To investigate ABIN-1’s potential roles in regulating TNF signals, we targeted the gene that encodes ABIN-1, tnip1
, in embryonic stem cells to generate mice deficient for ABIN-1 (Suppl. Fig. 1A–1C
). Deletion of exons 12 through 15 of tnip1
eliminated the full length (approximately 80 kD) ABIN-1 protein (Suppl. Fig. 1D
). While ABIN-1+/− mice appear normal, very few live born ABIN-1−/− mice were obtained from intercrossed ABIN-1+/− parents (). ABIN-1−/− embryos were found in Mendelian ratios at post coital days E12.5 through E18.5, but were only rarely obtained as live born mice ().
ABIN-1 is required for regulating TNF signals in utero.
To better understand how ABIN-1 sustains embryonic development, we noted that ABIN-1−/− embryos were smaller and paler than ABIN-1+/+ littermates at E16.5 and E18.5 (). Quantitation of fetal weights and hematocrits of these embryos revealed that ABIN−/− embryos were significantly smaller and anemic compared to control embryos (). Histological analyses of multiple tissues at various stages of development revealed that fetal livers of E16.5 ABIN-1−/− embryos contained multiple areas of hypocellularity (). Immunohistochemical analyses of caspase 3 cleavage indicated that these areas contained large number of apoptotic cells that were not detected in control embryos (). Thus, ABIN-1 is critical for preventing fetal liver apoptosis and sustaining fetal hematopoiesis.
ABIN-1 is required for embryonic development
The failure to protect embryonic fetal livers from TNF-induced PCD prevents successful development of mice (3
). We thus interbred ABIN-1+/− mice with TNF−/− mice to determine whether ABIN-1 supports embryonic development by regulating TNF-induced signals. Genotyping of live born mice from ABIN-1+/− TNF+/− intercrosses revealed that ABIN-1−/− animals were obtained in roughly Mendelian ratios when animals were homozygous TNF−/− mice (). Therefore, ABIN-1 sustains embryogenesis by regulating TNF signals, possibly by protecting cells against TNF-induced PCD.
To determine whether ABIN-1 directly protects cells from TNF-induced PCD, we tested the susceptibility of ABIN-1 deficient Jurkat T cells, HepG2 hepatoma cells and HT1080 fibrosarcoma cells as well as embryonic fibroblasts (MEFs) to undergo PCD. ABIN-1 deficient cells and ABIN-1−/− MEFS were more sensitive to TNF-induced PCD in the presence or absence of CHX (, Suppl. Figs. 2A, 2B, 2C
). As ABIN-1 is expressed in unstimulated cells, we performed subsequent experiments in the presence of CHX to distinguish ABIN-1’s anti-apoptotic functions from other translation dependent anti-apoptotic mechanisms. ABIN-1 deficient cells exhibited more caspase 8, caspase 3 and Bid cleavage when compared to control cells after treatment with TNF plus CHX ( and Suppl. Fig. 2D
). PCD in ABIN−/− cells was blocked by the caspase inhibitor ZVAD (), confirming that increased PCD in these cells was caspase mediated. Finally, GFP-FLAG-ABIN-1 protected ABIN-1−/− cells from TNF-induced PCD and prevented caspase 8, Bid and caspase 3 cleavage (). These results are consistent with a prior suggestion that ABIN-1 can protect hepatocytes from TNF-induced PCD (8
). Our experiments suggest that ABIN-1 directly protects multiple cell types against TNF-induced PCD.
ABIN-1 is required for protecting cells from TNF-induced PCD
Cells that exhibit deficient NFkB signaling are hypersensitive to TNF-induced PCD (4
). NFkB signaling was not decreased in ABIN-1−/− cells compared to control cells after TNF treatment (, Suppl Figs. 3A, 3B, 3C
). Levels of NFkB dependent survival proteins such as Bcl-xL
, XIAP and A20 were expressed at normal levels in ABIN-1−/− cells (). Prolonged JNK signaling has also been associated with exaggerated PCD after TNF treatment (9
). However, ABIN-1−/− MEFs and ABIN-1+/+ MEFS exhibited similar kinetics of phospho-JNK activation after TNF treatment, as well as normal p38 and ERK signaling (). These experiments suggest that ABIN-1 directly protects cells against TNF-induced PCD, and this function is not secondary to aberrant MAP kinase signaling.
Prior studies suggested that ABIN-1 binds A20, a potent restrictor of TNF-induced NFkB signaling, and that ABIN-1 inhibits NFkB signaling (1
). ABIN-1−/− cells exhibited slightly greater p-IkBa to IkBa ratios and IKKb kinase activity than control cells, while A20−/− cells displayed significantly prolonged NFkB signaling (Suppl. Figs. 3A, 3B
). ABIN-1−/− cells expressed similar levels of NFkB specific DNA binding activity, similar levels of NFkB dependent mRNAs and proteins (Suppl. Figs. 3C, 3D, 3E
). By contrast, A20−/− cells expressed significantly prolonged NFkB DNA binding activity and markedly higher levels of most NFkB target genes and proteins compared with either ABIN-1−/− or control cells (Suppl. Figs. 3C, 3D, and 3E
). Hence, ABIN-1 deficiency reveals a subtle role for ABIN-1 in restricting proximate NFkB signaling, which is associated with minimal affects on NFkB dependent gene transcription. Meanwhile, A20 deficiency leads to markedly prolonged NFkB signaling and increased NFkB dependent gene transcription.
After TNFR engagement, death inducing signaling complexes (DISCs) including TRADD, FADD, and caspase 8 may trigger caspase 8 cleavage and PCD (13
). ABIN-1 is expressed in resting cells, and ABIN-1 regulates TNF-induced PCD in the absence of protein synthesis. We thus hypothesized that ABIN-1 might directly regulate TNF-induced DISCs. We found that ABIN-1 indeed interacts with RIP1 and FADD under conditions that induce PCD (). To determine whether ABIN-1 directly regulates association of DISC proteins, we stimulated ABIN-1 deficient and control cells with TNF, CHX and ZVAD, immunoprecipitated endogenous caspase 8, and examined the association of DISC proteins. Endogenous RIP-1 was recruited normally to caspase 8 under these conditions (). By contrast, the amount of endogenous FADD associated with caspase 8 was significantly greater in ABIN-1 deficient cells, indicating that ABIN-1 is essential for inhibiting caspase 8’s interaction with FADD ().
To further investigate how ABIN-1 may regulate TNF-induced PCD, we noted that a peptide sequence overlapping the ABIN homology domain 2 (AHD2) of ABIN-1 is homologous to a larger, recently defined domain within IKKγ/NEMO that binds polyubiquitin chains and ubiquitylated RIP, called the N
inding (NUB) domain () (15
). In addition, ABIN-1 has a leucine zipper upstream of this domain (). Hypothesizing that ABIN-1, like IKKγ, may be a ubiquitin binding protein, we tested the ability of recombinant ABIN-1 proteins to bind His tagged ubiquitin chains. These studies showed that GST-ABIN-1 binds to His tagged polyubiquitin chains in a GST pull down assay (). GST-ABIN-1 binds both K48 linked as well as K63 linked polyubiquitin chains, and displays a preference for chains that are at least three ubiquitin moieties in length (). GST-ABIN-1 did not bind ubiquitin monomers (). These findings are consistent with a recent study demonstrating binding of ABIN-1 to ubiquitin chains (18
ABIN-1 is a ubiquitin sensor that uses a NUB domain to bind to DISC signaling complexes and protect cells from TNF-induced PCD
To further establish and localize ABIN-1’s ubiquitin binding domain, C-terminal and N-terminal truncation mutants of ABIN-1 were tested for ubiquitin binding. C-terminal GST-ABIN-1, which contains AHD2, binds K63 linked poly-ubiquitin chains while an N-terminal GST-ABIN-1 protein lacking this domain does not (). Several residues within IKKγ’s NUB domain that are required for ubiquitin binding are conserved in ABIN-1. C-terminal ABIN-1 proteins bearing either QQ (glutamine) to EE (glutamic acid) substitutions at positions 477/478 (“QQ477EE”), or a F (phenylalanine) to S (serine) mutation at position 482 (“F482S”) failed to bind ubiquitin chains (). Thus, ABIN-1 is a ubiquitin sensing protein that utilizes a NUB-like domain to bind polyubiquitin chains.
To determine whether ABIN-1’s ubiquitin binding activity mediates ABIN-1’s anti-apoptotic function, we introduced wild-type or mutant forms of FLAG tagged ABIN-1 into ABIN-1−/− 3T3s using a GFP expressing virus, purified productively infected cells by FACS sorting, and tested the cells for susceptibility to TNF-induced PCD. These experiments revealed that wild-type ABIN-1 and a leucine zipper mutant L397P protected ABIN-1−/− cells from TNF-induced PCD (). By contrast, ubiquitin binding domain mutants of ABIN-1 (QQ477EE, F482S, and D485N) failed to fully protect ABIN-1−/− cells from TNF-induced PCD (). To determine whether ABIN-1’s ubiquitin binding activity mediates ABIN-1’s capacity for regulating DISC formation, we tested whether a ubiquitin binding deficient form of ABIN-1 binds to this complex and whether it regulates the association of DISC proteins. Wild type, but not QQ477EE mutant, ABIN-1 was recruited to RIP1 and FADD (). Moreover, wild type, but not QQ477EE mutant, ABIN-1 blocked the recruitment of endogenous FADD to caspase 8 (). Therefore, ABIN-1 requires its ubiquitin binding domain to interact with DISC proteins, to inhibit FADD – caspase 8 binding, and to protect cells from TNF-induced PCD.
ABIN-1 binds to the ubiquitin editing enzyme A20, which also protects cells against TNF-induced PCD, so we investigated whether ABIN-1 might block TNF-induced PCD in an A20 dependent fashion (1
). A20 protein was induced by TNF in both ABIN-1+/+ and ABIN-1−/− MEFs, so ABIN-1 is not required for A20 expression (). A20 association with caspase 8 does not depend upon ABIN-1, arguing against a critical role for ABIN-1 in recruiting A20 to the DISC (). A20 was recruited normally to both wild type and QQ477EE forms of ABIN-1, indicating that ABIN-1 does not require ubiquitin binding to bind A20 (). Finally, wild type, but not QQ477EE mutant, ABIN-1 blocks TNF-induced PCD in A20−/− cells, showing that ABIN-1 does not require A20 to perform its anti-apoptotic function ().
In summary, we have demonstrated that ABIN-1 is a novel ubiquitin sensing protein that inhibits FADD-caspase 8 association in the DISC, protects cells against TNF-induced PCD, and sustains embryonic development. The recent unveiling of non-degradative ubiquitin modifications has led to the discovery of ubiquitin sensors that propagate
NFkB signals (16
). Much less is known about ubiquitin sensors and PCD signaling (22
). Our current studies provide the first evidence that ubiquitin sensors also restrict
signal transduction pathways, and that they regulate PCD signaling. These findings highlight the central roles of ubiquitin sensors in regulating physiological cell survival decisions in vivo.