IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis

Abstract

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.

Figure 1

IAPs carry an evolutionarily conserved UBA domain that mediates Ub binding. (a) Graph shows conservation of amino acid residues of IAPs of the cIAP- and XIAP subtype ranging from Drosophila to humans (see Supplementary Information, Fig. S1 for details). The UBA domain is highlighted in green. Shown below is a schematic representation of cIAPs indicating the location of the UBA domain. (b) Predicted 3D structure of the cIAP2 UBA domain. The 3D structure was modelled with the help of the solution structure of RSGI RUH-011, a UBA domain from Arabidopsis cDNA (PDB code 1vek), as it is the best modelling template for the 3D structure of cIAP2 UBA domain (statistically significant Pcons score 1.250), followed by three other structures (2cpw, 1whc, and 2dkl, see Supplementary Information for details) (c–l) Purified recombinant GST-tagged XIAP, cIAP1, cIAP2 and DIAP2 were immobilized on glutathione-sepharose resin. The indicated monoUb (c) Lys 48-linked (d–f, k) or Lys 63-linked (g–j, l) polyUb chains were added and Ub-binding was assayed by immunoblotting the bound fractions with an anti-Ub antibody. The presence of the GST–IAP was verified by immunoblotting with an anti-GST (c–k) or anti-XIAP (l) antibody.

Figure 2

RING-mediated dimerization of XIAP and cIAP1 is required for Ub binding. (a) Schematic representation of the constructs used. (b) Purified recombinant GST, GST-tagged monoUb and linear tetra-Ub (Ub₄) were immobilized on glutathione-sepharose resin. The indicated amounts of recombinant XIAP were added and the retention of XIAP was assayed by immunoblotting the bound fractions with an anti-XIAP antibody. The presence of GST, GST–Ub and GST–Ub₄ was verified by Coomassie staining. (c, d) Ub-binding assays were performed as described in Fig. 1. (e) Purified recombinant GST or linear Ub₄ were immobilized on glutathione-sepharose resin. Recombinant untagged cIAP1 fragments were added and the retention of cIAP1 was assayed by Coomassie staining and immunoblotting the bound fractions with an anti-cIAP1 antibody.

Figure 3

The UBA is required for IAP function. (a) Inducible cIAP1^WT, but not cIAP1^MF/AA, blocks sensitivity of cIAP1 knockout cells to TNF-α. A cIAP1 knockout MEF line was infected with inducible cIAP1 rescue constructs (iResc). Independent clones of cIAP1^−/− MEFs, reconstituted with either cIAP1^WT (n = 11) or cIAP1^MF/AA (n = 11) were tested for inducible expression of cIAP1 and sensitivity to TNF-α. Representative immunoblots of lysates from MEFs carrying the inducible-rescue constructs are shown to indicate normal levels of cIAP1. The inducible cIAP1 clones were left uninduced or induced for cIAP1 and then treated with TNF-α for 24 h. Cells were stained with PI and analysed by flow cytometry. Error bars are 2 × s.e.m. throughout. P = 0.0002 (two-tailed t-test) for cIAP1^MF/AA tested against cIAP1^WT in induced samples treated with TNF-α. (b–e) WT but not UBA domain mutant cIAP1 rescues the tomato (tom) phenotype caused by loss of z-cIAP1. Embryos from tom^s805 heterozygote intercrosses were injected at the one-cell stage with the indicated amounts of mRNA encoding z-cIAP1, z-cIAP1^MF/AA and z-cIAP1^F645A. Representative images of wild-type (b), tom mutants (c) and tom^s805 -mutants injected with z-cIAP1^WT (d) and z-cIAP1^MF/AA (e) are shown. A control mRNA for H2B–cherry (50 pg) was also used in each injection alone or in addition to the cIAP1 contructs. (f) Histograms show the percentage of rescued tom mutants identified 72 hpf after injection. P = 0.0010 (10 pg), P = 0.0006 (100 pg), P = 0.0051 (200 pg) for z-cIAP1^MF/AA tested against z-cIAP1^WT, and P = 0.0010 (10 pg), P = 0.00004 (100 pg), P = 0.0074 (200 pg) for z-cIAP1^F645A tested against z-cIAP1^WT. Scale bar, 0.25 mm. (g, h) cIAP1 UBA mutants fail to promote liver tumour formation. p53^−/−;Myc liver cells were injected subcutaneously into nude mice (g). Data are mean ± s.d. (n = 4). P = 0.022 (two-tailed t-test) for cIAP1^MF/AA tested against cIAP1^WT at 42 day time point after injection. Immunoblots of p53^−/−;Myc liver cells infected with retrovirus expressing cIAP1^WT or cIAP1^MF/AA mutant. (h) Histopathology of representative tumours from cIAP1^WT.

Figure 4

The ability of XIAP to induce NF-κB is dependent on its UBA domain. (a) Schematic representation of the XIAP constructs used. (b) Decreasing amounts of xiap plasmids (100 ng, 40 ng, 13 ng and 4 ng) and 50 ng of an NF-κB luciferase reporter were co-transfected into HEK293T cells. Data represent mean from one of three independent experiments. Expression levels of XIAP was determined by immunoblotting using anti-XIAP and anti-actin antibodies. The asterisk refers to a cross-reactive band. (c) 150 ng of xiap plasmids and 50 ng of a NF-κB luciferase reporter were co-transfected into HEK293T cells. Deletion of the UBA domain-like mutation of the MGF motif (MF/LL) abrogates XIAP-mediated NF-κB activation (P = 0.00005 and P = 0.0004, respectively). Similarly, mutation of the BIR1 domain (V80D) or RING finger region (F495A) also abrogated NF-κB signalling (P = 0.008 and P = 4.9 × 10⁻⁷, respectively). Of note, the F495A mutation abolishes E3 ligase activity of XIAP (see Supplementary Information, Fig. S3). Data represent mean ± s.d. of four (V80D and F495A) or five independent experiments. Two-tailed t-test was used to determine statistical significance (tested against XIAP^WT). (d–f) The UBA domain is essential for cIAP2–MALT1-mediated NF-κB activation. (d) Schematic representation of the position and frequency of the chromosomal breakpoints in cIAP2 and MALT1 observed in t(11:18)(q21:q21)-positive MALT-lymphomas. Arrowheads show the exon (E) boundaries and position of breakpoints. (e) Schematic representation of cIAP2–MALT1 (case4 variant) used in f. (f) The indicated cIAP2–MALT1 plasmids and a NF-κB luciferase reporter were co-transfected into HEK293T cells. Luciferase activities are expressed relative to wild-type cIAP2–MALT1. Data represent mean ± s.d. of four independent experiments. Two-tailed t-test was used to determine statistical significance (tested against cIAP2–MALT1^WT): MF/AA (P = 1.4 × 10⁻⁵), ΔBIR1 (P = 1.1 × 10⁻⁴), N-term (P = 9.7 × 10⁻¹¹), C-term (P = 1.6 × 10⁻⁷). Expression levels of cIAP2–MALT1 was determined by immunoblotting using anti-FLAG and anti-actin antibodies.

Figure 5

The UBA domain of cIAP2–MALT1 interacts with polyubiquitylated NEMO. (a) cIAP2–MALT1 interacts with Ub conjugates in a UBA-dependent manner. FLAG–cIAP2–MALT1 and FLAG–cIAP2–MALT1^MF/AA immobilized on anti-FLAG–agarose resin were used to pull down HA–Ub conjugates from HEK293T cell lysates expressing HA-tagged Ub. The asterisk denotes a cross-reactive band recognized by the HA-antibody. (b) The UBA domain is not required for cIAP2–MALT1 mediated polyubiquitylation of NEMO. The indicated cIAP2–MALT1 mutants were co-expressed with Strep-tagged-Ub and V5-tagged NEMO. Ubiquitylation of NEMO was determined by purifying Strep–Ub using Strep-Tactin-agarose resin and immunoblotting with an anti-V5 antibody. Note, Strep–Ub also immunoprecipitates non-ubiquitylated NEMO. This is due to the ability of NEMO to form homodimers, and, in this case, associates with ubiquitylated forms of NEMO. Levels of cIAP2–MALT1 and NEMO in lysates were determined using anti-FLAG and anti-V5 antibodies. (c) Self-oligomerization is normal in the cIAP2–MALT1^MF/AA mutant but impaired in cIAP2–MALT1^ΔBIR1. Oligomerization of cIAP2–MALT1 was determined by immunoprecipitation with anti-FLAG-agarose resin and immunoblotting with anti-V5. (d, f) cIAP2–MALT1 traps Lys 63-polyubiquitylated-NEMO. FLAG–cIAP2–MALT1 and FLAG–cIAP2–MALT1^MF/AA immobilized on anti-FLAG-agarose resin were used to pull down unmodified NEMO and ubiquitylated forms of NEMO from HEK293T cellular lysates (d). cIAP2–MALT^MF/AA fails to bind efficiently to polyubiquitylated NEMO. Note, in this assay (lane 8) cIAP2–MALT^MF/AA also binds less efficiently to non-ubiquitylated NEMO. This is probably due to the ability of NEMO to form homodimers whereby the non-modified form may have been co-purified through its association with polyubiquitylated NEMO. Mutation in the UBD of NEMO does not influence the binding of polyubiquitylated NEMO to cIAP2–MALT1. (e) cIAP2–MALT1 interacts with endogenous polyubiquitylated NEMO. The indicated constructs were expressed in HEK293T cells and binding to endogenous polyUb–NEMO was detected by immunoprecipitation with anti-FLAG–agarose resin and immunoblotting with anti-NEMO. (g) Model of cIAP2–MALT1-mediated NF-κB activation: the BIR1 domain of cIAP2 mediates oligomerization of cIAP2–MALT1 permitting recruitment of TRAF6 and unmodified NEMO, which weakly associates with cIAP2–MALT1. Following cIAP2–MALT1-assisted polyubiquitylation of NEMO, Lys 63-linked polyubiquitylated NEMO is tightly bound by cIAP2-MALT1 through UBA domain of cIAP2. This allows recruitment of TAK1, IKK activation and subsequent phosphorylation of IκBα, leading to its degradation and NF-κB-activation.