Irod/Ian5: an inhibitor of gamma-radiation- and okadaic acid-induced apoptosis

Abstract

Protein phosphatase-directed toxins such as okadaic acid (OA) are general apoptosis inducers. We show that a protein (inhibitor of radiation- and OA-induced apoptosis, Irod/Ian5), belonging to the family of immune-associated nucleotide binding proteins, protected Jurkat T-cells against OA- and gamma-radiation-induced apoptosis. Unlike previously described antiapoptotic proteins Irod/Ian5 did not protect against anti-Fas, tumor necrosis factor-alpha, staurosporine, UV-light, or a number of chemotherapeutic drugs. Irod antagonized a calmodulin-dependent protein kinase II-dependent step upstream of activation of caspase 3. Irod has predicted GTP-binding, coiled-coil, and membrane binding domains. Irod localized to the centrosomal/Golgi/endoplasmic reticulum compartment. Deletion of either the C-terminal membrane binding domain or the N-terminal GTP-binding domain did not affect the antiapoptotic function of Irod, nor the centrosomal localization. The middle part of Irod, containing the coiled-coil domain, was therefore responsible for centrosomal anchoring and resistance toward death. Being widely expressed and able to protect also nonimmune cells, the function of Irod may not be limited to the immune system. The function and localization of Irod indicate that the centrosome and calmodulin-dependent protein kinase II may have important roles in apoptosis signaling.

Figure 1.

Irod confers resistance selectively to cell death induced by OA and γ-radiation in Jurkat T-cells. (A) Expression, determined by RT-PCR, in stably transfected Jurkat T-cell clones, of pcDNA3.1-Irod (lane 2), pcDNA3.1-as-Irod (lane 3), and pcDNA3.1-empty vector (lane 4). (B) Percentage of apoptotic cells after 6-h treatment with 800 nM OA in the absence (open bars) or presence (filled bars) of the CaMKII inhibitor KN93 (20 μM). (C) Percentage of apoptotic cells 6 h after exposure to γ-radiation (25 Gy). The cells were incubated in the absence (open bars) or presence (filled bars) of KN93 (20 μM). Data in B and C represent the mean of at least four experiments ± SEM. The background, including KN93 treatment alone, was generally not >5% and was subtracted from all calculations. (D–I) Cells stained with the DNA binding fluorescent dye (Hoechst 33342) after 6 h of incubation with vehicle (D and G), 800 nM OA (E and H), or 800 nM OA + 20 μM KN93 (F and I). Wild-type cells are shown in D–F, and Irod-expressing cells in G–I. For further experimental details, see the MATERIALS AND METHODS.

Figure 2.

Irod protects against caspase 3 and PARP cleavage, but not HDM2 down-regulation. Jurkat T-cells, wild-type (WT), expressing empty vector (pcDNA3.1), Irod (IROD), or Irod in antisense direction (As-IROD) were irradiated with 25 Gy (A and B) and incubated for up to 6 h post-γ-radiation, or treated with 800 nM OA in the absence or presence (*) of 100 μM caspase inhibitor zVAD-fmk (C). Extracts were processed for Western blotting. The position and size of caspase 3, Bcl-2, Bax, PARP, HDM2, p53, and β-actin is indicated. Note stronger cleavage of caspase 3 (A) and PARP (B) in cells expressing as-Irod, and less cleavage in cells expressing Irod. Also note the rapid down-regulation of HDM2 in response to γ-radiation (B) and OA treatment (C). Similar results were obtained in at least three separate experiments.

Figure 3.

Effects of irod, Bcl-2, and specific CaMKII-inhibitors on OA- and daunorubicin-induced apoptosis in nonimmune cells. (A) Apoptotic response to OA (100 nM for 4 h) or 4 μM daunorubicine (DR) for 10 h, of wild-type LNCap cells (WT), or cells stably transfected with Bcl-2, Irod, as-Irod, or cDNA encoding a peptide containing the Irod^235–285 sequence (Oar2). (B) Apoptotic response to OA (600 nM for 6 h), or DR (4 μM for 10h), in 293T cells transiently transfected with constructs carrying Bcl-2, Irod, Oar2, or GFP reporter (GFP), compared with WT cells. (C) Effect on OA-induced 293T cell apoptosis of enforced expression of an active CaMKII peptide inhibitor AIP and an inactive control peptide AC3-C, alone or together with Irod. In some experiments of C, indicated on the abscissa, 20 μM KN93 was present together with OA. Data in A–C are shown with SEM (n = 4).

Figure 4.

Expression of Irod mRNA in human tissues and selected cell lines. (A) Multiple human tissue Northern blot. (B) Northern blot of Jurkat T-cells and two human AML cell lines. The positions of 28S and 18S rRNA is shown as reference. (C) Multiple human tissue expression array. The origin and position of RNA distributed on the array is indicated in the top subpanel. All blots were hybridized with a probe specific for exon 3 of Irod.

Figure 5.

N- and C-Terminally tagged Irod is expressed as an intact fusion protein with antiapoptotic activity. (A) Western blots of 293T cells transiently transfected with pJim-retrovirus vector carrying GFP alone (GFP) or encoding Irod tagged with HA at the N-terminal and fused with GFP at the C-terminal (HA-IROD-GFP), probed with either anti GFP (right lane) or anti HA (left lane). (B) Scanning confocal micrograph of 293T cells transfected with HA-IROD-GFP and illuminated to show GFP expression (left), or the immunofluorescent staining of HA-epitope (middle). The right subpanel is an overlay. The profile (bottom subpanel) represents the fluorescent intensity corresponding to GFP and anti-HA along a section of 13 μm (see bar in the right subpanel). (C) Apoptotic response to OA in 293T cells transiently transfected with HA-IROD-GFP (•), or the empty vector (GFP (○), compared with wild-type cells (▵). Data represents the mean of three separate experiments ± SEM.

Figure 6.

Subcellular localization of Irod. The top (A) panels show 293T cells transfected with GFP reporter alone. The other panels (B–E) show cells expressing HA-Irod-GFP. The left-hand subpanels show the endogenous GFP fluorescence, the middle subpanels show immunofluorescent staining of the HA-eiptope (A and B), of the ER marker calnexin (C), or the Golgi-marker mannosidase II (D). (E) Distribution of the mitochondrial marker MitoTracker. The right-hand subpanels represent merging of the left and middle images.

Figure 7.

Irod devoid of the predicted transmembrane domain localized to the centrosomal/Golgi region. The experiment was conducted as described in the legend to Figure 5B, except that the 293T cells were transfected with HA-Irod-ΔTM and GFP in a bicistronic vector. Whereas GFP was generally distributed in the cell (top), anti-HA immunostaining of HA-IROD-TM (middle) revealed a distinct, perinuclear labeling, suggesting centrosomal/Golgi localization. Photomicrographs and image processing was performed using either scanning confocal Leica NT software (A) or Openlab (Improvision) software packages (B).

Figure 8.

Antiapoptotic effect of Irod is confined to its central domain. (A) Exons 2 and 3 and the domain composition of Irod are shown, and the truncated Irod forms coded for by the constructs used in the experiments in C and D. The numbers refers to amino acid residues. The dotted box represents the leading sequence of Oar2. (B) Rat ortholog of Irod (rIrod/Ian5) and the truncated version, rIAN5(lyp) of it, resulting from the frameshift mutation of this gene in the diabetes-prone BB rat. The 19 residue tail of rIROD(lyp) is not encoded by the rIrod gene. (C) Apoptotic response to OA of 293T cells transiently tranfected with a pJIM-IRES-GFP vector for bicistronic expression of GFP and HA-Irod with deleted transmembrane domain (IROD-ΔTM, ▵), Irod with deleted GTP-binding domain (IROD-ΔGTP, ▴), or intact Irod (IROD, •). (D) Response to 4-h treatment with OA in 293T cells transiently transfected with Oar2 cDNA in the pBabeMN-retrovirus construct or with GFP reporter alone. Data represent the mean of three separate experiments ± SEM. (E) Amino acid sequence alignment of human (accession no. AK002158), rat (accession no. AY055776), and mouse (MacMurray et al., 2002) Irod in a predicted coiled-coil region. *, identical amino acids;:, conserved amino acids.