p53-mediated apoptosis requires inositol hexakisphosphate kinase-2

Abstract

Inositol pyrophosphates have been implicated in numerous biological processes. Inositol hexakisphosphate kinase-2 (IP6K2), which generates the inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), influences apoptotic cell death. The tumor suppressor p53 responds to genotoxic stress by engaging a transcriptional program leading to cell-cycle arrest or apoptosis. We demonstrate that IP6K2 is required for p53-mediated apoptosis and modulates the outcome of the p53 response. Gene disruption of IP6K2 in colorectal cancer cells selectively impairs p53-mediated apoptosis, instead favoring cell-cycle arrest. IP6K2 acts by binding directly to p53 and decreasing expression of proarrest gene targets such as the cyclin-dependent kinase inhibitor p21.

Fig. 1.

Gene disruption of IP6K2 in HCT116 cells. (A) Targeting scheme for disruption of IP6K2 gene in HCT116 cells. rAAV was employed to disrupt exon 6 of IP6K2 by inserting a cassette containing a splice acceptor (SA) followed by an internal ribosome entry sequence (IRES), the coding sequence of neomycin transferase (neo), and a polyadenylation signal (pA), flanked by recognition sites (LoxP) for Cre recombinase. Cre-expressing adenovirus (Ad-Cre) was used to excise this cassette, facilitating targeting of the second allele. The second round of rAAV targeting and Ad-Cre excision results in two disrupted IP6K2 alleles. (B) Confirmation of IP6K2 targeting by RT-PCR. (C) Gene disruption of IP6K2 reduces intracellular IP7 levels. Parental and IP6K2 Δ/Δ cells were labeled with [³H]myo-inositol, and intracellular inositol phosphates were resolved by HPLC.

Fig. 2.

IP6K2 is required for p53-mediated apoptosis. (A) Disruption of IP6K2 blocks 5-FU–induced apoptosis. HCT116 IP6K2 +/+ and Δ/Δ cells were treated with 5-FU 400 μM or DMSO control for 18 h. Immunoblotting for cleaved PARP and caspase 3 provides a measure of apoptosis, with β-actin as a loading control. (B) IP6K2 is required for p53-dependent, but not p53-independent, apoptosis. HCT116 IP6K2 +/+ and Δ/Δ cells were treated with 5-FU (400 μM), sulindac sulfide (120 μM), or DMSO control for 18 h and then immunoblotted for cleaved PARP. (C) Disruption of IP6K2 blocks 5-FU–induced apoptosis. HCT116 IP6K2 +/+ and Δ/Δ cells and p53 −/− cells were treated with 5-FU (400 μM) or DMSO control for 18 h. Annexin V/propidium iodide staining followed by flow cytometry was used to measure the apoptotic population. Bars represent means ± SD of two independent experiments, with results representative of results obtained with a second clone of IP6K2-disrupted cells. (D) Disruption of IP6K2 favors G1 cell-cycle arrest over apoptosis in response to 5-FU. HCT116 IP6K2 +/+ and Δ/Δ cells were treated with 5-FU (400 μM) or DMSO control for 18 h, fixed, stained with propidium iodide, and subjected to cell-cycle analysis by flow cytometry. (E) Disruption of IP6K2 selectively increases expression of pro-cell-cycle arrest p53 targets. HCT116 IP6K2 +/+ and Δ/Δ cells were treated with 5-FU (400 μM) or DMSO control for 18 h and immunoblotted for p53 targets as indicated.

Fig. 3.

IP6K2 down-regulates p21 expression in a kinase activity-dependent manner. (A) Tetracycline-inducible IP6K2 expression in U2OS cells sensitizes to etoposide-induced cytotoxicity. Cells were induced with tetracycline (1 μM) for 18 h and then treated with etoposide (20 μM) for 42 h, followed by MTT viability assay. Bars represent means ± SD of four independent experiments. (B) Tetracycline-inducible IP6K2 expression in U2OS cells leads to down-regulation of 5-FU–induced p21, but not PUMA, expression. Cells were induced with tetracycline (1 μM) for 18 h and then treated with 5-FU (400 μM) for 8 h. (C) Kinase activity of IP6K2 is required for p21 down-regulation. U2OS Tet-inducible cell lines expressing either myc-IP6K2 WT or kinase dead (KD) were induced with tetracycline (1 μM) for 18 h as indicated and then treated with 5-FU (400 μM) for 0, 4, 8, and 24 h.

Fig. 4.

IP6K2 directly binds p53 via the DNA-binding domain. (A) Binding of IP6K2 and p53 in a cooverexpression system. GST or GST-IP6K2 were coexpressed with myc-p53 in HCT116 p53 −/− cells. Glutathione-sepharose pulldown reveals binding of myc-p53 to GST-IP6K2 but not GST control. (B) Endogenous coimmunoprecipitation of p53 and IP6K2 from etoposide-treated U2OS cells. U2OS cells were treated with 20 μM etoposide or DMSO control for 18 h and then immunoprecipitated with control rabbit IgG or anti-IP6K2 polyclonal antibody. (C) Direct in vitro binding of IP6K2 and p53. Recombinant His-IP6K2, GST-p53, and GST control were purified from Escherichia coli and subjected to an in vitro binding reaction followed by glutathione-sepharose pulldown. (D) Mapping of the IP6K2 binding site on p53. Myc-tagged C-terminally truncated p53 constructs were coexpressed with GST or GST-IP6K2 in HCT116 p53 −/− cells, followed by glutathione-sepharose pulldown. p53 1–101 failed to bind IP6K2, whereas 1–186 was sufficient for binding. (E) Identification of dominant-negative fragments for IP6K2-p53 binding. In HCT116 p53 −/− cells, GST or GST-p53, myc-IP6K2, and myc-tagged IP6K2 fragment 1–67 or 68–143 were coexpressed as indicated. Fragment K2(1–67) but not K2(68–143) had a dominant-negative effect on the binding of full-length proteins. (F) IP6K2 fragment (1–67) disrupts full-length IP6K2-p53 binding. Increasing amounts of myc-IP6K2(1–67) DNA (0, 1, 2, 4 μg) were cotransfected with GST or GST-p53 and myc-IP6K2. IP6K2(1–67) disrupted p53-IP6K2 binding in a concentration-dependent manner. (G) Expression of dominant-negative fragment IP6K2(1–67) increases p21 induction by 5-FU. U2OS cells inducibly expressing the dominant-negative fragment myc-IP6K2(1–67) were induced with tetracycline (1 μM) for 24 h and then treated with 5-FU (400 μM) for 18 h.