPI. (B) Representative P-HSP90α-T5/7 and γ-H2AX confocal microscopy in a single TRAIL-treated HCT116 cell. P-HSP90α-T5/7 was labeled in green and γ-H2AX in red; the nucleus was stained blue with DAPI. (C) Table showing the highly significant relationship between P-HSP90α-T5/7 and γ-H2AX. Double-positive, single-positive, and double-negative cells were scored after 1 h TRAIL treatment. Expected numbers from the contingency table are in parentheses. Result of χ² test is shown at right (P < 0.0001). (D) Representative TUNEL experiment showing P-HSP90α-T5/7 staining in apoptotic nuclei (TRAIL 0.1 μg/mL, 1 h). P-HSP90α-T5/7 was labeled in red, and apoptotic nuclei were labeled in green. (E) Caspase requirement for TRAIL-induced P-HSP90α-T5/7. HCT116 cells were treated with Z-VAD-fmk at 100 μM for 1 h before exposure to TRAIL at 0.1 μg/mL for 3 h. P-HSP90α-T5/7 and γ-H2AX were analyzed by Western blotting. GAPDH was used as a loading control.

Fig. 3.

DNA-PK is the kinase phosphorylating HSP90α in apoptotic cells. (A) Effects of specific ATMi and DNA-PKi on the phosphorylation of HSP90α after TRAIL treatment. HCT116 cells were treated with the ATMi or DNA-PKi (10 μM, 1 h) before the addition of TRAIL (0.1 μg/mL, 3 h). P-HSP90α-T5/7 and total HSP90α were analyzed by Western blotting. GAPDH was used as a loading control. (B) DNA-PK–knockout cells fail to phosphorylate HSP90α and H2AX in response to TRAIL. DNA-PK^+/+ and DNA-PK^−/− HCT116 cells were treated as indicated. DNA-PK, P-HSP90α-T5/7, total HSP90α, and γ-H2AX were analyzed by Western blotting. GAPDH was used as a loading control. (C) P-HSP90α-T5/7 and P-DNA-PK-T2609 confocal immunofluorescence staining in HCT116 cells treated with DNA-PKi (10 μM, 1 h) before the addition of TRAIL (0.1 μg/mL, 1 h). P-HSP90α-T5/7 was labeled in green and P-DNA-PK-T2609 in red; nuclei were stained blue with DAPI. (D) Relative distribution of P-HSP90α-T5/7 and P-DNA-PK-T2609 in a single cell. (Left) Confocal microscopy images. (Right) Intensity tracing.

Fig. 4.

HSP90α down-regulation reduces the apoptotic DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. (A) HCT116 cells were treated with TRAIL 72 h after transfection with siRNA against HSP90α or negative control siRNA. HSP90α, P-HSP90α-T5/7, γ-H2AX, P-DNA-PK-T2609, total DNA-PK, and HSP90β were analyzed by Western blotting. GAPDH was used as a loading control. The numeric values indicated in italics under the blots were obtained by densitometry analysis (ImageQuant software) and represent the ratios of γ-H2AX or P-DNA-PK to GAPDH (taking the 3-h control siRNA as 1) (B) γ-H2AX and P-DNA-PK-T2609 confocal immunofluorescence staining in HCT116 cells treated with TRAIL (0.1 μg/mL, 1 h) 72 h after transfection with siRNA against HSP90α or negative control siRNA. P-DNA-PK was labeled in green and γ-H2AX in red; nuclei were stained blue with DAPI. (C) Quantification of the experiment presented in B. The percentages of γ-H2AX– and P-DNA-PK–positive cells are represented by red and green columns, respectively. Error bars indicate 95% confidence intervals. One thousand cells were counted. (D) Effect of HSP90α on DNA fragmentation measured by filter elution assay. HCT116 cells transfected with siRNA against HSP90α or negative control siRNA were treated with TRAIL (0.1 μg/mL) for the indicated times. The y-axis represents the percentage of DNA fragmentation. (E) Effect of HSP90α on apoptotic body formation measured as sub-G1 by FACS analysis. HCT116 cells transfected with siRNA against HSP90α or negative control siRNA were treated with TRAIL (0.1 μg/mL) for the indicated times. The y-axis represents the percentage of cells with sub-G1 DNA. *P < 0.5, unpaired t test.

Fig. 5.

Members of the apoptotic ring. 3D picture of HCT116 cells treated with TRAIL (0.1 μg/mL, 1 h). P-HSP90α was labeled in red, γ-H2AX in green, and the nucleus in gray. P-HSP90α-T5/7, γ-H2AX, P-ATM, P-Chk2, P-DNA-PK, and P-H2B have been identified in the apoptotic ring. See Movie S1.