An LRP16-containing preassembly complex contributes to NF-κB activation induced by DNA double-strand breaks

Abstract

The activation of NF-κB has emerged as an important mechanism for the modulation of the response to DNA double-strand breaks (DSBs). The concomitant SUMOylation and phosphorylation of IKKγ by PIASy and ATM, respectively, is a key event in this mechanism. However, the mechanism through which mammalian cells are able to accomplish these IKKγ modifications in a timely and lesion-specific manner remains unclear. In this study, we demonstrate that LRP16 constitutively interacts with PARP1 and IKKγ. This interaction is essential for efficient interactions among PARP1, IKKγ, and PIASy, the modifications of IKKγ, and the activation of NF-κB following DSB induction. The regulation of LRP16 in NF-κB activation is dependent on the DSB-specific sensors Ku70/Ku80. These data strongly suggest that LRP16, through its constitutive interactions with PARP1 and IKKγ, functions to facilitate the lesion-specific recruitment of PARP1 and IKKγ and, ultimately, the concomitant recruitment of PIASy to IKKγ in response to DSB damage. Therefore, the study has provided important new mechanistic insights concerning DSB-induced NF-κB activation.

Figure 1.

LRP16 physically interacts with IKKγ and PARP1. (A) A photograph of a representative silver-stained gel showing the profiles of proteins that were pulled down by IP. HeLa cells were treated with VP16 (50 μM) for 30 min. (B) HeLa cells were treated with 50 μM VP16 for the indicated times. Lysates were prepared for IP with an anti-LRP16 antibody or a rabbit IgG control. The IP products and 2% of the input were immunoblotted with the indicated antibodies. (C) Identical to B, except that total cell lysates from HeLa cells that were either exposed to IR (10 Gy) or treated with 5 μM doxorubicin (DOX) or 10μM camptothecin (CPT) for 30 min were used. (D) Identical to B, except that either the cytosolic or the nuclear fractions of the cell lysates that were derived from HeLa cells treated with or without 50 μM VP16 for 0 (-) or 30 (+) min were used. (E) Identical to B, except that ethidium bromide was added to a final concentration of 50 μg/ml when indicated. (F) HeLa cells were transfected with FLAG-tagged PARP1 deletion mutants and treated with VP16 for 30 min. The whole-cell extracts were prepared for Co-IP with an anti-FLAG antibody. (G) Identical to F, except that HeLa cells were transfected with FLAG-tagged IKKγ deletion mutants.

Figure 2.

LRP16 is required for NF-κB activation in response to genotoxic stress. (A) HeLa cells were transfected with the indicated siRNA and treated with or without VP16. The lysates were used for immunoblotting with the indicated antibodies. (B) HeLa cells were transfected with the indicated plasmids and siRNAs. After 48 h, the cells were induced by 50 μM VP16 for the indicated times. The lysates were used for immunoblotting. (C) HeLa cells were transfected with the indicated siRNAs and exposed to 10 Gy IR. The cytoplasmic and nuclear fractions were then prepared and immunoblotted with the indicated antibodies. (D) The RNA expression levels of cIAP2 and XIAP were determined by RT-PCR. β-actin served as a loading control. (E) The absorbance values of individual cultures were measured at various time points (0, 12, 24, 48 and 72 h) after exposure to IR to assess the relative growth rate of the cultures. (F) The transfected HeLa cells were treated with 10 Gy IR. Twenty hours after treatment, the percentage of apoptotic cells was detected by Annexin V staining followed by fluorescence-activated cell sorting analysis. (G) C33A cells were transfected with the LRP16 expression vector or the control vector. Forty-eight hours after transfection, the cells were processed as in A. (H) Identical to C, except that C33A cells were used for the transfection with the indicated plasmids. (I) C33A cells were transfected with the indicated plasmids and then processed as in E 42 h after transfection. (J) HeLa cells were transfected with the indicated siRNAs and treated with 10 ng/ml TNF-α for the indicated times. The relative levels of phosphorylated IKKβ, total IKKβ, IκBα and LRP16 were determined. β-actin was used as a control. (E, F, I) Data represent the means ± SD (error bars) of three biological replicates analyzed in triplicate. *P<0.01.

Figure 3.

Depletion of LRP16 diminishes the PARP1-IKKγ interaction and IKKγ SUMOylation, and phosphorylation. (A) HeLa cells were first transfected with control scramble (con) or LRP16 siRNA 374 (374) and then treated with 50 μM VP16 for various times (0, 10, 20 and 30 min). Lysates were prepared from the treated cells and used to perform an IP experiment with an anti-IKKγ antibody. (B) HeLa cells were first treated with VP16 as in A. The total cell extracts were then used for IP with an anti-IKKγ antibody, and the IP products were analyzed by western blot analysis using an anti-SUMO1 antibody. (C) HeLa cells were treated as in B, and the relative amounts of phosphorylated IKKγ in individual samples were then assessed by western blot analysis using an anti-p-IKKγ antibody. (D) Identical to C, except that HeLa cells were co-transfected with the indicated siRNAs and plasmids. (E) HeLa cell lysates were prepared as in B, and the cell lysates were then used for the IP experiments. The relative levels of PIASy, IKKγ and LRP16 were then assessed by western blot analysis.

Figure 4.

The LRP16-PARP1 and LRP16-IKKγ interactions are dependent on PAR. (A) HeLa cells were transfected with control or PARP1 siRNAs and the whole-cell extracts were examined with the indicated antibodies. (B) HeLa cells were transfected with the indicated siRNA and treated or not treated with VP16 (50 μM) for 30 min. IP was performed using an anti-LRP16 antibody. The samples were analyzed by immunoblotting with the indicated antibodies. (C) Identical to B except that lysates were derived from HeLa cells that were pretreated with either 10 mM 3-aminobenzamide (3-AB) or the solvent dimethyl sulfoxide (DMSO) for 0, 15 and 30 min were used. (D) A schematic illustration of LRP16 and its mutants. (E) GST, GST-LRP16 and GST-fusion LRP16 mutants were expressed using a prokaryotic expression system and the purified proteins were resolved by Coomassie staining. (F) PAR binding assays were performed with the indicated fusion proteins and synthesized PAR. ‘#’ represents the heat treatment. (G) HeLa cells were transfected with the indicated expression vectors of LRP16 mutants. Forty-eight hours after transfection, the cells were treated with VP16 and processed as in B, except that the antibody that was used for IP was anti-FLAG.

Figure 5.

LRP16, IKKγ and PARP1 could reside in the same complexes. (A) Various combinations of purified IKKγ, commercial PARP1 and GST-LRP16 (or GST) were mixed and incubated under in vitro conditions for 2 h at room temperature. A GST pull-down assay was then performed with GST-LRP16. (B) Identical to A, except that IKKγ and GST-LRP16 were replaced with GST-IKKγ and LRP16, respectively, to study the binding partners of IKKγ. (C) HeLa cells were transfected with either FLAG or FLAG-LRP16 expression vectors for 48 h and then treated with 50 mM VP16 for 30 min. Total cell lysates were then prepared for sequential IP experiments (with an anti-FLAG antibody followed by an anti-IKKγ antibody).

Figure 6.

LRP16 indirectly interacts with both Ku70 and Ku80. (A) Results of western blot experiments showing the physical interaction between endogenous LRP16 and Ku70/Ku80 in HeLa cells with (+) and without (-) VP16 treatment (50 μM). (B) Identical to A, except that ethidium bromide (50 μg/ml) was included in the lysates of the corresponding wells to test for DNA-mediated protein interactions. (C) Effects of siRNA-mediated depletion of Ku80 or Ku70 on the interaction of IKKγ with PARP1. (D) HeLa cells were co-transfected with the indicated plasmids and siRNA, and the lysates were immunoblotted with the indicated antibodies. (E and F) Effects of siRNA-mediated knockdown of Ku80 or Ku70 on DNA damage-induced NF-κB reporter gene activity (E) and the mRNA level of cIAP2 (F). Data represent the means ± SD (error bars) of three biological replicates analyzed in triplicate. *P<0.01. (G) Results of western blot experiments showing the dynamic interaction between endogenous LRP16 and Ku70 or Ku80 in HeLa cells that were treated with VP16 (50 μM).

Figure 7.

A schematic model illustrating the proposed interactions among the key components that are involved in the DSB-induced NF-κB activation. Briefly, weak constitutive interactions exist among LRP16, PARP1, IKKγ, Ku70 and Ku80 in unperturbed human cells. Following DSB damage induction, LRP16, PARP1 and IKKγ are recruited to DSB damage sites via their indirect association with Ku70 and Ku80. In contrast, DSB also cause the activation of ATM and the auto-poly-ribosylation of PARP1. The newly formed PAR-PARP1 then serves as a platform for recruiting the PAR-binding protein ATM and PIASy into the complex, which can ultimately result in the simultaneous phosphorylation and SUMOylation of IKKγ and the activation of NF-κB. In the absence of DSB damage, the activation of ATM or PARP1 and, accordingly, the phosphorylation or SUMOylation of IKKγ may occur. However, the simultaneous occurrence of these two modes of phosphorylation and SUMOylation of IKKγ modifications and, therefore, NF-κB activation either do not occur or occur only at low frequencies.