A novel zinc finger protein interacts with receptor-interacting protein (RIP) and inhibits tumor necrosis factor (TNF)- and IL1-induced NF-kappa B activation.

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.

RIP is a unique signal transducer in the TNF-R1-mediated NF-B activation pathway. RIP was first identified as a Fasinteracting protein by the yeast two-hybrid system (20). It was later demonstrated that RIP is a component of the TNF-R1 signaling complex (4,21). Gene knock-out experiments suggest that RIP is required for TNF-R1-mediated NF-B activation but is not required for Fas-and TNF-R1-mediated apoptosis (22,23). RIP is a serine/threonine kinase that contains three domains, including an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (4,20). RIP interacts with TRADD through their respective death domains. The intermediate domain of RIP interacts with the RING finger domain of TRAF2, and this interaction is required for RIP-mediated NF-B activation (21). Recently, it has been suggested that RIP directly interacts with IKK␥ and therefore recruits IKK to the TNF-R1 complex (24). However, studies with RIP-and TRAF2-deficient cells indicate that TRAF2, but not RIP, is required for recruitment of the IKK complex to TNF-R1, whereas RIP is required for activating IKK (25,26). Although RIP is a serine/threonine kinase, its kinase activity is not required for RIP-mediated NF-B activation (21)(22)(23)25). It has been proposed that RIP may activate IKK through a putative IKK kinase (25), which is probably MEKK3 (27). However, the precise mechanisms responsible for RIP-mediated IKK activation are not known. In addition, it is not known whether or how TNF-R1-mediated NF-B activation pathway is regulated at the level of RIP.

Reagents
FLAG (Sigma), Myc (Santa Cruz Biotechnology, Santa Cruz, CA), and the HA epitopes (Covance, Berkely, CA) were purchased from the indicated resources. The human embryonic kidney 293, the B lymphoma RPMI8226, and the T lymphoma Jurkat cells were purchased from ATCC (Manassas, VA). The rabbit polyclonal antiserum against human ZIN was raised against a 21-mer peptide having the following amino acid sequence: QKEAEEEQKRKNGENTFKRIG.
Constructs-The NF-B (Dr. Gary Johnson, University of Colorado Health Sciences Center) and IRF-1 (Dr. Uli Schindler, Tularik Inc.) luciferase reporter constructs were provided by the indicated investigators. Mammalian expression vectors for HA-or FLAG-tagged RIP, ZIN, and its deletion mutants were constructed by PCR amplification of the corresponding cDNA fragments and subsequently cloning into a CMV promoter-based vector containing a 5Ј-HA or FLAG tag.
Yeast Two-hybrid Screening-To construct a RIP bait vector, a cDNA fragment encoding full-length RIP was inserted in-frame into the Gal4 DNA-binding domain vector pGBT (CLONTECH, Palo Alto, CA). The human B cell cDNA library (ATCC, Manassas, VA) was screened as described (2,3,28).
5Ј RACE-5Ј RACE was performed using a mixture of several yeast two-hybrid cDNA libraries as template. The 5Ј primer corresponds to the sequence of the GAL4 activation domain: ACCGTCGACTGAAGAT-ACCCCACCAAACC. The 3Ј primer corresponds to the coding sequence of ZIN: AAGCGGCCGCCATCAGAAGCGATGC.
Northern Blot Hybridization-Human multiple tissue mRNA blots were purchased from CLONTECH. The cDNA probe was an ϳ1.0-kb fragment that encodes for amino acids 9 -363. The hybridization was performed with the radiolabeled ZIN cDNA probe in the Rapid Hybridization buffer (CLONTECH) under high stringency condition.
Cell Transfection and Reporter Gene Assays-293 cells (ϳ2 ϫ 10 5 ) were seeded on 6-well (35-mm) dishes and were transfected the following day by the standard calcium phosphate precipitation (29). Within the same experiment, each transfection was performed in triplicate, and where necessary, enough of an amount of empty control plasmid was added to ensure that each transfection continued to receive the same amount of total DNA. To normalize for transfection efficiency, 0.3 g of RSV-␤-gal plasmid was added to each transfection. Luciferase reporter assays were performed using a luciferase assay kit (BD PharMingen) and following the manufacturer's protocols. ␤-galactosidase activity was measured using the Galacto-Light chemiluminescent kit (TROPIX, Bedford, MA). Luciferase activities were normalized on the basis of ␤-galactosidase expression levels.
Co-immunoprecipitation and Western Blot Analysis-Transfected 293 cells from each 100-mm dish were lysed in 1 ml of lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation, 0.4-ml aliquots of lysates were incubated with 0.5 g of the indicated monoclonal antibody or control mouse IgG and 25 l of a 1:1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for at least 1 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were fractionated on SDS-PAGE, and subsequent Western blot analyses were performed as described (2,3,28).
Apoptosis Assays-␤-Galactosidase co-transfection assays for determination of cell death were performed as described previously (2,3,10,28,30). Briefly, 293 cells (ϳ2 ϫ 10 5 ) were seeded on 6-well (35-mm) dishes and were transfected the following day with 0.1 g of pCMV-␤galactosidase plasmid and the indicated testing plasmids. Within the same experiment, each transfection was performed in triplicate, and where necessary, enough of an amount of empty control plasmid was added to ensure that each transfection kept receiving the same amount of total DNA. Approximately 24 h after transfection, the cells were stained with X-gal as described previously (30). The numbers of survived blue cells from five representative viewing fields was determined microscopically. Data shown are averages and standard deviations of one representative experiment in which each transfection has been performed in triplicate.
Immunofluorescent Staining-293 cells cultured on glass coverslips were sequentially plunged into methanol and acetone at Ϫ20°C, each for 10 min. Cells were rehydrated in phosphate-buffered saline and stained with primary antibodies for 1 h at room temperature. Cells were then rinsed with phosphate-buffered saline and stained with either a CyTM3conjugated Affinipure donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) or Alexa FluorTM 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for 45 min at room temperature. The cells were rinsed with phosphate-buffered saline and mounted in Gel/Mount TM (Biomeda Corp., Foster City, CA). Cells were observed with a Leica DMR/XA immunofluorescence microscope using ϫ100 plan objective.

RESULTS
Identification of ZIN-To identify potential RIP-interacting proteins, we used the yeast two-hybrid system to screen a human B cell cDNA library with full-length RIP as bait. We screened a total of 5 ϫ 10 6 independent library clones and obtained 26 ␤-galactosidase-positive clones. The inserts of 9 of the 26 clones are not in-frame with the GAL4 activation domain in the library vector. Among the other 17 clones, two encode for FADD, a death domain-containing protein that has been reported to interact with RIP (21), and one encodes part of a novel RING-like zinc finger domain-containing protein, which we designated as ZIN. We further studied ZIN because some of the known RIP-interacting proteins, such as TRAF2 and A20, also contain RING or zinc finger domains.
Since the ZIN clone obtained from the yeast two-hybrid screening is not full-length, we obtained its full-length cDNA by a combination of GenBank TM data base searches for ZINencoding expressed sequence tag clones and 5Ј RACE. These efforts identified a ZIN cDNA of ϳ2.1 kb that is capable of encoding a 488-amino acid protein (Fig. 1A). The 5Ј of the putative start codon (ATG) has an in-frame stop codon, and the 3Ј of the cDNA has a poly(A) tail, suggesting that we obtained a cDNA fragment encoding full-length ZIN (data not shown).
Blast searches of the GenBank TM data bases indicate that ZIN has no significant homolog to known proteins except that the C-terminal part of ZIN is almost identical to an uncharacterized, hypothetical protein called TRIAD3 (GenBank TM accession number (NP_061884). Structural analysis suggests that ZIN contains four RING-like zinc finger domains (RLDs) at the middle (amino acids 137-352) and a proline-rich domain (PRD) at the C terminus (amino acids 396 -482) (Fig. 1A). The N terminus of ZIN has no detectable similarity with any other proteins. The structural properties suggest that ZIN is probably a zinc-binding protein.
Northern blot analysis suggests that RIN is ubiquitously expressed in all examined tissues as two transcripts of ϳ3.0 and ϳ6.0 kB, respectively (Fig. 1B). ZIN is expressed at relatively higher levels in peripheral blood leukocytes and testis (Fig. 1B).
Expression of ZIN Protein in Mammalian Cells-To determine whether ZIN is expressed in mammalian cells at protein level, we raised a rabbit polyclonal antiserum against a peptide corresponding to amino acids 370 -390 of ZIN. Western blot analysis suggests that ZIN is expressed as an ϳ56-kDa protein in all examined human cell lines, including B lymphoma PRMI8226, T lymphoma Jurkat, and embryonic kidney 293 cells (Fig. 2). The size of the endogenous ZIN protein is similar to that of overexpressed ZIN, confirming that the identified ZIN cDNA encodes a full-length protein (Fig. 2). In 293 cells, the ZIN antiserum also recognized a second higher molecular weight band, which may represent a post-translationally mod-ified or alternatively spliced form of ZIN or a different protein in 293 cells.
ZIN Interacts with RIP in Mammalian Cells-To determine whether full-length ZIN interacts with RIP in mammalian cells, we transfected 293 cells with expression plasmids for FLAG-tagged ZIN and HA-tagged RIP and performed co-immunoprecipitation experiments. These experiments suggest that ZIN interacts with RIP in 293 cells (Fig. 3).
To determine which domains of ZIN are required for interaction with RIP, we constructed three deletion mutants of ZIN. These include ZIN-(1-364) that contains the N-terminal domain and the RLDs, ZIN-(127-488) that contains the RLDs and the C-terminal PRD, and ZIN-(365-488) that contains only the C-terminal PRD (Fig. 3A). Transient transfection and coimmunoprecipitation experiments suggest that the two RLDcontaining mutants, ZIN-(1-364) and ZIN-(127-488), but not the RLD-lacking mutant ZIN-(365-488), interact with RIP (Fig. 3B). These data suggest that the RLDs of ZIN are required for interaction with RIP.

FIG. 3. RIP interacts with ZIN and its mutants in 293 cells. A,
construction of ZIN wild-type and deletion mutants. ND, N-terminal domain. FL, full-length. B, interaction between RIP and ZIN or its deletion mutants. 293 cells were transfected with 10 g of expression plasmid for HA-tagged RIP together with 10 g of expression plasmid for FLAG-tagged ZIN or its various mutants. 10 g of expression plasmid for crmA was also added to each transfection to inhibit RIP-induced cell death. Co-immunoprecipitation was performed with anti-FLAG antibody (␣F) or control IgG (C), and Western blot was performed with anti-HA antibody. Expression of RIP was confirmed by Western blot analysis of the lysates (L) with anti-HA antibody (lanes 1, 4, 7, and 10). Expression of ZIN and its mutants was confirmed by Western blot analysis of the lysates with anti-FLAG antibody (lower panel). The experiments were repeated three times, and similar results were obtained. IP, immunoprecipitation; Ab, antibody.

ZIN Inhibits RIP-and IKK␤-induced NF-B Activation-It
has been shown that RIP is absolutely required for TNF-R1induced NF-B activation (4,(21)(22)(23)(24)(25)(26). To determine whether ZIN has a similar function, we performed NF-B luciferase reporter gene assays. These experiments indicated that overexpression of ZIN could not activate NF-B in 293 cells (Fig. 4, A and C). Instead, overexpression of ZIN inhibited RIP-induced NF-B activation in a dose-dependent manner (Fig. 4A). To exclude the possibility that ZIN affects RIP expression but not RIP signaling, we examined RIP levels in the same lysates by Western blot. As shown in Fig. 4A, RIP levels were not significantly changed with the increased expression of ZIN. These data suggest that ZIN inhibits RIP-mediated NF-B activation.
ZIN Potentiates RIP-and TNF-induced Apoptosis-Previously, it has been suggested that overexpression of RIP potently induces apoptosis (20,21). Since ZIN is a RIP-interacting protein, we examined whether ZIN is involved in RIP-induced apoptosis. As shown in Fig. 6A, overexpression of ZIN did not induce apoptosis, but potentiated RIP-induced apoptosis in a dose-dependent manner.
ZIN Does Not Compete with TRAF2 for Binding to RIP-One of the possible explanations for inhibition of RIP-mediated NF-B activation by ZIN is that ZIN may dissociate TRAF2 from RIP. TRAF2 contains one RING finger domain and four zinc finger domains at its N terminus (43). It has been shown that the RING finger domain of TRAF2 interacts with the intermediate domain of RIP and that this interaction is important for TRAF2and RIP-mediated NF-B activation (21). Since the RLDs of ZIN are also responsible for interacting with RIP, we investigated the possibility that ZIN may compete with TRAF2 for binding to RIP. To do this, we transfected 293 cells with constant amounts of

FIG. 4. ZIN inhibits RIP-and IKK␤mediated NF-B activation.
A and B, full-length ZIN (A), but not ZIN-(365-488) (B), inhibits RIP-mediated NF-B activation. 293 cells were transfected with 0.3 g of NF-B-luciferase reporter plasmid, 0.3 g of RSV-␤-gal plasmid, and the indicated amounts (in g) of expression plasmids. 0.5 g of crmA expression plasmid was also added to each transfection to inhibit RIP-induced cell death. ϳ16 h after transfection, luciferase activities were measured and normalized based on ␤-gal levels. C and D, full-length ZIN (C), but not ZIN-(365-488) (D), inhibits IKK␤-mediated NF-B activation. Reporter gene assays were performed as in panels A and B except that RIP was replaced with IKK␤. Data shown are averages and standard deviations of relative luciferase activities from three independent experiments (transfection was performed in triplicate in each experiment). The levels of RIP expression in the transfected cells from one representative experiment are shown under the respective graphs. expression plasmids for TRAF2 and RIP and increased amounts of expression plasmid for ZIN. Co-immunoprecipitation experiments indicated that ZIN could not compete with TRAF2 for interaction with RIP (data not shown).
Colocalization of RIP and ZIN-ZIN has a putative nuclear localization signal sequence (amino acids 47-52). To determine the cellular localization of ZIN, we performed immunofluorescent microscopy. These experiments suggest that ZIN is mainly localized in the cytoplasm (Fig. 7). To determine whether RIP colocalizes with ZIN, we transfected 293 cells with an expression plasmid for HA-tagged RIP and performed double immunofluorescent staining. These experiments suggest that overexpressed RIP overlaps with endogenous ZIN (Fig. 7). In addition, we noticed that overexpression of RIP caused substantial aggregation of ZIN (Fig. 7), pointing to the possibility that overexpression of RIP leads to the formation of complexes that contain RIP, ZIN, and other molecules. DISCUSSION During the past several years, tremendous progress has been achieved on the molecular mechanisms of TNF-R1 signaling. TNF stimulation of TNF-R1 leads to recruitment of the adapter protein TRADD to the TNF-R1 signaling complex (2,4). TRADD recruits FADD and caspase-8 to activate caspase cascades, and this leads to mitochondria-dependent and -independent apoptosis (2, 6 -10, 31-35). TRADD also interacts with TRAF2 and RIP, and these interactions lead to NF-B activation through an IKK-dependent pathway and JNK activation through an MEKK1-MKK4-dependent pathway (2,4,(11)(12)(13)(14)(15)(16)(17)(18)(19). These models have now become paradigms of how all TNF receptor family members signal.
One of the major unsolved questions on TNF-R1 signaling is how TRAF2 and RIP activate downstream IKK. One group proposed a direct interaction between RIP and the IKK␥ subunit of the IKK complex (24). However, studies with RIP-and TRAF2-deficient cells indicate that TRAF2, but not RIP, is required for recruitment of the IKK complex to TNF-R1, whereas RIP is required for activating IKK, probably through MEKK3 (25)(26)(27). Currently, the precise mechanisms responsible for RIP-mediated IKK activation are not known.
We have used the yeast two-hybrid system to identify additional RIP-interacting proteins. This search identified ZIN as a novel RIP-interacting protein. ZIN contains four RLDs at the middle and a proline-rich domain at its C terminus. Overexpression of ZIN inhibits RIP-mediated NF-B activation, and the RLDs of ZIN are required for this inhibitory activity. Unexpectedly, overexpression of ZIN also inhibited IKK␤-mediated NF-B activation. In co-immunoprecipitation experiments, however, we failed to detect an interaction between IKK␤ and ZIN. The simplest explanation for this observation is that ZIN also targets a downstream signaling component of IKK␤.
ZIN can inhibit TNF-induced NF-B activation in 293 cells. Since only TNF-R1, but not TNF-R2, is expressed in 293 cells, these data suggest that ZIN inhibits TNF-R1-induced NF-B activation. This is consistent with the notion that RIP is required for TNF-R1-induced NF-B activation. Surprisingly, ZIN also inhibits IL1-induced NF-B activation. Inhibition of TNF-and IL1-induced NF-B activation is not due to a general inhibitory effect of transcription by ZIN because ZIN does not inhibit IFN-␥-induced IRF-1 activation. Our findings suggest that ZIN has multiple targets in TNF-and IL1-induced NF-B activation pathways. Currently, we do not know which protein(s) in the IL-1 signaling pathway are targeted by ZIN.
The structural and functional properties of ZIN are very similar to a previously characterized protein A20 (24, 36 -42). Although the sequence of ZIN has no significant homology to A20, both contain putative zinc finger structures. A20 can interact with multiple molecules, including TRAF1, -2, and -6, IKK␥/NEMO, and ABIN (24, 36 -42). Overexpression of A20 inhibits TNF-and IL-1-induced NF-B activation (36 -42). Gene knock-out studies have demonstrated a critical role for A20 in inhibition of TNF-induced NF-B activation and inflammation (41). Interestingly, it has been shown that the zinc finger domains of A20 are also required for its inhibition of TNF-and IL-1-induced NF-B activation (40).
Since the RLDs of ZIN are responsible for interacting with RIP, it is possible that ZIN may compete with TRAF2 for binding to RIP and therefore inhibit RIP-mediated NF-B activation. However, co-immunoprecipitation experiments indicate that this is not the case, suggesting that other mechanisms are involved in ZIN-mediated inhibition of RIP-induced NF-B activation.
Overexpression of ZIN potentiates RIP-and TNF-induced apoptosis in 293 cells. Previously, it has been shown that NF-B activation can prevent cells from apoptosis induced by TNF and other stimuli (13, 44 -46). The simplest explanation for ZINЈs potentiation of RIP-and TNF-induced apoptosis is that ZIN inhibits RIP-induced NF-B activation and thus sensitizes cells to apoptosis.
Sequence analysis suggests that a bipartite nuclear localization signal sequence exists at amino acids 36 -53 of ZIN. This raises the possibility that ZIN is a nuclear protein. However, our immunofluorescent staining experiments suggest that ZIN is mainly localized to the cytoplasm. Moreover, these experiments indicate that overexpressed RIP colocalizes with ZIN and causes the aggregation of ZIN. These data provide additional evidences that ZIN is functionally associated with RIP.
In conclusion, we have identified a novel RING-like zinc finger protein that is capable of inhibiting TNF-and IL1induced NF-B activation. The identification of ZIN, like A20, may shed new light on the negative regulation of TNF-and IL1-induced NF-B activation pathways. However, the data provided in this study were mostly from protein overexpression; a physiological role for ZIN needs to be defined by experiments dealing with endogenous protein and/or gene knock-out studies. FIG. 7. ZIN colocalizes with RIP. In the upper panels, untransfected 293 cells were stained with preimmune serum (A), anti-ZIN (B), or peptide antigen preincubated anti-ZIN (C). In the lower panels, 293 cells were transfected with an expression plasmid for HA-tagged RIP. (crmA plasmid was also added to inhibit RIPinduced cell death.) Double immunofluorescent staining was performed with anti-HA (D) and anti-ZIN (E). Panel F is the overlay image of panels D and E. Nuclei were stained with 4Ј,6Ј-diamidino-2-phenylindole.